ALBERT

Description: Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9 and 4 of sialic acid. Recently, a dispute has been opened on its association to autoimmunity. In order to get new insights on human SIAE biology and to clarify its seemingly contradictory molecular properties, we combined in silico characterization, phylogenetic analysis and homology modeling with cellular studies in COS7 cells. Genomic and phylogenetic analysis revealed that in most tissues only the "long" isoform, originally referred to lysosomal sialic acid esterase, is detected. Using the homology modeling approach, we predicted a model of SIAE 3D structure, which fulfills the topological features of SGNH-hydrolase family. In addition, the model and site-directed mutagenesis experiments allowed the definition of the residues involved in catalysis. SIAE transient expression revealed that the protein is glycosylated and is active in vitro as an esterase with a pH optimum corresponding to 8.4–8.5. Moreover, glycosylation influences the biological activity of the enzyme and is essential for release of SIAE into the culture medium. According to these findings, co-localization experiments demonstrated the presence of SIAE in membranous structures corresponding to endoplasmic reticulum and Golgi complex. Thus, at least in COS7 cells, SIAE behaves as a typical secreted enzyme, subjected to glycosylation and located along the classical secretory route or in the extracellular space. In these environments, the enzyme could act on 9- O -acetylated sialic acid residues, contributing to the fine-tuning of the various functions played by this acidic sugar.

Description: Vaccination against the ubiquitous parasite Toxoplasma gondii would provide the most efficient prevention against toxoplasmosis-related congenital, brain and eye diseases in humans. We investigated the immune response elicited by pathogen-specific glycosylphosphatidylinositol (GPI) glycoconjugates using carbohydrate microarrays in a BALB/c mouse model. We further examined the protective properties of the glycoconjugates in a lethal challenge model using the virulent T. gondii RH strain. Upon immunization, mice raised antibodies that bind to the respective GPIs on carbohydrate microarrays, but were mainly directed against an unspecific GPI epitope including the linker. The observed immune response, though robust, was unable to provide protection in mice when challenged with a lethal dose of viable tachyzoites. We demonstrate that anti-GPI antibodies raised against the here described semi-synthetic glycoconjugates do not confer protective immunity against T. gondii in BALB/c mice.

Description: Carbohydrate antigens are valuable as components of vaccines for bacterial infectious agents and human immunodeficiency virus (HIV), and for generating immunotherapeutics against cancer. The crystal structures of anti-carbohydrate antibodies in complex with antigen reveal the key features of antigen recognition and provide information that can guide the design of vaccines, particularly synthetic ones. This review summarizes structural features of anti-carbohydrate antibodies to over 20 antigens, based on six categories of glyco-antigen: (i) the glycan shield of HIV glycoproteins; (ii) tumor epitopes; (iii) glycolipids and blood group A antigen; (iv) internal epitopes of bacterial lipopolysaccharides; (v) terminal epitopes on polysaccharides and oligosaccharides, including a group of antibodies to Kdo-containing Chlamydia epitopes; and (vi) linear homopolysaccharides.

Description: Sialic acids (SAs) are widely expressed on immune cells and their levels and linkages named as sialylation status vary upon cellular environment changes related to both physiological and pathological processes. In this study, we performed a global profiling of the sialylation status of macrophages and their release of SAs in the cell culture medium by using flow cytometry, confocal microscopy and liquid chromatography tandem mass spectrometry (LC-MS/MS). Both flow cytometry and confocal microscopy results showed that cell surface α-2,3-linked SAs were predominant in the normal culture condition and changed slightly upon treatment with atorvastatin for 24 h, whereas α-2,6-linked SAs were negligible in the normal culture condition but significantly increased after treatment. Meanwhile, the amount of total cellular SAs increased about three times (from 369 ± 29 to 1080 ± 50 ng/mL) upon treatment as determined by the LC-MS/MS method. On the other hand, there was no significant change for secreted free SAs and conjugated SAs in the medium. These results indicated that the cell surface α-2,6 sialylation status of macrophages changes distinctly upon atorvastatin stimulation, which may reflect on the biological functions of the cells.

Description: Prion diseases are transmissible neurodegenerative disorders associated with the conversion of the cellular prion protein, PrP C , to a misfolded isoform called PrP Sc . Although PrP Sc is a necessary component of the infectious prion, additional factors, or cofactors, have been shown to contribute to the efficient formation of transmissible PrP Sc . Glycosaminoglycans (GAGs) are attractive cofactor candidates as they can be found associated with PrP Sc deposits, have been shown to enhance PrP misfolding in vitro, are found in the same cellular compartments as PrP C and have been shown to be disease modifying in vivo. Here we investigated the effects of the sulfated GAGs, heparin and heparan sulfate (HS), on disease associated misfolding of full-length recombinant PrP. More specifically, the degree of sulfation of these molecules was investigated for its role in modulating the disease-associated characteristics of PrP. Both heparin and HS induced a β-sheet conformation in recombinant PrP that was associated with the formation of aggregated species; however, the biochemical properties of the aggregates formed in the presence of heparin or HS varied in solubility and protease resistance. Furthermore, these properties could be modified by changes in GAG sulfation, indicating that subtle changes in the properties of prion disease cofactors could initiate disease associated misfolding.

Description: The display of cell-surface glycolipids and glycoproteins is essential for the motility, adhesion and colonization of pathogenic bacteria such as Campylobacter jejuni . Recently, the cell-surface display of C. jejuni glycoconjugates has been the focus of considerable attention; however, our understanding of the roles that glycosylation plays in bacteria still pales in comparison with our understanding of mammalian glycosylation. One of the reasons for this is that carbohydrate metabolic labeling, a powerful tool for studying mammalian glycans, is difficult to establish in bacterial systems and has a significantly more limited scope. Herein, we report the development of an alternative strategy that can be used to study bacterial cell-surface glycoconjugates. Galactose oxidase (GalO) is used to generate an aldehyde at C-6 of terminal GalNAc residues of C. jejuni glycans. This newly generated aldehyde can be conjugated with aminooxy-functionalized purification tags or fluorophores. The label can be targeted towards specific glycoconjugates using C. jejuni mutant strains with N -glycan or lipo-oligosaccharides (LOS) assembly defects. GalO-catalyzed labeling of cell-surface glycoproteins with biotin, allowed for the purification and identification of known extracellular N-linked glycoproteins as well as a recently identified O-linked glycan modifying PorA. To expand the scope of the GalO reaction, live-cell fluorescent labeling of C. jejuni was used to compare the levels of surface-exposed LOS to the levels of N-glycosylated, cell-surface proteins. While this study focuses on the GalO-catalyzed labeling of C. jejuni , it can in principle be used to evaluate glycosylation patterns and identify glycoproteins of interest in any bacteria.

Description: Human sialidases (NEUs) catalyze the removal of N -acetyl neuraminic acids from the glycome of the cell and regulate a diverse repertoire of nominal cellular functions, such as cell signaling and adhesion. A greater understanding of their substrate permissivity is of interest in order to discern their physiological functions in disease states and in the design of specific and effective small molecule inhibitors. Towards this, we have synthesized soluble fluorogenic reporters of mammalian sialidase activity bearing unnatural sialic acids commonly incorporated into the cellular glycocalyx via metabolic glycoengineering. We found cell-surface sialidases in Jurkat capable of cleaving unnatural sialic acids with differential activities toward a variety of R groups on neuraminic acid. In addition, we observed modulated structure–activity relationships when cell-surface sialidases were presented glycans with unnatural bulky, hydrophobic or fluorinated moieties incorporated directly via glycoengineering. Our results confirm the importance of cell-surface sialidases in glycoengineering incorporation data. We demonstrate the flexibility of human NEUs toward derivatized sugars and highlight the importance of native glycan presentation to sialidase binding and activity. These results stand to inform not only metabolic glycoengineering efforts but also inhibitor design.

Description: A defect in the assembly of the oligosaccharide donor (Dol-PP-GlcNAc 2 Man 9 Glc 3 ) for N-linked glycosylation causes hypoglycosylation of proteins by the oligosaccharyltransferase (OST). Mammalian cells express two OST complexes that have different catalytic subunits (STT3A or STT3B). We monitored glycosylation of proteins in asparagine-linked glycosylation 6 (ALG6) deficient cell lines that assemble Dol-PP-GlcNAc 2 Man 9 as the largest oligosaccharide donor. Based upon pulse labeling experiments, 30–40% of STT3A-dependent glycosylation sites and 20% of STT3B-dependent sites are skipped in ALG6-congenital disorders of glycosylation fibroblasts supporting previous evidence that the STT3B complex has a relaxed preference for the fully assembled oligosaccharide donor. Glycosylation of STT3B-dependent sites was more severely reduced in the ALG6 deficient MI8-5 cell line. Protein immunoblot analysis and RT–PCR revealed that MI8-5 cells express 2-fold lower levels of STT3B than the parental Chinese hamster ovary cells. The combination of reduced expression of STT3B and the lack of the optimal Dol-PP-GlcNAc 2 Man 9 Glc 3 donor synergize to cause very severe hypoglycosylation of proteins in MI8-5 cells. Thus, differences in OST subunit expression can modify the severity of hypoglycosylation displayed by cells with a primary defect in the dolichol oligosaccharide assembly pathway.

Description: Xenopus laevis (African clawed frog) has two types of proto-type galectins that are similar to mammalian galectin-1 in amino acid sequence. One type, comprising xgalectin-Ia and -Ib, is regarded as being equivalent to galectin-1, and the other type, comprising xgalectin-Va and -Vb, is expected to be a unique galectin subgroup. The latter is considerably abundant in frog skin; however, its biological function remains unclear. We determined the crystal structures of two proto-type galectins, xgalectin-Ib and -Va. The structures showed that both galectins formed a mammalian galectin-1-like homodimer, and furthermore, xgalectin-Va formed a homotetramer. This tetramer structure has not been reported for other galectins. Gel filtration and other experiments indicated that xgalectin-Va was in a dimer–tetramer equilibrium in solution, and lactose binding enhanced the tetramer formation. The residues involved in the dimer–dimer association were conserved in xgalectin-Va and -Vb, and one of the Xenopus (Silurana) tropicalis proto-type galectins, but not in xgalectin-Ia and -Ib, and other galectin-1-equivalent proteins. Xgalectin-Va preferred Galβ1-3GalNAc and not Galβ1-4GlcNAc, while xgalectin-Ib preferred Galβ1-4GlcNAc as well as human galectin-1. Xgalectin-Va/Vb would have diverged from the galectin-1 group with accompanying acquisition of the higher oligomer formation and altered ligand selectivity.

Description: Legionaminic acids (Leg) are bacterial analogs of neuraminic acid, with the same stereochemistry but different substituents at C5, C7 and C9. Hence they may be incorporated into useful analogs of sialoglycoconjugates, and we previously reported two sialyltransferases that could utilize cytidine monophosphate (CMP)-Leg5Ac7Ac for preparation of Leg glycoconjugates, which were resistant to sialidases [Watson DC, Leclerc S, Wakarchuk WW, Young NM. 2011. Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid. Glycobiology . 21:99–108.]. These were the porcine ST3Gal1 and Pasteurella multocida sialyltransferases. We now report two additional sialyltransferases with superior Leg-transferase properties to the previous two. These are (i) a truncated form of a Photobacterium α2,6-sialyltransferase with an Ala-Met mutation in its active site, and (ii) an α2,3-sialyltransferase from Neisseria meningitidis MC58 with a higher transferase activity than the P. multocida enzyme, with either CMP-Neu5Ac or CMP-Leg5Ac7Ac as the donor. These enzymes will enable the production of useful Leg5Ac7Ac glycoconjugate derivatives with either α2,6 or α2,3 linkages and unique biological properties.

Description: The rare N-unsubstituted glucosamine ( $$\hbox{ GlcN }{{\hbox{ H }}_{3}}^{+}$$ ) residues in heparan sulfate (HS) have important biological and pathophysiological roles. Because of their low natural abundance, the use of chemically generated, structurally defined, N-unsubstituted heparin/HS oligosaccharides can greatly contribute to the investigation of their natural role in HS. However, the sequencing of mixtures of chemically generated oligosaccharides presents major challenges due to the difficulties in separating isomers and the available detection methods. In this study, we developed and validated a simple and sensitive method for the sequence analysis of N-unsubstituted heparin/HS oligosaccharides. This protocol involves pH 4 nitrous acid (HNO 2 ) degradation, size-exclusion HPLC and ion-pair reversed-phase liquid chromatography-ion trap/time-of-flight mass spectrometry (IPRP-LC-ITTOF MS). We unexpectedly found that absorbance at 232 nm (normally used for specific detection of C4–C5 unsaturated oligosaccharides) was, in most cases, still sufficiently sensitive to also simultaneously detect saturated oligosaccharides during HPLC, thus simplifying the positional analysis of $$\hbox{ GlcN }{{\hbox{ H }}_{3}}^{+}$$ residues. The IPRP-LC-ITTOF MS system can supply further structural information leading to full sequence determination of the original oligosaccharide. This new methodology has been used to separate and sequence a variety of chemically generated, N-unsubstituted dp6 species containing between 1 and 3 $$\hbox{ GlcN }{{\hbox{ H }}_{3}}^{+}$$ residues per oligosaccharide in different positional combinations. This strategy offers possibilities for the sequencing of natural N-unsubstituted oligosaccharides from HS and should also be applicable, with minor modification, for sequencing at N-sulfated residues using alternative pH 1.5 HNO 2 scission.

Description: A major aspect of carbohydrate-dependent galectin functionality is their cross-linking capacity. Using a cell surface as biorelevant platform for galectin binding and a panel of 40 glycans as sensor part of a fluorescent polyacrylamide neoglycopolymer for profiling galectin reactivity, properties of related proteins can be comparatively analyzed. The group of the chicken galectins (CGs) is an especially suited system toward this end due to its relatively small size, compared with mammalian galectins. The experiments reveal particularly strong reactivity toward N -acetyllactosamine repeats for all tested CGs and shared reactivity of CG-1A and CG-2 to histo-blood group ABH determinants. In cross-species comparison, CG-1B's properties closely resembled those of human galectin-1, as was the case for the galectin-2 (but not galectin-3) ortholog pair. Although binding-site architectures are rather similar, reactivity patterns can well differ.

Description: The Mediterranean region is a hot spot of climate change vulnerable to increased droughts and heat waves. Scaling carbon fluxes from leaf to landscape levels is particularly challenging under drought conditions. We aimed to improve the mechanistic understanding of the seasonal acclimation of photosynthesis and morphology in sunlit and shaded leaves of four Mediterranean trees ( Quercus ilex L., Pinus halepensis Mill., Arbutus unedo L. and Quercus pubescens Willd.) under natural conditions. V c,max and J max were not constant, and mesophyll conductance was not infinite, as assumed in most terrestrial biosphere models, but varied significantly between seasons, tree species and leaf position. Favourable conditions in winter led to photosynthetic recovery and growth in the evergreens. Under moderate drought, adjustments in the photo/biochemistry and stomatal/mesophyllic diffusion behaviour effectively protected the photosynthetic machineries. Severe drought, however, induced early leaf senescence mostly in A. unedo and Q. pubescens , and significantly increased leaf mass per area in Q. ilex and P. halepensis . Shaded leaves had lower photosynthetic potentials but cushioned negative effects during stress periods. Species-specificity, seasonal variations and leaf position are key factors to explain vegetation responses to abiotic stress and hold great potential to reduce uncertainties in terrestrial biosphere models especially under drought conditions.

Description: Plants experiencing drought stress are frequently more susceptible to pathogens, likely via alterations in physiology that create favorable conditions for pathogens. Common plant responses to drought include the production of reactive oxygen species (ROS) and the accumulation of free amino acids (AAs), particularly proline. These same phenomena also frequently occur during pathogenic attack. Therefore, drought-induced perturbations in AA and ROS metabolism could potentially contribute to the observed enhanced susceptibility. Furthermore, nitrogen (N) availability can influence AA accumulation and affect plant resistance, but its contributions to drought-induced susceptibility are largely unexplored. Here we show that drought induces accumulation of hydrogen peroxide (H 2 O 2 ) in Austrian pine ( Pinus nigra Arnold) shoots, but that shoot infection by the blight and canker pathogen Diplodia sapinea (Fr.) Fuckel leads to large reductions in H 2 O 2 levels in droughted plants. In in vitro assays, H 2 O 2 was toxic to D. sapinea , and the fungus responded to this oxidative stress by increasing catalase and peroxidase activities, resulting in substantial H 2 O 2 degradation. Proline increased in response to drought and infection when examined independently, but unlike all other AAs, proline further increased in infected shoots of droughted trees. In the same tissues, the proline precursor, glutamate, decreased significantly. Proline was found to protect D. sapinea from H 2 O 2 damage, while also serving as a preferred N source in vitro. Fertilization increased constitutive and drought-induced levels of some AAs, but did not affect plant resistance. A new model integrating interactions of proline and H 2 O 2 metabolism with drought and fungal infection of plants is proposed.

Description: Small differences in the sensitivity of stomatal conductance to light intensity on leaf surfaces may lead to large differences in total canopy transpiration ( E C ) with increasing canopy leaf area ( L ). Typically, the increase of L would more than compensate for the decrease of transpiration per unit of leaf area ( E L ), resulting in concurrent increase of E C . However, highly shade-intolerant species, such as Larix principis-rupprechtii Mayr., may be so sensitive to increased shading that such compensation is not complete. We hypothesized that in such a stand, windfall-induced spatial variation at a decameter scale would result in greatly reduced E L in patches of high L leading to lower E C than low competition patches of sparse canopy. We further hypothesized that quicker extraction of soil moisture in patches of lower competition will result in earlier onset of drought symptoms in these patches. Thus, patches of low L will transition from light to soil moisture as the factor dominating E L . This process should progressively homogenize E C in the stand even as the variation of soil moisture is increasing. We tested the hypotheses utilizing sap flux of nine trees, and associated environmental and stand variables. The results were consistent with only some of the expectations. Under non-limiting soil moisture, E L was very sensitive to the spatial variation of L , decreasing sharply with increasing L and associated decrease of mean light intensity on leaf surfaces. Thus, under the conditions of ample soil moisture maximum E C decreased with increasing patch-scale L . Annual E C and biomass production also decreased with L , albeit more weakly. Furthermore, variation of E C among patches decreased as average stand soil moisture declined between rain events. However, contrary to expectation, high L plots which transpired less showed a greater E L sensitivity to decreasing stand-scale soil moisture, suggesting a different mechanism than simple control by decreasing soil moisture. We offer potential explanations to the observed phenomenon. Our results demonstrate that spatial variation of L at decameter scale, even within relatively homogeneous, single-species, even-aged stands, can produce large variation of transpiration, soil moisture and biomass production and should be considered in 1-D soil–plant–atmosphere models.

Description: The main goal of this study was to develop a method for the extraction and indirect estimation of the quantity of calcium oxalate (CaOx) in the foliage of trees. Foliar tissue was collected from a single tree of each species (five conifers and five hardwoods) for comparison of extractions in different solvents using 10 replicates per species from the same pool of tissue. For each species, calcium (Ca) and oxalate were extracted sequentially in double deionized water and 2N acetic acid, and finally, five replicate samples were extracted in 5% (0.83N) perchloric acid (PCA) and the other five in 2N hydrochloric acid (HCl); three cycles of freezing and thawing were used for each solvent. Total ions were extracted by microwave digestion. Calcium was quantified with an inductively coupled plasma emission spectrophotometer method and oxalate was eluted and quantified using a high performance liquid chromatography method. This experiment was repeated again with two conifer and two hardwood species using four trees per species, and two analytical replicates for each tree. We report here that, regardless of age of individual trees within a species, time of collection or species type, the third extraction in PCA or HCl resulted in near equimolar quantities of Ca and oxalate ( r 2  ≥ 0.99). This method provides an easy estimate of the quantity of CaOx crystals using a small sample of foliar tissue. An additional benefit of PCA is that it precipitates the nucleic acids and proteins, allowing the quantification of several free/soluble metabolites such as amino acids, polyamines, organic acids and inorganic elements all from a single sample extract.

Description: Glycosaminoglycans (GAG) play a ubiquitous role in tissues and cells. In eukaryotic cells, heparan sulfate (HS) is initially degraded by an endo-β-glucuronidase called heparanase-1 (HPSE). HS oligosaccharides generated by the action of HPSE intensify the activity of signaling molecules, activating inflammatory response, tumor metastasis, and angiogenesis. The aim of the present study was to understand if sulfated GAG could modulate HPSE, since the mechanisms that regulate HPSE have not been completely defined. CHO-K1 cells were treated with 4-methylumbelliferone (4-MU) and sodium chlorate, to promote total inhibition of GAG synthesis, and reduce the sulfation pattern, respectively. The GAG profile of the wild CHO-K1 cells and CHO-745, deficient in xylosyltransferase, was determined after [ 35 S]-sulfate labeling. HPSE expression was determined via real-time quantitative polymerase chain reaction. Total ablation of GAG with 4-MU in CHO-K1 inhibited HPSE expression, while the lack of sulfation had no effect. Interestingly, 4-MU had no effect in CHO-745 cells for these assays. In addition, a different enzyme location was observed in CHO-K1 wild-type cells, which presents HPSE mainly in the extracellular matrix, in comparison with the CHO-745 mutant cells, which is found in the cytoplasm. In view of our results, we can conclude that GAG are essential modulators of HPSE expression and location. Therefore, GAG profile could impact cell behavior mediated by the regulation of HPSE.

Description: The white-rot fungus Heterobasidion parviporum Niemelä & Korhonen establishes a necrotrophic interaction with Norway spruce ( Picea abies (L.) H.Karst.) causing root and butt rot and growth losses in living trees. The interaction occurs first with the bark and the outer sapwood, as the pathogen enters the tree via wounds or root-to-root contacts. Later, when the fungus reaches the heartwood, it spreads therein creating a decay column, and the interaction mainly occurs in the inner sapwood where the tree creates a reaction zone. While bark and outer sapwood interactions are well studied, little is known about the nature of the transcriptional responses leading to the creation of a reaction zone. In this study, we sampled bark and sapwood both proximal and distal to the reaction zone in artificially inoculated and naturally infected trees. We quantified gene expression levels of candidate genes in secondary metabolite, hormone biosynthesis and signalling pathways using quantitative polymerase chain reaction. An up-regulation of mainly the phenylpropanoid pathway and jasmonic acid biosynthesis was found at the inoculation site, when inoculations were compared with wounding. We found that transcriptional responses in inner sapwood were similar to those reported upon infection through the bark. Our data suggest that the defence mechanism is induced due to direct fungal contact irrespective of the tissue type. Understanding the nature of these interactions is important when considering tree breeding-based resistance strategies to reduce the spread of the pathogen between and within trees.

Description: Global warming and associated decreases in summer rainfall may threaten tree vitality and forest productivity in many regions of the temperate zone in the future. One option for forestry to reduce the risk of failure is to plant genotypes which combine high productivity with drought tolerance. Growth experiments with provenances from different climates indicate that drought exposure can trigger adaptive drought responses in temperate trees, but it is not well known whether and to what extent regional precipitation reduction can increase the drought resistance of a species. We conducted a common garden growth experiment with five European beech ( Fagus sylvatica L.) populations from a limited region with pronounced precipitation heterogeneity (816–544 mm year –1 ), where phylogenetically related provenances grew under small to large water deficits. We grew saplings of the five provenances at four soil moisture levels (dry to moist) and measured ~30 morphological (leaf and root properties, root : shoot ratio), physiological (leaf water status parameters, leaf conductance) and growth-related traits (above- and belowground productivity) with the aim to examine provenance differences in the drought response of morphological and physiological traits and to relate the responsiveness to precipitation at origin. Physiological traits were more strongly influenced by provenance (one-third of the studied traits), while structural traits were primarily affected by water availability in the experiment (two-thirds of the traits). The modulus of leaf tissue elasticity reached much higher values late in summer in plants from moist origins resulting in more rapid turgor loss and a higher risk of hydraulic failure upon drought. While experimental water shortage affected the majority of morphological and productivity-related traits in the five provenances, most parameters related to leaf water status were insensitive to water shortage. Thus, plant morphology, and root growth in particular, did respond to reduced water availability with higher phenotypic plasticity than did physiology. We conclude that beech provenances exposed to different precipitation regimes have developed some genotypic differences with respect to leaf water status regulation, but these adaptations are associated with only minor adaptation in plant morphology and they do not affect the growth rate of the saplings.

Description: Many studies have demonstrated linkages between the occurrence of fog and ecophysiological functioning in cloud forests, but few have investigated hydraulic functioning as a determining factor that explains sharp changes in vegetation. The objective of this study was to compare the plant water status during cloud-immersed and non-immersed conditions and hydraulic vulnerability in branches and roots of species across a temperate, mountain fog ecotone. Because cloud forests are often dark, cool and very moist, we expected cloud forest species to have less drought-tolerant characteristics (i.e., lower P e and P 50 —the pressures required to induce a 12 and 50% loss in hydraulic conductivity, respectively) relative to non-cloud forest species in adjacent (lower elevation) forests. Additionally, due to the ability of cloud forest species to absorb cloud-fog water, we predicted greater improvements in hydraulic functioning during fog in cloud forest species relative to non-cloud forest species. Across the cloud forest ecotone, most species measured were very resistant to losses in conductivity with branch P 50 values from –4.5 to –6.0 MPa, hydraulic safety margins ( min – P 50 ) 〉1.5 MPa and low calculated hydraulic conductivity losses. Roots had greater vulnerabilities, with P 50 values ranging from –1.4 to –2.5 MPa, leading to greater predicted losses in conductivity (~20%). Calculated values suggested strong losses of midday leaf hydraulic conductance in three of the four species, supporting the hydraulic segmentation hypothesis. In both cloud forest and hardwood species, s were greater on foggy days than sunny days, demonstrating the importance of fog periods to plant water balance across fog regimes. Thus, frequent fog did not result in systemic changes in hydraulic functioning or vulnerability to embolism across our temperate cloud forest ecotone. Finally, roots functioned with lower hydraulic conductivity than branches, suggesting that they may serve as more sensitive indicators of hydraulic functioning in these mesic, foggy ecosystems.

Description: Climate warming is having an impact on distribution, acclimation and defence capability of plants. We compared the emission rate and composition of volatile organic compounds (VOCs) from silver birch ( Betula pendula (Roth)) provenances along a latitudinal gradient in a common garden experiment over the years 2012 and 2013. Micropropagated silver birch saplings from three provenances were acquired along a gradient of 7° latitude and planted at central (Joensuu 62°N) and northern (Kolari 67°N) sites. We collected VOCs emitted by shoots and assessed levels of herbivore damage of three genotypes of each provenance on three occasions at the central site and four occasions at the northern site. In 2012, trees of all provenances growing at the central site had higher total VOC emission rates than the same provenances growing at the northern site; in 2013 the reverse was true, thus indicating a variable effect of latitude. Trees of the southern provenance had lower VOC emission rates than trees of the central and northern provenances during both sampling years. However, northward or southward translocation itself had no significant effect on the total VOC emission rates, and no clear effect on insect herbivore damage. When VOC blend composition was studied, trees of all provenances usually emitted more green leaf volatiles at the northern site and more sesquiterpenes at the central site. The monoterpene composition of emissions from trees of the central provenance was distinct from that of the other provenances. In summary, provenance translocation did not have a clear effect in the short-term on VOC emissions and herbivory was not usually intense at the lower latitude. Our data did not support the hypothesis that trees growing at lower latitudes would experience more intense herbivory, and therefore allocate resources to chemical defence in the form of inducible VOC emissions.

Description: Virus-induced gene silencing (VIGS) has been shown to be an effective tool for investigating gene functions in herbaceous plant species, but has rarely been tested in trees. The establishment of a fast and reliable transformation system is especially important for woody plants, many of which are recalcitrant to transformation. In this study, we established a tobacco rattle virus (TRV)-based VIGS system for two Populus species, Populus euphratica and P.   x   canescens . Here, TRV constructs carrying a 266 bp or a 558 bp fragment of the phytoene desaturase (PDS) gene were Agrobacterium -infiltrated into leaves of the two poplar species. Agrobacterium -mediated delivery of the shorter insert, TRV2 -PePDS 266 , into the host poplars resulted in expected photobleaching in both tree species, but not the longer insert, PePDS 558 . The efficiency of VIGS was temperature-dependent, increasing by raising the temperature from 18 to 28 °C. The optimized TRV–VIGS system at 28 °C resulted in a high silencing frequency and efficiency up to 65–73 and 83–94%, respectively, in the two tested poplars. Moreover, syringe inoculation of Agrobacterium in 100 mM acetosyringone induced a more efficient silencing in the two poplar species, compared with other agroinfiltration methods, e.g., direct injection, misting and agrodrench. There were plant species-related differences in the response to VIGS because the photobleaching symptoms were more severe in P.   x   canescens than in P. euphratica. Furthermore, VIGS-treated P. euphratica exhibited a higher recovery rate (50%) after several weeks of the virus infection, compared with TRV-infected P.   x   canescens plants (20%). Expression stability of reference genes was screened to assess the relative abundance of PePDS mRNA in VIGS-treated P. euphratica and P.   x   canescens. PeACT7 was stably expressed in P. euphratica and UBQ-L was selected as the most suitable reference gene for P.   x   canescens using three different statistical approaches, geNorm, NormFinder and BestKeeper. Quantitative real-time PCR showed significant reductions in PDS transcripts (55–64%) in the photobleached leaves of both VIGS-treated poplar species. Our results demonstrate that the TRV-based VIGS provides a practical tool for gene functional analysis in Populus sp., especially in those poplar species which are otherwise recalcitrant to transformation.

Description: Latex, the cytoplasm of laticiferous cells localized in the inner bark of rubber trees ( Hevea brasiliensis Müll. Arg.), is collected by tapping the bark. Following tapping, latex flows out of the trunk and is regenerated, whereas in untapped trees, there is no natural exudation. It is still unknown whether the carbohydrates used for latex regeneration in tapped trees is coming from recent photosynthates or from stored carbohydrates, and in the former case, it is expected that latex carbon isotope composition of tapped trees will vary seasonally, whereas latex isotope composition of untapped trees will be more stable. Temporal variations of carbon isotope composition of trunk latex ( 13 C-L), leaf soluble compounds ( 13 C-S) and bulk leaf material ( 13 C-B) collected from tapped and untapped 20-year-old trees were compared. A marked difference in 13 C-L was observed between tapped and untapped trees whatever the season. Trunk latex from tapped trees was more depleted (1.6 on average) with more variable 13 C values than those of untapped trees. 13 C-L was higher and more stable across seasons than 13 C-S and 13 C-B, with a maximum seasonal difference of 0.7 for tapped trees and 0.3 for untapped trees. 13 C-B was lower in tapped than in untapped trees, increasing from August (middle of the rainy season) to April (end of the dry season). Differences in 13 C-L and 13 C-B between tapped and untapped trees indicated that tapping affects the metabolism of both laticiferous cells and leaves. The lack of correlation between 13 C-L and 13 C-S suggests that recent photosynthates are mixed in the large pool of stored carbohydrates that are involved in latex regeneration after tapping.

Description: Ferritins and other cage proteins have been utilized as models to understand the fundamentals of protein folding and self-assembly. The bacterioferritin (BFR) from Escherichia coli, a maxi-ferritin made up of 24 subunits, was chosen as the basis for a mutagenesis study to investigate the role of electrostatic intermolecular interactions mediated through charged amino acids. Through structural and computational analyses, three charged amino acids R30, D56 and E60 which involved in an electrostatic interaction network were mutated to the opposite charge. Four mutants, R30D, D56R, E60H and D56R-E60H, were expressed, purified and characterized. All of the mutants fold into α-helical structures. Consistent with the computational prediction, they all show a lowered thermostability; double mutant D56R-E60H was found to be 16°C less stable than the wild type. Except for the mutant E60H, all the other mutations completely shut down the formation of protein cages to favour the dimer state in solution. The mutants, however, retain their ability to form cage-like nanostructures in the dried, surface immobilized conditions of transmission electron microscopy. Our findings confirm that even a single charge-inversion mutation at the 2-fold interface of BFR can affect the quaternary structure of its dimers and their ability to self-assemble into cage structures.

Description: Most of bacteria can swim by rotating flagella bidirectionally. The C ring, located at the bottom of the flagellum and in the cytoplasmic space, consists of FliG, FliM and FliN, and has an important function in flagellar protein secretion, torque generation and rotational switch of the motor. FliG is the most important part of the C ring that interacts directly with a stator subunit. Here, we introduced a three-amino acids in-frame deletion mutation (PSA) into FliG from Vibrio alginolyticus , whose corresponding mutation in Salmonella confers a switch-locked phenotype, and examined its phenotype. We found that this FliG mutant could not produce flagellar filaments in a fliG null strain but the FliG(PSA) protein could localize at the cell pole as does the wild-type protein. Unexpectedly, when this mutant was expressed in a wild-type strain, cells formed flagella efficiently but the motor could not rotate. We propose that this different phenotype in Vibrio and Salmonella might be due to distinct interactions between FliG mutant and FliM in the C ring between the bacterial species.

Description: Sulphation is known to be critically involved in the metabolism of acetaminophen in vivo . This study aimed to systematically identify the major human cytosolic sulfotransferase (SULT) enzyme(s) responsible for the sulphation of acetaminophen. A systematic analysis showed that three of the twelve human SULTs, SULT1A1, SULT1A3 and SULT1C4, displayed the strongest sulphating activity towards acetaminophen. The pH dependence of the sulphation of acetaminophen by each of these three SULTs was examined. Kinetic parameters of these three SULTs in catalysing acetaminophen sulphation were determined. Moreover, sulphation of acetaminophen was shown to occur in HepG2 human hepatoma cells and Caco-2 human intestinal epithelial cells under the metabolic setting. Of the four human organ samples tested, liver and intestine cytosols displayed considerably higher acetaminophen-sulphating activity than those of lung and kidney. Collectively, these results provided useful information concerning the biochemical basis underlying the metabolism of acetaminophen in vivo previously reported.

Description: In this study, the physicochemical and enzymatic properties of recombinant human ubiquitin (Ub)-specific protease (USP) 47, a novel member of the C19 family of de-ubiquitinating enzymes (DUB), were characterized for the first time. Recombinant human USP47 was expressed in a baculovirus expression system and purified to homogeneity. The purified protein was shown to be a monomeric protein with a molecular mass of ~146 kDa on sodium dodecyl sulphate—polyacrylamide gel electrophoresis. USP47 released Ub from Ub-aminoacyl-4-metheylcoumaryl-7-amide and Ub-tagged granzyme B. The substitution of the potential nucleophile Cys109 with Ser severely abrogated the Ub-releasing activity of USP47, indicating that USP47 is indeed a cysteine DUB. An assay using Ub dimer substrates showed that the enzyme cleaved a variety of isopeptide bonds between 2 Ub molecules, including the Lys48- and Lys63-linked isopeptide bonds. USP47 also released a Ub moiety from Lys48- and Lys63-linked polyUb chains. Of the inhibitors tested, N -ethylmaleimide, Zn ion and Ub aldehyde revealed a dose-dependent inhibition of USP47. In this study, clear differences in the enzymatic properties between USP47 and USP7 (the most closely related proteins among DUBs) were also found. Therefore, our results suggest that USP47 may play distinct roles in Ub-mediated cellular processes via DUB activity.

Description: P24 antigen is the main structural protein of HIV-1, its detection provide a means to aid the early diagnosis of HIV-1 infection. The aim of this study was to improve the selectivity and sensitivity of the HIV P24 diagnostic assay by developing a cohort of 9E8 affinity-matured antibodies through in vitro phage affinity maturation which was performed by complementarity determining region (CDR)-hot spot mutagenesis strategy. Antibody 9E8-491 had an affinity constant of 5.64 x 10 –11 M, which was 5.7-fold higher than that of the parent antibody (9E8). Furthermore, the affinity, sensitivity and specificity of 9E8-491 were higher than those of 9E8, which indicate that 9E8-491 is a good candidate detection antibody for HIV P24 assay. Structure analysis of matured variants revealed that most hydrogen bonds resided in HCDR3. Among the antibody–antigen predicted binding residues, Tyr 100A/100B was the original conserved residue that was commonly present in HCDR3 of 9E8 and variants. Arg 100 /Asp 100C was the major variant substitution that most likely influenced the binding differences among variants and 9E8 monoclonal antibody. Both efficient library panning and predicted structural data were in agreement that the binding residues were mostly located in HCDR3 and enabled identification of key residues that influence antibody affinity.

Description: Trees contain non-structural carbon (NSC), but it is unclear for how long these reserves are stored and to what degree they are used to support plant activity. We used radiocarbon ( 14 C) to show that the carbon (C) in stemwood NSC can achieve ages of several decades in California oaks. We separated NSC into two fractions: soluble (~50% sugars) and insoluble (mostly starch) NSC. Soluble NSC contained more C than insoluble NSC, but we found no consistent trend in the amount of either pool with depth in the stem. There was no systematic difference in C age between the two fractions, although ages increased with stem depth. The C in both NSC fractions was consistently younger than the structural C from which they were extracted. Together, these results indicate considerable inward mixing of NSC within the stem and rapid exchange between soluble and insoluble pools, compared with the timescale of inward mixing. We observed similar patterns in sympatric evergreen and deciduous oaks and the largest differences among tree stems with different growth rates. The 14 C signature of carbon dioxide (CO 2 ) emitted from tree stems was higher than expected from very recent photoassimilates, indicating that the mean age of C in respiration substrates included a contribution from C fixed years previously. A simple model that tracks NSC produced each year, followed by loss (through conversion to CO 2 ) in subsequent years, matches our observations of inward mixing of NSC in the stem and higher 14 C signature of stem CO 2 efflux. Together, these data support the idea of continuous accumulation of NSC in stemwood and that ‘vigor’ (growth rate) and leaf habit (deciduous vs evergreen) control NSC pool size and allocation.

Description: Gibberellins (GAs) are important regulators of plant shoot biomass growth, and GA 20-oxidase (GA20ox) is one of the major regulatory enzymes in the GA biosynthetic pathway. Previously, we showed that the expression levels of a putative GA20ox1 (i.e., PdGA20ox1 ) in stem tissue of 3-month-old seedlings of 12 families of Pinus densiflora were positively correlated with stem diameter growth across those same families growing in an even-aged 32-year-old pine forest (Park EJ, Lee WY, Kurepin LV, Zhang R, Janzen L, Pharis RP (2015) Plant hormone-assisted early family selection in Pinus densiflora via a retrospective approach. Tree Physiol 35:86–94). To further investigate the molecular function of this gene in the stem wood growth of forest trees, we produced transgenic poplar lines expressing PdGA20ox1 under the control of the 35S promoter (designated as 35S::PdGA20ox1). By age 3 months, most of the 35S::PdGA20ox1 poplar trees were showing an exceptional enhancement of stem wood growth, i.e., up to fourfold increases in stem dry weight, compared with the nontransformed control poplar plants. Significant increases in endogenous GA 1 , its immediate precursor (GA 20 ) and its catabolite (GA 8 ) in elongating internode tissue accompanied the increased stem growth in the transgenic lines. Additionally, the development of gelatinous fibers occurred in vertically grown stems of the 35S::PdGA20ox1 poplars. An analysis of the cell wall monosaccharide composition of the 35S::PdGA20ox1 poplars showed significant increases in xylose and glucose contents, indicating a qualitative increase in secondary wall depositions. Microarray analyses led us to find a total of 276 probe sets that were upregulated (using threefold as a threshold) in the stem tissues of 35S::PdGA20ox1 poplars relative to the controls. ‘Cell organization or biogenesis’- and ‘cell wall’-related genes were overrepresented, including many of genes that are involved in cell wall modification. Several transcriptional regulators, which positively regulate cell elongation through GA signaling, were also upregulated. In contrast, genes involved in defense signaling were appreciably downregulated in the 35S::PdGA20ox1 stem tissues, suggesting a growth versus defense trade-off. Taken together, our results suggest that PdGA20ox1 functions to promote stem growth and wood formation in poplar, probably by activating GA signaling while coincidentally depressing defense signaling.

Description: Bark beetles (Coleoptera: Curculionidae, Scolytinae) cause widespread tree mortality in coniferous forests worldwide. Constitutive and induced host defenses are important factors in an individual tree’s ability to survive an attack and in bottom-up regulation of bark beetle population dynamics, yet quantifying defense levels is often difficult. For example, in Pinus spp., resin flow is important for resistance to bark beetles but is extremely variable among individuals and within a season. While resin is produced and stored in resin ducts, the specific resin duct metrics that best correlate with resin flow remain unclear. The ability and timing of some pine species to produce induced resin is also not well understood. We investigated (i) the relationships between ponderosa pine ( Pinus ponderosa Lawson & C. Lawson) resin flow and axial resin duct characteristics, tree growth and physiological variables, and (ii) if mechanical wounding induces ponderosa pine resin flow and resin ducts in the absence of bark beetles. Resin flow increased later in the growing season under moderate water stress and was highest in faster growing trees. The best predictors of resin flow were nonstandardized measures of resin ducts, resin duct size and total resin duct area, both of which increased with tree growth. However, while faster growing trees tended to produce more resin, models of resin flow using only tree growth were not statistically significant. Further, the standardized measures of resin ducts, density and duct area relative to xylem area, decreased with tree growth rate, indicating that slower growing trees invested more in resin duct defenses per unit area of radial growth, despite a tendency to produce less resin overall. We also found that mechanical wounding induced ponderosa pine defenses, but this response was slow. Resin flow increased after 28 days, and resin duct production did not increase until the following year. These slow induced responses may allow unsuccessfully attacked or wounded trees to resist future bark beetle attacks. Forest management that encourages healthy, vigorously growing trees will also favor larger resin ducts, thereby conferring increased constitutive resistance to bark beetle attacks.

Description: Many skeletal diseases have common pathological phenotype of defective osteogenesis of bone marrow stromal cells (BMSCs), in which histone modifications play an important role. However, few studies have examined the dynamics of distinct histone modifications during osteogenesis. In this study, we examined the dynamics of H3K9/K14 and H4K12 acetylation; H3K4 mono-, di- and tri-methylation; H3K9 di-methylation and H3K27 tri-methylation in osteogenic genes, runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase, bone sialoprotein and osteocalcin, during C3H10T1/2 osteogenesis. H3 and H4 acetylation and H3K4 di-methylation were elevated, and H3K9 di-methylation and H3K27 tri-methylation were reduced in osteogenic genes during C3H10T1/2 osteogenesis. C3H10T1/2 osteogenesis could be modulated by altering the patterns of H3 and H4 acetylation and H3K27 tri-methylation. In a glucocorticoid-induced osteoporosis mouse model, we observed the attenuation of osteogenic potential of osteoporotic BMSCs in parallel with H3 and H4 hypo-acetylation and H3K27 hyper-tri-methylation in Runx2 and Osx genes. When H3 and H4 acetylation was elevated, and H3K27 tri-methylation was reduced, the attenuated osteogenic potential of osteoporotic BMSCs was rescued effectively. These observations provide a deeper insight into the mechanisms of osteogenic differentiation and the pathophysiology of osteoporosis and can be used to design new drugs and develop new therapeutic methods to treat skeletal diseases.

Description: Dihydrouridine (D) is formed by tRNA dihydrouridine synthases (Dus). In mesophiles, multiple Dus enzymes bring about D modifications at several positions in tRNA. The extreme-thermophilic eubacterium Thermus thermophilus , in contrast, has only one dus gene in its genome and only two D modifications (D20 and D20a) in tRNA have been identified. Until now, an in vitro assay system for eubacterial Dus has not been reported. In this study, therefore, we constructed an in vitro assay system using purified Dus. Recombinant T. thermophilus Dus lacking bound tRNA was successfully purified. The in vitro assay revealed that no other factors in living cells were required for D formation. A dus gene disruptant ( dus ) strain of T. thermophilus verified that the two D20 and D20a modifications in tRNA were derived from one Dus protein. The dus strain did not show growth retardation at any temperature. The assay system showed that Dus modified tRNA Phe transcript at 60°C, demonstrating that other modifications in tRNA are not essential for Dus activity. However, a comparison of the formation of D in native tRNA Phe purified from the dus strain and tRNA Phe transcript revealed that other tRNA modifications are required for D formation at high temperatures.

Description: Human lactate dehydrogenase (LDH) has attracted attention as a potential target for cancer therapy and contraception. In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae , with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even though LDHC did not generate lactate at the levels seen with LDHA. In contrast, the human LDHB did not complement the yeast pdc null mutant, although LDHB did generate lactate in yeast cells. Expression of LDHB as a red fluorescent protein (RFP) fusion yielded blebs in yeast, whereas LDHA-RFP and LDHC-RFP fusion proteins exhibited cytosolic distribution. Thus, LDHB exhibits several unique features when expressed in yeast cells. Because yeast cells are amenable to genetic analysis and cell-based high-throughput screening, our pdc /LDH strains are expected to be of use for versatile analyses of human LDH.

Description: RelB is activated by the non-canonical NF-B pathway, which is crucial for immunity by establishing lymphoid organogenesis and B-cell and dendritic cell (DC) maturation. To elucidate the mechanism of the RelB-mediated immune cell maturation, a precise understanding of the relationship between cell maturation and RelB expression and activation at the single-cell level is required. Therefore, we generated knock-in mice expressing a fusion protein between RelB and fluorescent protein (RelB-Venus) from the Relb locus. The Relb Venus / Venus mice developed without any abnormalities observed in the Relb –/– mice, allowing us to monitor RelB-Venus expression and nuclear localization as RelB expression and activation. Relb Venus / Venus DC analyses revealed that DCs consist of RelB – , RelB low and RelB high populations. The RelB high population, which included mature DCs with projections, displayed RelB nuclear localization, whereas RelB in the RelB low population was in the cytoplasm. Although both the RelB low and RelB – populations barely showed projections, MHC II and co-stimulatory molecule expression were higher in the RelB low than in the RelB – splenic conventional DCs. Taken together, our results identify the RelB low population as a possible novel intermediate maturation stage of cDCs and the Relb Venus / Venus mice as a useful tool to analyse the dynamic regulation of the non-canonical NF-B pathway.

Description: Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea . Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles.

Description: Temperature responses and sensitivity of photosynthesis ( A n _ T ) and respiration for leaves at different ages are crucial to modeling ecosystem carbon (C) cycles and productivity of evergreen forests. Understanding the mechanisms and processes of temperature sensitivity may further shed lights on temperature acclimation of photosynthesis and respiration with leaf aging. The current study examined temperature responses of photosynthesis and respiration of young leaves (YLs) (fully expanded in current growth season) and old leaves (OLs) (fully expanded in last growth season) of Quercus aquifolioides Rehder and E.H. Wilson in an alpine oak forest, southwestern China. Temperature responses of dark respiration ( R dark ), net assimilation ( A n ), maximal velocity of carboxylation ( V cmax ) and maximum rate of electron transport ( J max ) were significantly different between the two leaf ages. Those differences implied different temperature response parameters should be used for leaves of different ages in modeling vegetation productivity and ecosystem C cycles in Q. aquifolioides forests and other evergreen forests. We found that RuBP carboxylation determined the downward shift of A n _ T in OLs, while RuBP regeneration and the balance between Rubisco carboxylation and RuBP regeneration made little contribution. Sensitivity of stomatal conductance to vapor pressure deficit changed in OLs and compensated part of the downward shift. We also found that OLs of Q. aquifolioides had lower A n due to lower stomatal conductance, higher stomatal conductance limitation and deactivation of the biochemical processes. In addition, the balance between R dark and A n changed between OLs and YLs, which was represented by a higher R dark / A n ratio for OLs.

Description: Plants allocate carbon (C) to sink tissues depending on phenological, physiological or environmental factors. We still have little knowledge on C partitioning into various cellular compounds and metabolic pathways at various ecophysiological stages. We used compound-specific stable isotope analysis to investigate C partitioning of freshly assimilated C into tree compartments (needles, branches and stem) as well as into needle water-soluble organic C (WSOC), non-hydrolysable structural organic C (stOC) and individual chemical compound classes (amino acids, hemicellulose sugars, fatty acids and alkanes) of Norway spruce ( Picea abies ) following in situ 13 C pulse labelling 15 days after bud break. The 13 C allocation within the above-ground tree biomass demonstrated needles as a major C sink, accounting for 86% of the freshly assimilated C 6 h after labelling. In needles, the highest allocation occurred not only into the WSOC pool (44.1% of recovered needle 13 C) but also into stOC (33.9%). Needle growth, however, also caused high 13 C allocation into pathways not involved in the formation of structural compounds: (i) pathways in secondary metabolism, (ii) C-1 metabolism and (iii) amino acid synthesis from photorespiration. These pathways could be identified by a high 13 C enrichment of their key amino acids. In addition, 13 C was strongly allocated into the n -alkyl lipid fraction (0.3% of recovered 13 C), whereby 13 C allocation into cellular and cuticular exceeded that of epicuticular fatty acids. 13 C allocation decreased along the lipid transformation and translocation pathways: the allocation was highest for precursor fatty acids, lower for elongated fatty acids and lowest for the decarbonylated n -alkanes. The combination of 13 C pulse labelling with compound-specific 13 C analysis of key metabolites enabled tracing relevant C allocation pathways under field conditions. Besides the primary metabolism synthesizing structural cell compounds, a complex network of pathways consumed the assimilated 13 C and kept most of the assimilated C in the growing needles.

Description: Non-structural carbohydrates (NSC) in plant tissue are frequently quantified to make inferences about plant responses to environmental conditions. Laboratories publishing estimates of NSC of woody plants use many different methods to evaluate NSC. We asked whether NSC estimates in the recent literature could be quantitatively compared among studies. We also asked whether any differences among laboratories were related to the extraction and quantification methods used to determine starch and sugar concentrations. These questions were addressed by sending sub-samples collected from five woody plant tissues, which varied in NSC content and chemical composition, to 29 laboratories. Each laboratory analyzed the samples with their laboratory-specific protocols, based on recent publications, to determine concentrations of soluble sugars, starch and their sum, total NSC. Laboratory estimates differed substantially for all samples. For example, estimates for Eucalyptus globulus leaves (EGL) varied from 23 to 116 (mean = 56) mg g –1 for soluble sugars, 6–533 (mean = 94) mg g –1 for starch and 53–649 (mean = 153) mg g –1 for total NSC. Mixed model analysis of variance showed that much of the variability among laboratories was unrelated to the categories we used for extraction and quantification methods (method category R 2 = 0.05–0.12 for soluble sugars, 0.10–0.33 for starch and 0.01–0.09 for total NSC). For EGL, the difference between the highest and lowest least squares means for categories in the mixed model analysis was 33 mg g –1 for total NSC, compared with the range of laboratory estimates of 596 mg g –1 . Laboratories were reasonably consistent in their ranks of estimates among tissues for starch ( r = 0.41–0.91), but less so for total NSC ( r = 0.45–0.84) and soluble sugars ( r = 0.11–0.83). Our results show that NSC estimates for woody plant tissues cannot be compared among laboratories. The relative changes in NSC between treatments measured within a laboratory may be comparable within and between laboratories, especially for starch. To obtain comparable NSC estimates, we suggest that users can either adopt the reference method given in this publication, or report estimates for a portion of samples using the reference method, and report estimates for a standard reference material. Researchers interested in NSC estimates should work to identify and adopt standard methods.

Description: Glycomics may assist in uncovering the structure–function relationships of protein glycosylation and identify glycoprotein markers in colorectal cancer (CRC) research. Herein, we performed label-free quantitative glycomics on a carbon-liquid chromatography–tandem mass spectrometry-based analytical platform to accurately profile the N-glycosylation changes associated with CRC malignancy. N -Glycome profiling was performed on isolated membrane proteomes of paired tumorigenic and adjacent non-tumorigenic colon tissues from a cohort of five males (62.6 ± 13.1 y.o.) suffering from colorectal adenocarcinoma. The CRC tissues were typed according to their epidermal growth factor receptor (EGFR) status by western blotting and immunohistochemistry. Detailed N -glycan characterization and relative quantitation identified an extensive structural heterogeneity with a total of 91 N -glycans. CRC-specific N-glycosylation phenotypes were observed including an overrepresentation of high mannose, hybrid and paucimannosidic type N -glycans and an under-representation of complex N -glycans ( P 〈 0.05). Sialylation, in particular α2,6-sialylation, was significantly higher in CRC tumors relative to non-tumorigenic tissues, whereas α2,3-sialylation was down-regulated ( P 〈 0.05). CRC stage-specific N-glycosylation was detected by high α2,3-sialylation and low bisecting β1,4-GlcNAcylation and Lewis-type fucosylation in mid-late relative to early stage CRC. Interestingly, a novel link between the EGFR status and the N-glycosylation was identified using hierarchical clustering of the N -glycome profiles. EGFR-specific N -glycan signatures included high bisecting β1,4-GlcNAcylation and low α2,3-sialylation (both P 〈 0.05) relative to EGFR-negative CRC tissues. This is the first study to correlate CRC stage and EGFR status with specific N -glycan features, thus advancing our understanding of the mechanisms causing the biomolecular deregulation associated with CRC.

Description: Fucosylated chondroitin sulfate (FCS) is a glycosaminoglycan found in sea cucumbers. It has a backbone like that of mammalian chondroitin sulfate (4-β- d -GlcA-1-〉3-β- d -GalNAc-1) n but substituted at the 3rd position of the β- d -glururonic acid residues with α-fucose branches. The structure of these branches varies among FCSs extracted from different species of sea cucumbers, as revealed by solution NMR spectroscopy. Some species ( Isostichopus badionotus and Patalus mollis ) contain branches formed by single α-fucose residues but with variable sulfation patterns (2,4-, 3,4- and 4-sulfation). FCS from Ludwigothurea grisea is distinguished because it contains preponderant branches formed by disaccharide units containing non-sulfated and 3-sulfated α-fucose units at the reducing and non-reducing ends, respectively. Despite the structural variability on their α-fucose branches, these FCSs have similar anticoagulant action on assays using purified reagents. They have serpin-dependent and serpin-independent effects. Pharmacological assays using experimental animals showed that the three types of FCSs have similar antithrombotic effect and bleeding tendency. They also activate factor XII on the same range of concentration. Based on these observations, we proposed that only few sulfated α-fucose branches along the FCS chain are enough to assure the binding of this glycosaminoglycan to proteins of the coagulation system. Substitution with additional sulfated α-fucose does not increase further the activity. Overall, the use of FCSs with marked variability on their branches of α-fucose allowed us to establish correlations between structures vs biological effects of these glycosaminoglycans on a more refined basis. It opens new avenues for therapeutic intervention using FCSs.

Description: Enzymes that affect glycoproteins of the human immune system, and thereby modulate defense responses, are abundant among bacterial pathogens. Two endoglycosidases from the human pathogen Streptococcus pyogenes , EndoS and EndoS2, have recently been shown to hydrolyze N-linked glycans of human immunoglobulin G. However, detailed characterization and comparison of the hydrolyzing activities have not been performed. In the present study, we set out to characterize the enzymes by comparing the activities of EndoS and EndoS2 on a selection of therapeutic monoclonal antibodies (mAbs), cetuximab, adalimumab, panitumumab and denosumab. By analyzing the glycans hydrolyzed by EndoS and EndoS2 from the antibodies using matrix-assisted laser desorption ionization time of flight, we found that both the enzymes cleaved complex glycans and that EndoS2 hydrolyzed hybrid and oligomannose structures to a greater extent compared with EndoS. A comparison of ultra-high-performance liquid chromatography (LC) profiles of the glycan pool of cetuximab hydrolyzed with EndoS and EndoS2 showed that EndoS2 hydrolyzed hybrid and oligomannose glycans, whereas these peaks were missing in the EndoS chromatogram. We utilized this difference in glycoform selectivity, in combination with the IdeS protease, and developed a LC separation method to quantify high mannose content in the Fc fragments of the selected mAbs. We conclude that EndoS and EndoS2 hydrolyze different glycoforms from the Fc-glycosylation site on therapeutic mAbs and that this can be used for rapid quantification of high mannose content.

Description: Calnexin (CNX), known as a lectin chaperone located in the endoplasmic reticulum (ER), specifically recognizes G 1 M 9 GN 2 -proteins and facilitates their proper folding with the assistance of ERp57 in mammalian cells. However, it has been left unidentified how CNX works in Aspergillus oryzae , which is a filamentous fungus widely exploited in biotechnology. In this study, we found that a protein disulfide isomerase homolog TigA can bind with A. oryzae CNX (AoCNX), which was revealed to specifically recognize monoglucosylated glycans, similarly to CNX derived from other species, and accelerate the folding of G 1 M 9 GN 2 -ribonuclease (RNase) in vitro. For refolding experiments, a homogeneous monoglucosylated high-mannose-type glycoprotein G 1 M 9 GN 2 -RNase was chemoenzymatically synthesized from G 1 M 9 GN-oxazoline and GN-RNase. Denatured G 1 M 9 GN 2 -RNase was refolded with highest efficiency in the presence of both soluble form of AoCNX and TigA. TigA contains two thioredoxin domains with CGHC motif, mutation analysis of which revealed that the one in N-terminal regions is involved in binding to AoCNX, while the other in catalyzing protein refolding. The results suggested that in glycoprotein folding process of A. oryzae , TigA plays a similar role as ERp57 in mammalian cells, as a partner protein of AoCNX.

Description: Polysialic acid (polySia) is a linear polymer of sialic acid that modifies neural cell adhesion molecule (NCAM) in the vertebrate brain. PolySia is a large and exclusive molecule that functions as a negative regulator of cell–cell interactions. Recently, we demonstrated that polySia can specifically bind fibroblast growth factor 2 (FGF2) and BDNF; however, the protective effects of polySia on the proteolytic cleavage of these proteins remain unknown, although heparin/heparan sulfate has been shown to impair the cleavage of FGF2 by trypsin. Here, we analyzed the protective effects of polySia on the proteolytic cleavage of FGF2 and proBDNF/BDNF. We found that polySia protected intact FGF2 from tryptic activity via the specific binding of extended polySia chains on NCAM to FGF2. Oligo/polySia also functioned to impair the processing of proBDNF by plasmin via binding of oligo/polySia chains on NCAM. In addition, the polySia structure synthesized by mutated polysialyltransferase, ST8SIA2/STX(SNP7), which was previously identified from a schizophrenia patient, was impaired for these functions compared with polySia produced by normal ST8SIA2. Taken together, these data suggest that the protective effects of polySia toward FGF2 and proBDNF may be involved in the regulation of the concentrations of these neurologically active molecules.

Description: The scaffolding protein Salvador (Sav) plays a key role in the Hippo (Hpo) signalling pathway, which controls tissue growth by inhibiting cell proliferation and promoting apoptosis. Dysregulation of the Hippo pathway contributes to cancer development. Since the identification of the first Sav gene in 2002, very little is known regarding the molecular basis of Sav-SARAH mediating interactions due to its insolubility. In this study, refolding of the first Sav (known as WW45)-SARAH provided insight into the biochemical and biophysical properties, indicating that WW45-SARAH exhibits properties of a disordered protein, when the domain was refolded at a neutral pH. Interestingly, WW45-SARAH shows folded and rigid conformations relative to the decrease in pH. Further, diffracting crystals were obtained from protein refolded under acidic pH, suggesting that the refolded WW45 protein at low pH has a homogeneous and stable conformation. A comparative analysis of molecular properties found that the acidic-stable fold of WW45-SARAH enhances a heterotypic interaction with Mst2-SARAH. In addition, using an Mst2 mutation that disrupts homotypic dimerization, we showed that the monomeric Mst2-SARAH domain could form a stable complex of 1:1 stoichiometric ratio with WW45 refolded under acidic pH.

Description: Hypercholesterolemia is one of the factors contributing to cardiovascular problems. Erythrocytes are known to contribute its cholesterol to atherosclerotic plaque. Our earlier study showed that erythrocytes overexpress chondroitin sulphate/dermatan sulphate (CS/DS), a linear co-polymer, during diabetes which resulted in increased cytoadherence to extracellular matrix (ECM) components. This study was carried out to determine whether diet-induced hypercholesterolemia had any effect on erythrocyte CS/DS and impacted cytoadherence to ECM components. Unlike in diabetes, diet-induced hypercholesterolemia did not show quantitative changes in erythrocyte CS/DS but showed difference in proportion of un-sulphated and 4- O -sulphated disaccharides. Erythrocytes from hypercholesterolemic rats showed increased adhesion to ECM components which was abrogated to various extents when subjected to chondroitinase ABC digestion. However, isolated CS/DS chains showed a different pattern of binding to ECM components indicating that orientation of CS/DS chains could be playing a role in binding.

Description: The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 x 10 7 from cDNA of peripheral blood mononuclear cells of an alpaca ( Lama pacos ) immunized with a fragment of IZUMO1 (IZUMO1 PFF ) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (〉93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning.

Description: The autophosphorylation of specific tyrosine residues occurs in the cytoplasmic region of the insulin receptor (IR) upon insulin binding, and this in turn initiates signal transduction. The R3 subfamily (Ptprb, Ptprh, Ptprj and Ptpro) of receptor-like protein tyrosine phosphatases (RPTPs) is characterized by an extracellular region with 6–17 fibronectin type III-like repeats and a cytoplasmic region with a single phosphatase domain. We herein identified the IR as a substrate for R3 RPTPs by using the substrate-trapping mutants of R3 RPTPs. The co-expression of R3 RPTPs with the IR in HEK293T cells suppressed insulin-induced tyrosine phosphorylation of the IR. In vitro assays using synthetic phosphopeptides revealed that R3 RPTPs preferentially dephosphorylated a particular phosphorylation site of the IR: Y960 in the juxtamembrane region and Y1146 in the activation loop. Among four R3 members, only Ptprj was co-expressed with the IR in major insulin target tissues, such as the skeletal muscle, liver and adipose tissue. Importantly, the activation of IR and Akt by insulin was enhanced, and glucose and insulin tolerance was improved in Ptprj -deficient mice. These results demonstrated Ptprj as a physiological enzyme that attenuates insulin signalling in vivo , and indicate that an inhibitor of Ptprj may be an insulin-sensitizing agent.

Description: The diazotrophic cyanobacterium Anabaena sp. strain PCC 7120 (A.7120) differentiates into specialized heterocyst cells that fix nitrogen under nitrogen starvation conditions. Although reducing equivalents are essential for nitrogen fixation, little is known about redox systems in heterocyst cells. In this study, we investigated thioredoxin (Trx) networks in Anabaena using TrxM, and identified 16 and 38 candidate target proteins in heterocysts and vegetative cells, respectively, by Trx affinity chromatography (Motohashi et al. (Comprehensive survey of proteins targeted by chloroplast thioredoxin. Proc Natl Acad Sci USA , 2001; 98 , 11224–11229)). Among these, the Fe–S cluster scaffold protein NifU that facilitates functional expression of nitrogenase in heterocysts was found to be a potential TrxM target. Subsequently, we observed that the scaffold activity of N-terminal catalytic domain of NifU is enhanced in the presence of Trx-system, suggesting that TrxM is involved in the Fe–S cluster biogenesis.

Description: In this study, we examined the role of aminopeptidases with reference to endoplasmic reticulum aminopeptidase 1 (ERAP1) in nitric oxide (NO) synthesis employing murine macrophage cell line RAW264.7 cells activated by lipopolysaccharide (LPS) and interferon (IFN)- and LPS-activated peritoneal macrophages derived from ERAP1 knockout mouse. When NO synthesis was measured in the presence of peptides having N-terminal Arg, comparative NO synthesis was seen with that measured in the presence of Arg. In the presence of an aminopeptidase inhibitor amastatin, NO synthesis in activated RAW264.7 cells was significantly decreased. These results suggest that aminopeptidases are involved in the NO synthesis in activated RAW264.7 cells. Subsequently, significant reduction of NO synthesis was observed in ERAP1 knockdown cells compared with wild-type cells. This reduction was rescued by exogenously added ERAP1. Furthermore, when peritoneal macrophages prepared from ERAP1 knockout mouse were employed, reduction of NO synthesis in knockout mouse macrophages was also attributable to ERAP1. In the presence of amastatin, further reduction was observed in knockout mouse-derived macrophages. Taken together, these results suggest that several aminopeptidases play important roles in the maximum synthesis of NO in activated macrophages in a substrate peptide-dependent manner and ERAP1 is one of the aminopeptidases involved in the NO synthesis.

Description: Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits. Each subunit has two distinct maltooligosaccharide binding sites: a storage site and a catalytic site. Our characterization of the properties of these sites suggested that GP activity consists of two activities: (i) binding to the glycogen molecule and (ii) phosphorolysis of the non-reducing-end glucose residues. Activity (i) is mainly due to the activities of the two storage sites, which depended on the ionic strength of the medium and were directly inhibited by cyclodextrins (CDs). Activity (i) is of benefit to GP because a high concentration of non-reducing-end glucose residues is localized on the surface of the glycogen molecule. Activity (ii), the total activity of the two catalytic sites, exhibited relatively little ionic strength dependence. Because the combined activity of (i) and (ii) is deduced using glycogen as an assay substrate, the sole activity of (ii) must be measured using small maltooligosyl-substrates. By using a very low concentration of pyridylaminated maltohexaose, we demonstrated that the GP catalytic sites are active even in the presence of CDs, and that the actions of the catalytic site and the storage site are independent of each other.

Description: O -GlcNAcylation is a ubiquitous, dynamic and reversible post-translational protein modification in metazoans, and it is catalysed and removed by O -GlcNAc transferase (OGT) and O -GlcNAcase, respectively. Prokaryotes lack endogenous OGT activity. It has been reported that coexpression of mammalian OGT with its target substrates in Escherichia coli produce O -GlcNAcylated recombinant proteins, but the plasmids used were not compatible, and the expression of both OGT and its target protein were induced by the same inducer. Here, we describe a compatible dual plasmid system for coexpression of OGT and its target substrate for O -GlcNAcylated protein production in E. coli . The approach was validated using the CKII and p53 protein as control. This compatible dual plasmid system contains an arabinose-inducible OGT expression vector with a pUC origin and an isopropyl β - d -thiogalactopyranoside-inducible OGT target substrate expression vector bearing a p15A origin. The dual plasmid system produces recombinant proteins with varying O -GlcNAcylation levels by altering the inducer concentration. More importantly, the O -GlcNAcylation efficiency was much higher than the previously reported system. Altogether, we established an adjustable compatible dual plasmid system that can effectively yield O -GlcNAcylated proteins in E. coli .

Description: Active equi-paritioning of the F plasmid is achieved by its sopABC gene. SopA binds to the sopAB promoter region and SopB binds to sopC . SopA also polymerizes in the presence of ATP and Mg(II), which is stimulated by SopB. Non-specific DNA is known to inhibit SopA polymerization and disassemble SopA polymer. This study followed kinetics of polymerization and de-polymerization of SopA by turbidity measurement and found new effects by DNA and SopB. Plasmid DNA, at low concentrations, shortened the lag (nucleation) phase of SopA polymerization and also caused an initial ‘burst’ of turbidity. Results with two non-specific 20-bp DNAs indicated sequence/length dependence of these effects. sopAB operator DNA only showed inhibition of SopA polymerization. Results of turbidity decrease of pre-formed SopA polymer in the presence of ethylenediaminetetraacetic acid showed that SopB also accelerates disassembly of the SopA polymer. The steady-state level of turbidity in the presence of SopB and plasmid DNA indicated synergy between SopB and DNA in the disassembly. SopB protein showed no effect on SopA polymerization, when SopB was specifically bound to DNA. This result and others with truncation mutants of SopB suggested that a proper configuration of the domains of SopB is important for SopA-SopB interactions.

Description: Influenza A virus (IAV) has been raising public health and safety concerns worldwide. Cyanovirin-N (CVN) is a prominent anti-IAV candidate, but both cytotoxicity and immunogenicity have hindered the development of this protein as a viable therapy. In this article, linker-CVN (LCVN) with a flexible and hydrophilic polypeptide at the N-terminus was efficiently produced from the cytoplasm of Escherichia coli at a 〉15-l scale. PEGylation at the N-terminal α-amine of LCVN was also reformed as 20 kDa PEGylated linkered Cyanovirin-N (PEG 20k –LCVN). The 50% effective concentrations of PEG 20k –LCVN were 0.43 ± 0.11 µM for influenza A/HK/8/68 (H3N2) and 0.04 ± 0.02 µM for A/Swan/Hokkaido/51/96 (H5N3), dramatically lower than that of the positive control, Ribavirin (2.88 ± 0.66 x 10 3 µM and 1.79 ± 0.62 x 10 3 µM, respectively). A total of 12.5 µM PEG 20k –LCVN effectively inactivate the propagation of H3N2 in chicken embryos. About 2.0 mg/kg/day PEG 20k –LCVN increased double the survival rate (66.67%, P = 0.0378) of H3N2 infected mice, prolonged the median survival period, downregulated the mRNA level of viral nuclear protein and decreased (attenuated) the pathology lesion in mice lung. A novel PEGylated CVN derivative, PEG 20k –LCVN, exhibited potent and strain-dependent anti-IAV activity in nanomolar concentrations in vitro, as well as in micromolar concentration in vivo .

Description: The semi-filamentous multicellular cyanobacterium Limnothrix / Pseudanabaena sp. strain ABRG5-3 undergoes autolysis, which involves the accumulation of polyphosphate compounds and disintegration of thylakoid membranes in cells, as a unique feature that occurs due to growth conditions. In this study, the overexpression and easy recovery of alkane (a saturated hydrocarbon, C 17 H 36 ) as a biofuel were examined in recombinants of the cyanobacteria ABRG5-3 and Synechocystis sp. strain PCC6803. The results obtained indicated that the accumulated mass of alkane accounted for ~50 or 60% of the dry weight of ABRG5-3 or PCC6803 recombinant cells, respectively. Furthermore, cultivating cells in liquid medium BG11 in which the nitrogen resource had been depleted promoted the production of alkane and cell lysis, resulting in the easy recovery of target products from the supernatant.

Description: Leucine-rich repeat kinase 2 (LRRK2) has been identified as a causative gene for Parkinson’s disease (PD). LRRK2 contains a kinase and a GTPase domain, both of which provide critical intracellular signal-transduction functions. We showed previously that Rab5b, a small GTPase protein that regulates the motility and fusion of early endosomes, interacts with LRRK2 and co-regulates synaptic vesicle endocytosis. Using recombinant proteins, we show here that LRRK2 phosphorylates Rab5b at its Thr6 residue in in vitro kinase assays with mass spectrophotometry analysis. Phosphorylation of Rab5b by LRRK2 on the threonine residue was confirmed by western analysis using cells stably expressing LRRK2 G2019S. The phosphomimetic T6D mutant exhibited stronger GTPase activity than that of the wild-type Rab5b. In addition, phosphorylation of Rab5b by LRRK2 also exhibited GTPase activity stronger than that of the unphosphorylated Rab5b protein. Two assays testing Rab5’s activity, neurite outgrowth analysis and epidermal growth factor receptor degradation assays, showed that Rab5b T6D exhibited phenotypes that were expected to be observed in the inactive Rab5b, including longer neurite length and less degradation of EGFR. These results suggest that LRRK2 kinase activity functions as a Rab5b GTPase activating protein and thus, negatively regulates Rab5b signalling.

Description: The cellular Src (c-Src) tyrosine kinase is upregulated and believed to play a pivotal role in various human cancers. However, the molecular mechanism underlying c-Src-mediated tumour progression remains elusive. Recent studies have revealed that several microRNAs (miRNAs) function as tumour suppressors by regulating the malignant expression of signalling molecules. Aberrant expression of miRNAs is frequently observed in human cancers and should be exploited to seek related molecular targets. In this review, we focus on miRNAs found to be involved in Src signalling in various cancers. We summarize recent findings on Src-related miRNAs, their target genes, mechanisms behind their interplay and their implications for cancer therapeutics.

Description: Tail-anchored (TA) proteins, a class of membrane proteins having an N-terminal cytoplasmic region anchored to the membrane by a single C-terminal transmembrane domain, are posttranslationally inserted into the endoplasmic reticulum (ER) membrane. In yeasts, the posttranslational membrane insertion is mediated by the Guided Entry of TA Proteins (GET) complex. Get3, a cytosolic ATPase, targets newly synthesized TA proteins to the ER membrane, where Get2 and Get3 constitute the Get3 receptor driving the membrane insertion. While mammalian cells employ TRC40 and WRB, mammalian homologs of Get3 and Get1, respectively, they lack the gene homologous to Get2. We recently identified calcium-modulating cyclophilin ligand (CAML) as a TRC40 receptor, indicating that CAML was equivalent to Get2 in the context of the membrane insertion. On the other hand, CAML has been well characterized as a signaling molecule that regulates various biological processes, raising the question of how the two distinct actions of CAML, the membrane insertion and the signal transduction, are assembled. In this review, we summarize recent progress of the molecular mechanism of the membrane insertion of TA proteins and discuss the possibility that CAML could sense the various signals at the ER membrane, thereby controlling TA protein biogenesis.

Description: In bacterial organisms, the oriC -independent primosome plays an essential role in replication restart after dissociation of the replication DNA-protein complex following DNA damage. PriC is a key protein component in the oriC -independent replication restart primosome. Our previous study suggested that PriC was divided into an N-terminal domain and a C-terminal domain, with the latter domain being the major contributor to single-stranded DNA (ssDNA) binding capacity. In this study, we prepared several PriC mutants in which basic and aromatic amino acid residues were mutated to alanine. Five of these residues, Arg107, Lys111, Phe118, Arg121 and Lys165 in the C-terminal domain, were shown to be involved in ssDNA binding. Moreover, we evaluated the binding of the PriC mutants to the ssDNA-binding protein (SSB) complex. Five residues, Phe118, Arg121, Arg129, Tyr152 and Arg155 in the C-terminal domain of PriC, were shown to be involved in SSB binding in the presence of ssDNA. On the basis of these results, we propose a structural model of the C-terminal domain of PriC and discuss how the interactions of PriC with SSB and ssDNA may contribute to the regulation of PriC-dependent replication restart.

Description: L -Lysine α-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L -lysine to form α-keto--aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 Å resolution revealed that the overall structure is similar to that of snake venom L -amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX.

Description: For a multistep pre-targeting method using antibodies, a streptavidin mutant with low immunogenicity, termed low immunogenic streptavidin mutant No. 314 (LISA-314), was produced previously as a drug delivery tool. However, endogenous biotins (BTNs) with high affinity ( K d 〈 10 –10 M) for the binding pocket of LISA-314 prevents access of exogenous BTN-labelled anticancer drugs. In this study, we improve the binding pocket of LISA-314 to abolish its affinity for endogenous BTN species, therefore ensuring that the newly designed LISA-314 binds only artificial BTN analogue. The replacement of three amino acid residues was performed in two steps to develop a mutant termed V212, which selectively binds to 6-(5-((3a S ,4 S ,6a R )-2-iminohexahydro-1 H -thieno[3,4- d ]imidazol-4-yl)pentanamido)hexanoic acid (iminobiotin long tail, IMNtail). Surface plasmon resonance results showed that V212 has a K d value of 5.9 x 10 –7 M towards IMNtail, but no binding affinity for endogenous BTN species. This V212/IMNtail system will be useful as a novel delivery tool for anticancer therapy.

Description: A number of gene mutations are detected in cells derived from human cancer tissues, but roles of these mutations in cancer cell development are largely unknown. We examined G364R mutation of MCM4 detected in human skin cancer cells. Formation of MCM4/6/7 complex is not affected by the mutation. Consistent with this notion, the binding to MCM6 is comparable between the mutant MCM4 and wild-type MCM4. Nuclear localization of this mutant MCM4 expressed in HeLa cells supports this conclusion. Purified MCM4/6/7 complex containing the G364R MCM4 exhibited similar levels of single-stranded DNA binding and ATPase activities to the complex containing wild-type MCM4. However, the mutant complex showed only 30–50% of DNA helicase activity of the wild-type complex. When G364R MCM4 was expressed in HeLa cells, it was fractionated into nuclease-sensitive chromatin fraction, similar to wild-type MCM4. These results suggest that this mutation does not affect assembly of MCM2-7 complex on replication origins but it interferes some step at function of MCM2-7 helicase. Thus, this mutation may contribute to cancer cell development by disturbing DNA replication.

Description: We employed the warm temperate conifer Cunninghamia lanceolata (Lamb.) Hook. as a model of plantation forest species to investigate ecophysiological responses to root treatments (control (0%), and ~25, 50 or 75% of the initial root mass) under well-watered and water-limited conditions. Our results indicated that total root dry mass accumulation was negatively associated with the severity of root pruning, but there was evidence of multiple compensatory responses. The plants exhibited higher instantaneous and long-term (assessed by carbon isotope composition, 13 C) water-use efficiency in pruning treatments, especially under low water availability. Root pruning also increased the fine root/total root mass ratio, specific root length and fine root vitality in both water availability treatments. As a result of the compensatory responses, under well-watered conditions, height, stem dry mass accumulation, leaf/fine root biomass ratio (L/FR), transpiration rate, photosynthetic capacity and photosynthetic nitrogen-use efficiency ( E N ) were the highest under 25% pruning. Yet, all these traits except L/FR and foliage nitrogen content were severely reduced under 75% pruning. Drought negatively affected growth and leaf gas exchange rates, and there was a greater negative effect on growth, water potential, gas exchange and E N when 〉25% of total root biomass was removed. The stem/aboveground mass ratio was the highest under 25% pruning in both watering conditions. These results indicate that the responses to root severance are related to the excision intensity and soil moisture content. A moderate root pruning proved to be an effective means to improve stem dry mass accumulation.

Description: The timing of wood formation is crucial to determine how environmental factors affect tree growth. The long-lived bristlecone pine ( Pinus longaeva D. K. Bailey) is a foundation treeline species in the Great Basin of North America reaching stem ages of about 5000 years. We investigated stem cambial phenology and radial size variability to quantify the relative influence of environmental variables on bristlecone pine growth. Repeated cellular measurements and half-hourly dendrometer records were obtained during 2013 and 2014 for two high-elevation stands included in the Nevada Climate-ecohydrological Assessment Network. Daily time series of stem radial variations showed rehydration and expansion starting in late April–early May, prior to the onset of wood formation at breast height. Formation of new xylem started in June and lasted until mid-September. There were no differences in phenological timing between the two stands, or in the air and soil temperature thresholds for the onset of xylogenesis. A multiple logistic regression model highlighted a separate effect of air and soil temperature on xylogenesis, the relevance of which was modulated by the interaction with vapor pressure and soil water content. While air temperature plays a key role in cambial resumption after winter dormancy, soil thermal conditions coupled with snowpack dynamics also influence the onset of wood formation by regulating plant–soil water exchanges. Our results help build a physiological understanding of climate–growth relationships in P. longaeva , the importance of which for dendroclimatic reconstructions can hardly be overstated. In addition, environmental drivers of xylogenesis at the treeline ecotone, by controlling the growth of dominant species, ultimately determine ecosystem responses to climatic change.

Description: Seasonal analyses of cambial cell production and day-by-day stem radial increment can help to elucidate how climate modulates wood formation in conifers. Intra-annual dynamics of wood formation were determined with microcores and dendrometers and related to climatic signals in Norway spruce ( Picea abies (L.) Karst.). The seasonal dynamics of these processes were observed at two sites of different altitude, Savignano (650 m a.s.l.) and Lavazè (1800 m a.s.l.) in the Italian Alps. Seasonal dynamics of cambial activity were found to be site specific, indicating that the phenology of cambial cell production is highly variable and plastic with altitude. There was a site-specific trend in the number of cells in the wall thickening phase, with the maximum cell production in early July (DOY 186) at Savignano and in mid-July (DOY 200) at Lavazè. The formation of mature cells showed similar trends at the two sites, although different numbers of cells and timing of cell differentiation were visible in the model shapes; at the end of ring formation in 2010, the number of cells was four times higher at Savignano (106.5 cells) than at Lavazè (26.5 cells). At low altitudes, microcores and dendrometers described the radial growth patterns comparably, though the dendrometer function underlined the higher upper asymptote of maximum growth in comparison with the cell production function. In contrast, at high altitude, these functions exhibited different trends. The best model was obtained by fitting functions of the Gompertz model to the experimental data. By combining radial growth and cambial activity indices we defined a model system able to synchronize these processes. Processes of adaptation of the pattern of xylogenesis occurred, enabling P. abies to occupy sites with contrasting climatic conditions. The use of daily climatic variables in combination with plant functional traits obtained by sensors and/or destructive sampling could provide a suitable tool to better investigate the effect of disturbances on response strategies in trees and, consequently, contribute to improving our prediction of tree growth and species resilience based on climate scenarios.

Description: In deciduous trees growing in temperate forests, bud break and growth in spring must rely on intrinsic carbon (C) reserves. Yet it is unclear whether growth and C storage occur simultaneously, and whether starch C in branches is sufficient for refoliation. To test in situ the relationships between growth, phenology and C utilization, we monitored stem growth, leaf phenology and stem and branch nonstructural carbohydrate (NSC) dynamics in three deciduous species: Carpinus betulus L., Fagus sylvatica L. and Quercus petraea (Matt.) Liebl. To quantify the role of NSC in C investment into growth, a C balance approach was applied. Across the three species, 〉95% of branchlet starch was consumed during bud break, confirming the importance of C reserves for refoliation in spring. The C balance calculation showed that 90% of the C investment in foliage (7.0–10.5 kg tree –1 and 5–17 times the C needed for annual stem growth) was explained by simultaneous branchlet starch degradation. Carbon reserves were recovered sooner than expected, after leaf expansion, in parallel with stem growth. Carpinus had earlier leaf phenology (by ~25 days) but delayed cambial growth (by ~15 days) than Fagus and Quercus , the result of a competitive strategy to flush early, while having lower NSC levels.

Description: Fungal infections result in decreases in photosynthesis, induction of stress and signaling volatile emissions and reductions in constitutive volatile emissions, but the way different physiological processes scale with the severity of infection is poorly known. We studied the effects of infection by the obligate biotrophic fungal pathogen Melampsora larici-populina Kleb., the causal agent of poplar leaf rust disease, on photosynthetic characteristics, and constitutive isoprene and induced volatile emissions in leaves of Populus balsamifera var. suaveolens (Fisch.) Loudon. exhibiting different degrees of damage. The degree of fungal damage, quantified by the total area of chlorotic and necrotic leaf areas, varied between 0 (noninfected control) and ~60%. The rates of all physiological processes scaled quantitatively with the degree of visual damage, but the scaling with damage severity was weaker for photosynthetic characteristics than for constitutive and induced volatile release. Over the whole range of damage severity, the net assimilation rate per area ( A A ) decreased 1.5-fold, dry mass per unit area 2.4-fold and constitutive isoprene emissions 5-fold, while stomatal conductance increased 1.9-fold and dark respiration rate 1.6-fold. The emissions of key stress and signaling volatiles (methanol, green leaf volatiles, monoterpenes, sesquiterpenes and methyl salicylate) were in most cases nondetectable in noninfested leaves, and increased strongly with increasing the spread of infection. The moderate reduction in A A resulted from the loss of photosynthetically active biomass, but the reduction in constitutive isoprene emissions and the increase in induced volatile emissions primarily reflected changes in the activities of corresponding biochemical pathways. Although all physiological alterations in fungal-infected leaves occurred in a stress severity-dependent manner, modifications in primary and secondary metabolic pathways scaled differently due to contrasting operational mechanisms.

Description: Current knowledge of the genetic mechanisms underlying the inheritance of photosynthetic activity in forest trees is generally limited, yet it is essential both for various practical forestry purposes and for better understanding of broader evolutionary mechanisms. In this study, we investigated genetic variation underlying selected chlorophyll a fluorescence (ChlF) parameters in structured populations of Scots pine ( Pinus sylvestris L.) grown on two sites under non-stress conditions. These parameters were derived from the OJIP part of the ChlF kinetics curve and characterize individual parts of primary photosynthetic processes associated, for example, with the exciton trapping by light-harvesting antennae, energy utilization in photosystem II (PSII) reaction centers (RCs) and its transfer further down the photosynthetic electron-transport chain. An additive relationship matrix was estimated based on pedigree reconstruction, utilizing a set of highly polymorphic single sequence repeat markers. Variance decomposition was conducted using the animal genetic evaluation mixed-linear model. The majority of ChlF parameters in the analyzed pine populations showed significant additive genetic variation. Statistically significant heritability estimates were obtained for most ChlF indices, with the exception of DI 0 /RC, D0 and P0 ( F v / F m ) parameters. Estimated heritabilities varied around the value of 0.15 with the maximal value of 0.23 in the ET 0 /RC parameter, which indicates electron-transport flux from Q A to Q B per PSII RC. No significant correlation was found between these indices and selected growth traits. Moreover, no genotype  x  environment interaction (G  x  E) was detected, i.e., no differences in genotypes’ performance between sites. The absence of significant G  x  E in our study is interesting, given the relatively low heritability found for the majority of parameters analyzed. Therefore, we infer that polygenic variability of these indices is selectively neutral.

Description: The ethylene response factor (ERF) family is one of the largest plant-specific transcription factor families, playing an important role in plant development and response to stresses. The ERF76 gene is a member of the poplar ERF transcription factor gene family. First, we validated that the ERF76 gene expressed in leaf and root tissues is responsive to salinity stress. We then successfully cloned the ERF76 cDNA fragment containing an open reading frame from di-haploid Populus simonii   x   Populus nigra and proved that ERF76 protein is targeted to the nucleus. Finally, we transferred the gene into the same poplar clone by the Agrobacterium -mediated leaf disc method. Using both RNA-Seq and reverse transcription-quantitative polymerase chain reaction, we validated that expression level of ERF76 is significantly higher in transgenic plants than that in the nontransgenic control. Using RNA-Seq data, we have identified 375 genes that are differentially expressed between the transgenic plants and the control under salt treatment. Among the differentially expressed genes, 16 are transcription factor genes and 45 are stress-related genes, both of which are upregulated significantly in transgenic plants, compared with the control. Under salt stress, the transgenic plants showed significant increases in plant height, root length, fresh weight, and abscisic acid (ABA) and gibberellin (GA) concentration compared with the control, suggesting that overexpression of ERF76 in transgenic poplar upregulated the expression of stress-related genes and increased the ability of ABA and GA biosynthesis, which resulted in stronger tolerance to salt stress.

Description: Summer droughts are likely to increase in frequency and intensity across Europe, yet long-lived trees may have a limited ability to tolerate drought. It is therefore critical that we improve our understanding of phenotypic plasticity to drought in natural populations for ecologically and economically important trees such as Populus nigra L. A common garden experiment was conducted using ~500 wild P. nigra trees, collected from 11 river populations across Europe. Phenotypic variation was found across the collection, with southern genotypes from Spain and France characterized by small leaves and limited biomass production. To examine the relationship between phenotypic variation and drought tolerance, six genotypes with contrasting leaf morphologies were subjected to a water deficit experiment. ‘North eastern’ genotypes were collected at wet sites and responded to water deficit with reduced biomass growth, slow stomatal closure and reduced water use efficiency (WUE) assessed by 13 C. In contrast, ‘southern’ genotypes originating from arid sites showed rapid stomatal closure, improved WUE and limited leaf loss. Transcriptome analyses of a genotype from Spain (Sp2, originating from an arid site) and another from northern Italy (Ita, originating from a wet site) revealed dramatic differences in gene expression response to water deficit. Transcripts controlling leaf development and stomatal patterning, including SPCH , ANT , ER , AS1 , AS2 , PHB , CLV1 , ERL1–3 and TMM , were down-regulated in Ita but not in Sp2 in response to drought.

Description: Isoprene is the most abundant type of nonmethane, biogenic volatile organic compound in the atmosphere, and it is produced mainly by terrestrial plants. The tropical tree species Ficus septica Burm. F. (Rosales: Moraceae) has been shown to cease isoprene emissions when exposed to temperatures of 12 °C or lower and to re-induce isoprene synthesis upon subsequent exposure to temperatures of 30 °C or higher for 24 h. To elucidate the regulation of genes underlying the disabling and then induction of isoprene emission during acclimatization to ambient temperature, we conducted gene expression analyses of F. septica plants under changing temperature using quantitative real-time polymerase chain reaction and western blotting. Transcription levels were analyzed for 17 genes that are involved in metabolic pathways potentially associated with isoprene biosynthesis, including isoprene synthase ( ispS ). The protein levels of ispS were also measured. Changes in transcription and protein levels of the ispS gene, but not in the other assessed genes, showed identical temporal patterns to isoprene emission capacity under the changing temperature regime. The ispS protein levels strongly and positively correlated with isoprene emission capacity ( R 2  = 0.92). These results suggest that transcriptional regulation of ispS gave rise to the temporal variation in isoprene emission capacity in response to changing temperature.

Description: Clonal integration between ramets can be an ecological advantage of clonal plant species in environments where resources are patchily distributed. We investigated physiological integration among Populus balsamifera L. ramets under drought stress in order to demonstrate water sharing between connected ramets. Pairs of connected ramets were grown in separate pots in the greenhouse where half of ramets had the parental root connection severed and half were left intact. Drought stress was applied to one ramet, and growth, specific leaf area (SLA), net photosynthesis, stomatal conductance, leaf water potential and carbon isotopic composition ( 13 C) were measured after an 8-week growing period. Droughted ramets connected to watered ramets were able to maintain high gas exchange activity and water potential, similar to watered ramets. Leaf water potential and SLA results showed that the root connection was more beneficial for proximal compared with distal ramets. The parental root connection also allowed droughted ramets to discriminate more against 13 C compared with severed ramets. In conclusion, this study shows compelling evidence of physiological integration of connected P. balsamifera ramets through water sharing.

Description: Importin α performs the indispensable role of ferrying proteins from the cytoplasm into the nucleus with a transport carrier, importin β1. Mammalian cells from mouse or human contain either six or seven importin α subtypes, respectively, each with a tightly regulated expression. Therefore, the combination of subtype expression in a cell defines distinct signaling pathways to achieve progressive changes in gene expression essential for cellular events, such as differentiation. Recent studies reveal that, in addition to nucleocytoplasmic transport, importin αs also serve non-transport functions. In this review, we first discuss the physiological significance of importin α as a nuclear transport regulator, and then focus on the functional diversities of importin αs based on their specific subcellular and cellular localizations, such as the nucleus and plasma membrane. These findings enrich our knowledge of how importin αs actively contribute to various cellular events.

Description: The structure of the complex of maize sulfite reductase (SiR) and ferredoxin (Fd) has been determined by X-ray crystallography. Co-crystals of the two proteins prepared under different conditions were subjected to the diffraction analysis and three possible structures of the complex were solved. Although topological relationship of SiR and Fd varied in each of the structures, two characteristics common to all structures were found in the pattern of protein-protein interactions and positional arrangements of redox centres; (i) a few negative residues of Fd contact with a narrow area of SiR with positive electrostatic surface potential and (ii) [2Fe-2S] cluster of Fd and [4Fe-4S] cluster of SiR are in a close proximity with the shortest distance around 12 Å. Mutational analysis of a total of seven basic residues of SiR distributed widely at the interface of the complex showed their importance for supporting an efficient Fd-dependent activity and a strong physical binding to Fd. These combined results suggest that the productive electron transfer complex of SiR and Fd could be formed through multiple processes of the electrostatic intermolecular interaction and this implication is discussed in terms of the multi-functionality of Fd in various redox metabolisms.

Description: Analysis of replicating mammalian mitochondrial DNA (mtDNA) suggested that initiation of the replication occurs not only at the specific position, Ori-H but also across a broad zone in mtDNA. We investigated relationship of mitochondrial transcription initiation which takes place upstream of Ori-H and mtDNA replication initiation through analysing the effect of knockdown of mitochondrial transcription factor B2, TFB2M and mitochondrial RNA polymerase, POLRMT, components of the transcription initiation complexes in cultured human cells. Under the conditions where suppression of the transcription initiation complexes was achieved by simultaneous depletion of TFB2M and POLRMT, decrease of replication intermediates of mtDNA RITOLS replication mode accompanied reduction in mtDNA copy number. On the other hand, replication intermediates of coupled leading and lagging strand DNA replication, another proposed replication mode, appeared to be less affected. The findings support the view that the former mode involves transcription from the light strand promoter (LSP), and suggest that initiation of the latter mode is independent from the transcription and has distinct regulation. Further, knockdown of TFB2M alone caused significant decrease of 7S DNA, which implies that transcription initiation complexes formed at the LSP engage 7S DNA synthesis more frequently than the initiation of productive replication and transcription.

Description: Cycas revoluta leaf lectin (CRLL) of mannose-recognizing jacalin-related lectin (mJRL) has two tandem repeated carbohydrate recognition domains, and shows the characteristic sugar-binding specificity toward high mannose-glycans, compared with other mJRLs. We expressed the N-terminal domain and C-terminal domain (CRLL-N and CRLL-C) separately, to determine the fine sugar-binding specificity of each domain, using frontal affinity chromatography, glycan array and equilibrium dialysis. The specificity of CRLL toward high mannose was basically derived from CRLL-N, whereas CRLL-C had affinity for α1-6 extended mono-antennary complex-type glycans. Notably, the affinity of CRLL-N was most potent to one of three Man 8 glycans and Man 9 glycan, whereas the affinity of CRLL-C decreased with the increase in the number of extended α1-2 linked mannose residue. The recognition of the Man 8 glycans by CRLL-N has not been found for other mannose recognizing lectins. Glycan array reflected these specificities of the two domains. Furthermore, it was revealed by equilibrium dialysis method that the each domain had two sugar-binding sites, similar with Banlec, banana mannose-binding Jacalin-related lectin.

Description: O -GlcNAcylation is an inducible, highly dynamic and reversible post-translational modification, mediated by a unique enzyme named O -linked N -acetyl- d -glucosamine ( O -GlcNAc) transferase (OGT). In response to nutrients, O -GlcNAc levels are differentially regulated on many cellular proteins involved in gene expression, translation, immune reactions, protein degradation, protein–protein interaction, apoptosis and signal transduction. In contrast to eukaryotic cells, little is known about the role of O -GlcNAcylation in the viral life cycle. Here, we show that the overexpression of the OGT reduces the replication efficiency of Kaposi's sarcoma-associated herpesvirus (KSHV) in a dose-dependent manner. In order to investigate the global impact of O -GlcNAcylation in the KSHV life cycle, we systematically analyzed the 85 annotated KSHV-encoded open reading frames for O -GlcNAc modification. For this purpose, an immunoprecipitation (IP) strategy with three different approaches was carried out and the O -GlcNAc signal of the identified proteins was properly controlled for specificity. Out of the 85 KSHV-encoded proteins, 18 proteins were found to be direct targets for O -GlcNAcylation. Selected proteins were further confirmed by mass spectrometry for O -GlcNAc modification. Correlation of the functional annotation and the O -GlcNAc status of KSHV proteins showed that the predominant targets were proteins involved in viral DNA synthesis and replication. These results indicate that O -GlcNAcylation plays a major role in the regulation of KSHV propagation.

Description: Galectins are potent adhesion/growth-regulatory effectors with characteristic expression profiles. Understanding the molecular basis of gene regulation in each case requires detailed information on copy number of genes and sequence(s) of their promoter(s). Our report reveals plasticity in this respect between galectins and species. We here describe occurrence of a two-gene constellation for human galectin (Gal)-7 and define current extent of promoter-sequence divergence. Interestingly, cross-species genome analyses also detected single-copy display. Because the regulatory potential will then be different, extrapolations of expression profiles are precluded between respective species pairs. Gal-4 coding in chromosomal vicinity was found to be confined to one gene, whereas copy-number variation also applied to Gal-9. The example of rat Gal-9 teaches the lesson that the presence of multiple bands in Southern blotting despite a single-copy gene constellation is attributable to two pseudogenes. The documented copy-number variability should thus be taken into consideration when studying regulation of galectin genes, in a species and in comparison between species.

Description: In studying the molecular basis for the potent immune activity of previously described gamma and delta inulin particles and to assist in production of inulin adjuvants under Good Manufacturing Practice, we identified five new inulin isoforms, bringing the total to seven plus the amorphous form. These isoforms comprise the step-wise inulin developmental series amorphous -〉 alpha-1 (AI-1) -〉 alpha-2 (AI-2) -〉 gamma (GI) -〉 delta (DI) -〉 zeta (ZI) -〉 epsilon (EI) -〉 omega (OI) in which each higher isoform can be made either by precipitating dissolved inulin or by direct conversion from its precursor, both cases using regularly increasing temperatures. At higher temperatures, the shorter inulin polymer chains are released from the particle and so the key difference between isoforms is that each higher isoform comprises longer polymer chains than its precursor. An increasing trend of degree of polymerization is confirmed by end-group analysis using 1 H nuclear magnetic resonance spectroscopy. Inulin isoforms were characterized by the critical temperatures of abrupt phase-shifts (solubilizations or precipitations) in water suspensions. Such (aqueous) "melting" or "freezing" points are diagnostic and occur in strikingly periodic steps reflecting quantal increases in noncovalent bonding strength and increments in average polymer lengths. The (dry) melting points as measured by modulated differential scanning calorimetry similarly increase in regular steps. We conclude that the isoforms differ in repeated increments of a precisely repeating structural element. Each isoform has a different spectrum of biological activities and we show the higher inulin isoforms to be more potent alternative complement pathway activators.

Description: The methylotrophic yeast, Pichia pastoris , is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N -linked glycans but up to now no one has addressed engineering the O -linked glycosylation pathway. Typically, O -linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with β- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O -linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O -linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein- O -linked-mannose β-1,2- N -acetylglucosaminyltransferase 1, resulted in the capping of the single O -linked mannose residues with N -acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O -linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N -linked glycosylated biotherapeutics to include molecules possessing O -linked glycans.

Description: A requirement for advancing antibody-based medicine is the development of proteins that can bind with high affinity to a specific epitope related to a critical protein activity site. As a part of generating such proteins, we have succeeded in creating a binding protein without changing epitope by complementarity-determining region 3 (CDR3) grafting (Inoue et al. , Affinity transfer to a human protein by CDR3 grafting of camelid VHH. Protein Sci. 20, 1971–1981). However, the affinity of the target-binding protein was low. In this manuscript, the affinity maturation of a target-binding protein was examined using CDR3-grafted camelid single domain antibody (VHH) as a model protein. Several amino acids in the CDR1 and CDR2 regions of VHH were mutated to tyrosines and/or serines and screened for affinity-matured proteins by using in silico analysis. The mutation of two amino acids in the CDR2 region to arginine and/or aspartic acid increased the affinity by decreasing the dissociation rate. The affinity of designed mutant increased by ~20-fold over that of the original protein. In the present study, candidate mutants were narrowed down using in silico screening and computational modelling, thus avoiding much in vitro analytical effort. Therefore, the method used in this study is expected to be one of the useful for promoting affinity maturation of antibodies.

Description: A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ~30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.

Description: The tripartite motif (TRIM) or RBCC proteins are characterized by the TRIM composed of a RING finger, B-box and coiled-coil domains. TRIM proteins often play roles in the post-translational protein modification, including ubiquitylation and other ubiquitin-like modifications. Evidence has accumulated in regard to the contribution of TRIM proteins to diverse cellular processes, including such as cell cycle progression, apoptosis, immunity and transcriptional regulation. In particular, some of the TRIM proteins have been characterized to exert oncogenic or tumour suppressor-like functions depending on the context. A recent report by Inoue and his colleagues has revealed that Terf/TRIM17 stimulates the degradation of a kinetochore protein ZWINT and regulates the proliferation of breast cancer cells. Terf has also been paid attention as a factor promoting neuronal apoptosis, by degrading a Bcl2-like anti-apoptotic protein Mcl-1. Like aircraft trim tabs, TRIM proteins trim the balance of homoeostasis by modulating various biological pathways through protein–protein interactions.

Description: Cyclin-dependent kinase (CDK) that plays a central role in preventing re-replication of DNA phosphorylates several replication proteins to inactivate them. MCM4 in MCM2-7 and RPA2 in RPA are phosphorylated with CDK in vivo . There are inversed correlations between the phosphorylation of these proteins and their chromatin binding. Here, we examined in vitro phosphorylation of human replication proteins of MCM2-7, RPA, TRESLIN, CDC45 and RECQL4 with CDK2/cyclinE, CDK2/cyclinA, CDK1/cyclinB, CHK1, CHK2 and CDC7/DBF4 kinases. MCM4, RPA2, TRESLIN and RECQL4 were phosphorylated with CDKs. Effect of the phosphorylation by CDK2/cyclinA on DNA-binding abilities of MCM2-7 and RPA was examined by gel-shift analysis. The phosphorylation of RPA did not affect its DNA-binding ability but that of MCM4 inhibited the ability of MCM2-7. Change of six amino acids of serine and threonine to alanines in the amino-terminal region of MCM4 rendered the mutant MCM2-7 insensitive to the inhibition with CDK. These biochemical data suggest that phosphorylation of MCM4 at these sites by CDK plays a direct role in dislodging MCM2-7 from chromatin and/or preventing re-loading of the complex to chromatin.

Description: To determine the effects of alcohols on the low-frequency local motions that control slow changes in structural dynamics of native-like compact states of proteins, we have studied the effects of alcohols on structural fluctuation of M80-containing -loop by measuring the rate of thermally driven CO dissociation from a natively folded carbonmonoxycytochrome c under varying concentrations of alcohols (methanol, ethanol, 1-propanol, 2-propanol, 3°-butanol, 2,2,2-trifluoroethanol). As alcohol is increased, the rate coefficient of CO dissociation ( k diss ) first decreases in subdenaturing region and then increases on going from subdenaturing to denaturing milieu. This decrease in k diss is more for 2,2,2-trifluroethanol and 1-propanol and least for methanol, indicating that the first phase of motional constraint is due to the hydrophobicity of alcohols and intramolecular protein cross-linking effect of alcohols, which results in conformational entropy loss of protein. The thermal denaturation midpoint for ferrocytochrome c decreases with increase in alcohol, indicating that alcohol decrease the global stability of protein. The stabilization free energy ( G ) in alcohols’ solution was calculated from the slope of the Wyman–Tanford plot and water activity. The m -values obtained from the slope of G versus alcohols plot were found to be more negative for longer and linear chain alcohols, indicating destabilization of proteins by alcohols through disturbance of hydrophobic interactions and hydrogen bonding.

Description: Ubiquitination is a post-translational modification involved in the regulation of a broad variety of cellular functions, such as protein degradation and signal transduction, including nuclear factor-B (NF-B) signalling. NF-B is crucial for inflammatory and immune responses, and aberrant NF-B signalling is implicated in multiple disorders. We found that linear ubiquitin chain assembly complex (LUBAC), composed of HOIL-1L, HOIP and SHARPIN, generates a novel type of Met1 (M1)-linked linear polyubiquitin chain and specifically regulates the canonical NF-B pathway. Moreover, specific deubiquitinases, such as CYLD, A20 (TNFAIP3) and OTULIN/gumby, inhibit LUBAC-induced NF-B activation by different molecular mechanisms, and several M1-linked ubiquitin-specific binding domains have been structurally defined. LUBAC and these linear ubiquitination-regulating factors contribute to immune and inflammatory processes and apoptosis. Functional impairments of these factors are correlated with multiple disorders, including autoinflammation, immunodeficiencies, dermatitis, B-cell lymphomas and Parkinson’s disease. This review summarizes the molecular basis and the pathophysiological implications of the linear ubiquitination-mediated NF-B activation pathway regulation by LUBAC.

Description: We screened circadian-regulated genes in rat cartilage by using a DNA microarray analysis. In rib growth-plate cartilage, numerous genes showed statistically significant circadian mRNA expression under both 12:12 h light–dark and constant darkness conditions. Type II collagen and aggrecan genes—along with several genes essential for post-translational modifications of collagen and aggrecan, including prolyl 4-hydroxylase 1, lysyl oxidase, lysyl oxidase-like 2 and 3'-phosphoadenosine 5'-phosphosulphate synthase 2—showed the same circadian phase. In addition, the mRNA level of SOX9, a master transcription factor for the synthesis of type II collagen and aggrecan, has a similar phase of circadian rhythms. The circadian expression of the matrix-related genes may be critical in the development and the growth of various cartilages, because similar circadian expression of the matrix-related genes was observed in hip joint cartilage. However, the circadian phase of the major matrix-related genes in the rib permanent cartilage was almost the converse of that in the rib growth-plate cartilage under light–dark conditions. We also found that half of the oscillating genes had conserved clock-regulatory elements, indicating contribution of the elements to the clock outputs. These findings suggest that the synthesis of the cartilage matrix macromolecules is controlled by cell-autonomous clocks depending upon the in vivo location of cartilage.

Description: Human chromosome 7 open reading frame 24 (C7orf24)/-glutamyl cyclotransferase has been suggested to be a potential diagnostic marker for several cancers, including carcinomas in the bladder urothelium, breast and endometrial epithelium. We here investigated the epigenetic regulation of the human C7orf24 promoter in normal diploid ARPE-19 and IMR-90 cells and in the MCF-7 and HeLa cancer cell lines to understand the transcriptional basis for the malignant-associated high expression of C7orf24. Chromatin immunoprecipitation analysis revealed that histone modifications associated with active chromatin were enriched in the proximal region but not in the distal region of the C7orf24 promoter in HeLa and MCF-7 cells. In contrast, elevated levels of histone modifications leading to transcriptional repression and accumulation of heterochromatin proteins in the C7orf24 promoter were observed in the ARPE-19 and IMR-90 cells, compared to the levels in HeLa and MCF-7 cancer cells. In parallel, the CpG island of the C7orf24 promoter was methylated to a greater extent in the normal cells than in the cancer cells. These results suggest that the transcriptional silencing of the C7orf24 gene in the non-malignant cells is elicited through heterochromatin formation in its promoter region; aberrant expression of C7orf24 associated with malignant alterations results from changes in chromatin dynamics.

Description: Abscisic acid (ABA) is a stress-inducible plant hormone comprising an inevitable component of the human diet. Recently, stress-induced accumulation of autocrine ABA was shown in humans, as well as ABA-mediated modulation of a number of disease-associated systems. Now, the application of a chemical proteomics approach to gain further insight into ABA mechanisms of action in mammalian cells is reported. An ABA mimetic photoaffinity probe was applied to intact mammalian insulinoma and embryonic cells, leading to the identification of heat shock protein 70 (HSP70) family members, (including GRP78 and HSP70-2) as putative human ABA-binding proteins. In vitro characterization of the ABA–HSP70 interactions yielded K d s in the 20–60 µM range, which decreased several fold in the presence of co-chaperone. However, ABA was found to have only variable- and co-chaperone-independent effects on the ATPase activity of these proteins. The potential implications of these ABA–HSP70 interactions are discussed with respect to the intracellular protein folding and extracellular receptor-like activities of these stress-inducible proteins. While mechanistic and functional relevance remain enigmatic, we conclude that ABA can bind to human HSP70 family members with physiologically relevant affinities and in a co-chaperone-dependent manner.

Description: At weaning, the intestinal mucosa surface glycans change from predominantly sialylated to fucosylated. Intestinal adaptation from milk to solid food is regulated by intrinsic and extrinsic factors. The contribution by glucocorticoid, an intrinsic factor, and colonization by microbiota, an extrinsic factor, was measured as the induction of α1,2/3-fucosyltransferase and sucrase-isomaltase (SI) activity and gene expression in conventionally raised, germ-free, and bacteria-depleted mice. In conventionally raised mice, cortisone acetate (CA) precociously accelerated SI gene expression up to 3 weeks and fut2 to 4 weeks of age. In germ-free mice, CA treatment induces only SI expression but not fucosyltransferase. In post-weaning bacteria-deficient (germ-free and bacteria-depleted) mice, fut2 expression remains at low suckling levels. In microbiota deficient mice, intestinal fut2 (but not fut1 , fut4 or fut7 ) was induced only by adult microbiota, but not immature microbiota or CA. Fut2 induction could also be restored by colonization by Bacteroides fragilis , but not by a B. fragilis mutant unable to utilize fucose. Restoration of fut2 expression (by either microbiota or B. fragilis ) in bacteria-depleted mice is necessary for recovery from dextran sulfate sodium-induced mucosal injury. Thus, glucocorticoids and microbes regulate distinct aspects of gut ontogeny: CA precociously accelerates SI expression and, only in colonized mice, fut2 early expression. The adult microbiota is required for the fut2 induction responsible for the highly fucosylated adult gut phenotype and is necessary for recovery from intestinal injury. Fut2 -dependent recovery from inflammation may explain the high incidence of inflammatory disease (Crohn's and necrotizing enterocolitis) in populations with mutant FUT2 polymorphic alleles.

Description: Selectins and their carbohydrate ligands mediate the homing of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow. We have previously shown that ex vivo fucosylation of selectin ligands on HSPCs by α1,3 fucosyltransferase VI (FUT6) leads to improved human cord blood (CB)-HSPC engraftment in non-obese diabetic (NOD)/severe combined immune deficient (SCID) mice. In the present study, we determined whether surface fucosylation with α1,3 fucosyltransferase VII (FUT7), which is primarily expressed by hematopoietic cells, improves the function of selectin ligands on CB-HSPCs in comparison with FUT6. A saturating amount of either FUT6 or FUT7, which generates comparable levels of expression of fucosylated epitopes on CB CD34 + cells, was used for these experiments. In vitro, FUT7-treated CB CD34 + cells exhibited greater binding to P- or E-selectin than that of FUT6-treated CB CD34 + cells under static or physiological flow conditions. In vivo, FUT7 treatment, like FUT6, improved the early engraftment of CB CD34 + cells in the bone marrow of sublethally irradiated NOD/SCID interleukin (IL)-2R null (NSG) mice. FUT7 also exhibited marginally—yet statistically significant—increased engraftment at 4 and 6 weeks after transplantation. In addition, FUT7-treated CB CD34 + cells exhibited increased homing to the bone marrow of irradiated NSG mice relative to sham-treated cells. These data indicate that FUT7 is effective at improving the function of selectin ligands on CB-HSPCs in vitro and enhancing early engraftment of treated CB-HSPCs in the bone marrow of recipients.

Description: Glycosphingolipids are expressed on the cell membrane and act as important factors in various events that occur across the plasma membrane. Lactosylceramide (LacCer) is synthesized from glucosylceramide and is a common precursor of various glycosphingolipids existing in whole body. Based on the enzyme purification, β1,4-galactosyltransferase 6 ( B4galt6 ) cDNA was isolated as a LacCer synthase-coding gene in the rat brain. We generated B4galt6 gene knockout (KO) mice and analyzed their phenotypes to examine roles of β4GalT6. B4galt6 KO mice were born and grew up apparently normal. LacCer synthase activity and the composition of acidic glycosphingolipids in the brain were almost equivalent or minimally different between wild-type and KO mice. Studies by mouse embryonic fibroblasts (MEFs) revealed that the silencing of B4galt5 gene resulted in the marked reduction in LacCer synthase activity and this reduction was more severe in MEFs derived from B4galt6 KO mice than those from wild-type mice. These results suggested that β4GalT6 plays a role as a LacCer synthase, whereas β4GalT5 acts as a main enzyme for LacCer biosynthesis in these tissues and cells.

Description: Endoplasmic reticulum (ER) α-glucosidase I is an enzyme that trims the distal α1,2-linked glucose (Glc) residue from the Glc 3 Man 9 GlcNAc 2 oligosaccharide following its addition to nascent glycoproteins in the initial step of processing. This reaction is critical to the subsequent processing of N-glycans and thus defects in α-glucosidase I gene in human cause congenital disorder of glycosylation (CDG) type IIb. We identified the Caenorhabditis elegans α-glucosidase I gene (F13H10.4, designated agl-1 ) that encodes a polypeptide with 36% identity to human α-glucosidase I. The agl-1 cDNA restored the expression of complex-type N-glycans on the cell surface of α-glucosidase I-defective Chinese hamster ovary Lec23 cells. RNAi knockdown of agl-1 [ agl-1 (RNAi)] produced worms that were visibly similar to wild-type, but lifespan was reduced to about half of the control. Analyses of N -glycosylation in agl-1 (RNAi) animals by western blotting and mass spectrometry showed reduction of paucimannose and complex-type glycans and dramatic increase of glucosylated oligomannose glycans. In addition, a significant amount of unusual terminally fucosylated N-glycans were found in agl-1 (RNAi) animals. ER stress response was also provoked, leading to the accumulation of large amounts of triglucosylated free oligosaccharides (FOSs) (Glc 3 Man 4–5 GlcNAc 1–2 ) in agl-1 (RNAi) animals. Acceleration of ER-associated degradation in response to the accumulation of unfolded glycoproteins and insufficient interaction with calnexin/calreticulin in the ER lumen likely accounts for the increase of FOSs. Taken together, these studies in C. elegans demonstrate that decreased ER α-glucosidase I affects the entire N-glycan profile and induces chronic ER stress, which may contribute to the pathophysiology of CDG-IIb in humans.