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  • Immunocytochemistry  (44)
  • gene expression
  • Springer  (71)
  • American Meteorological Society
  • Periodicals Archive Online (PAO)
  • 1995-1999
  • 1990-1994  (71)
  • 1935-1939
  • 1991  (71)
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  • Springer  (71)
  • American Meteorological Society
  • Periodicals Archive Online (PAO)
  • Wiley-Blackwell  (23)
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  • 1995-1999
  • 1990-1994  (71)
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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Cellular and molecular life sciences 47 (1991), S. 321-331 
    ISSN: 1420-9071
    Keywords: Symbiosis ; ectomycorrhiza ; ectomycorrhiza development ; gene expression ; ectomycorrhizins ; protein patterns
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary An ectomycorrhiza, a specialized root organ, is the result of a complex interaction leading to a finely-tuned symbiosis between a plant and a compatible ectomycorrhizal fungus. Ultrastructural observations combined with cytochemical and biochemical studies reveal that structural and metabolic changes in the symbiont cells lead to the final phenotype of the active ectomycorrhiza. In the present review these changes are interpreted as changes in gene expression and discussed within the context of ectomycorrhiza development. Recent genetic data indicate that the continued vegetative growth of the ectomycorrhizal hyphae and the root tissues, and their ability to switch to symbiotic organ formation, is basically controlled by developmentally critical genes. The activity of these ‘symbiotic genes’ during the differentiation of ectomycorrhizas is associated with extensive changes in the concentration of particular polypeptides and protein biosynthesis. The present state of knowledge about the developmental biology of ectomycorrhizas allows only speculation about the events during their development.
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  • 2
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    Antonie van Leeuwenhoek 59 (1991), S. 65-76 
    ISSN: 1572-9699
    Keywords: anaerobic respiration ; FNR protein ; oxygen regulation ; gene expression ; E. coli
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Molecular oxygen is an important regulatory signal in facultative anaerobic bacteria and controles the expression of a great variety of genes positively or negatively. The expression of anaerobic respiration and of related functions of E. coli is controlled by the positive gene regulator FNR, which activates transcription in the absence of O2. The regulated genes carry a FNR consensus sequence upstream of the promoter. Under the same conditions FNR represses some of the genes of aerobic respiration. The binding to the DNA occurs by an α-helix-turn-α-helix DNA-binding domain. FNR contains 5 cysteine residues, four of which are arranged in a cluster close to the N-terminal end. For the function of FNR as a O2-dependent regulator three of the cysteine residues in the cluster and the residue outside the cluster are essential. FNR binds iron as a cofactor which most likely is involved in the O2-sensing by the protein. The experiments indicate that the cysteine residues are responsible for the binding of the iron. From the protein in vivo two functional states can be differentiated, an aerobic or metal-depleted form and an anaerobic form. Only the anaerobic form acts as a gene activator or repressor. Sensing of O2 or of positive redox potentials by the iron ion is thought to cause the conversion of the two functional states. The FNR protein in addition contains a potential nucleotide binding domain. The significance and function of this site is not clear.
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  • 3
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    Cellular and molecular life sciences 47 (1991), S. 22-31 
    ISSN: 1420-9071
    Keywords: Atherosclerosis ; cellular differentiation ; gene expression ; foam cells ; lipoproteins ; phorbol esters ; transcription factors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary THP-1 is a human monocytic leukemia cell line. After treatment with phorbol esters, THP-1 cells differentiate into macrophage-like cells which mimic native monocyte-derived macrophages in several respects. Compared to other human myeloid cell lines, such as HL-60, U937, KG-1, or HEL cell lines, differentiated THP-1 cells behave more like native monocyte-derived macrophages. Because of these characteristics, the THP-1 cell line provides a valuable model for studying the mechanisms involved in macrophage differentiation, and for exploring the regulation of macrophage-specific genes as they relate to physiological functions displayed by these cells.
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  • 4
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    Cellular and molecular life sciences 47 (1991), S. 905-912 
    ISSN: 1420-9071
    Keywords: Gene transfer ; gene modification ; gene expression ; livestock ; transgenic animal ; pharmaceutical proteins ; milk composition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract There is every reason to expect that it will be possible within the next few years to begin to use farm animals to produce large quantities of some of the human proteins that are needed for the treatment of disease. Revolutionary new opportunities for the production of novel proteins in milk have been created by the development of methods for gene transfer. Exploitation of these opportunities depends upon selection and cloning of milk protein genes and identification of the sequences that govern tissue specific hormonally induced expression in the mammary gland. Studies with three genes, ovine β-lactoglobulin, rat β-casein and whey acidic protein of rat and mouse, suggest that they may all meet this requirement. Fragments of the ovine β-lactoglobulin, murine whey acidic protein and rabbit β-casein genes have directed production of novel proteins in the milk of transgenic mice, sheep, rabbits and pigs. The proteins were biologically active and usually co-migrated with authentic proteins. In early experiments, protein concentration was low, but our recent observations suggest that fusion genes containing genomic clones direct production of concentrations of protein that are suitable for commercial exploitation. In the longer term, two approaches may offer the potential of more reliable expression. Control elements capable of directing expression that is independent of site of insertion of the gene, but dependent on the number of copies of the gene, have been identified for a small number of genes. The availability of such elements for the milk protein genes would increase the reliability of gene expression considerably. Alternatively, targeted mutation of genes may allow the insertion of coding sequences within an existing gene so avoiding position effects.
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  • 5
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    Springer
    Cellular and molecular life sciences 47 (1991), S. 866-877 
    ISSN: 1420-9071
    Keywords: Transgenic mice ; microinjection ; recombinant DNA ; gene expression ; transcription factors ; chromatin ; homologous recombination ; episomal maintenance ; embryonic stem cells ; germ line ; position-effect ; mosaicism ; globin genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract This chapter is an attempt to summarize some commonly accepted and some more subjective opinions about the regulation of transgene expression in laboratory animals. After a short historical introduction, I present some general notions regarding gene structure/function. The spotlight shifts then to the description of the most popular techniques for gene transfer, including the targeted gene replacement. The different approaches are briefly discussed in terms of intrinsic advantages and limitations regarding gene expression patterns. Furthermore, the role of enhancers, promoters and othercis-acting elements such as silencers and dominant control regions as well as their involvement in the chromatin on-off state are discussed on the basis of a specific example studied in our laboratory. The review concludes by presenting recent results and the new perspectives opening in the field of ‘surrogate’ (also called ‘reversed’) genetics. Some problems which remain to be solved both at the technical as well as at the social-ethical level are also briefly presented.
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  • 6
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    Plant cell reports 10 (1991), S. 308-314 
    ISSN: 1432-203X
    Keywords: Agrobacterium tumefaciens ; Brassica juncea ; genetic transformation ; gene expression ; transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO3 and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All Ro transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R1 seed progeny and showed co-segregation.
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  • 7
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    Journal of comparative physiology 169 (1991), S. 39-50 
    ISSN: 1432-1351
    Keywords: Photoreception ; Retinally degenerate ; Mouse ; Circadian ; Rods ; Cones ; 11-cis retinaldehyde ; Immunocytochemistry ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary We have examined the effects of light on circadian locomotor rhythms in retinally degenerate mice (C57BL/6J mice homozygous for the rd allele: rd/rd). The sensitivity of circadian photoreception in these mice was determined by varying the irradiance of a 15 min light pulse (515 nm) given at circadian time 16 and meauring the magnitude of the phase shift of the locomotor rhythm. Experiments were performed on animals 80 days of age. Despite the loss of visual photoreceptors in the rd/rd retina, animals showed circadian responses to light that were indistinguishable from mice with normal retinas (rd/+ and +/+). While no photoreceptor outersegments were identified in the retina of rd/rd animals (80–100 days of age), we did identify a small number of perikarya that were immunoreactive for cone opsins, and even fewer cells that contained rod opsin. Using HPLC, we demonstrated the presence and photoisomerization of the rhodopsin chromophore 11-cis retinaldehyde. The rd/rd retinas contained about 2% of 11-cis retinaldehyde found in +/+ retinas. We have yet to determine whether the opsin immunoreactive perikarya or some other unidentified cell type mediate circadian light detection in the rd/rd retina.
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  • 8
    ISSN: 1573-4919
    Keywords: mitochondrial biogenesis ; cytochrome c oxidase ; mRNA quantitation ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Summary Cytochrome c oxidase (COX), like other multi-subunit components of the respiratory chain, is controlled by both the nuclear and the mitochondrial genome. In order to find wether there is a close relationship between mRNAs encoded by the nucleus and by the mitochondrion, and between these mRNAs and enzyme activity, we compared six rat tissues (ventricle, liver, m. soleus, m. plantaris, and the white and red portions of m. gastrocnemius). We found a tenfold range for COX activity, a tenfold range for the contents of mRNA III (mitochondrial) and mRNA VIc (nuclear), a threefold range for total [poly(A)+] mRNA content and a sevenfold range for total RNA content in these tissues. The ratio of mRNA III to mRNA VIc was equal in each tissue, indicating the presence of a mechanism that coordinates the two genomes. There was a good correlation between mRNA content and COX activity (r = 0.78 for VIc, r = 0.77 for 111; p 〈 0.0001), demonstrating that the expression of this enzyme is mainly under pretranslational control.
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  • 9
    ISSN: 1573-4919
    Keywords: Rb and p53 genes ; gene expression ; colorectal cancers ; colon carcinoma cell lines ; cell cycle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We have compared the expression of the retinoblastoma (Rb) and p53 genes in normal human fibroblasts, colon carcinoma cell lines, matched pairs of colorectal tumor tissues and adjacent normal mucosa and in synchronized human diploid fibroblast cell line W138. The increased expression of Rb and p53 RNA was observed in a majority of colorectal cancers in comparison to adjacent normal mucosa and is accompanied by proportional increase in the expression of histone H3 gene. The Rb and p53 RNA levels varied significantly between the various colon carcinoma cell lines. However, we found that the expression of Rb and p53 RNA is regulated differently in cell cycle synchronized normal human fibroblasts. The Rb mRNA level did not change with the position in the cell cycle and did not differ significantly whether the cells were serum deprived or in 10% serum. But p53 mRNA expression follows the same pattern as histone H3 mRNA.
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  • 10
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    Molecular and cellular biochemistry 103 (1991), S. 149-154 
    ISSN: 1573-4919
    Keywords: EGF-receptor gene ; gene expression ; cycloheximide ; dexamethasone and A431 carcinoma cell line
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Human A431 carcinoma cell line is known to have 30 fold amplified epidermal growth factor receptor (EGF-R) gene. We have studied the effect of steroid hormone dexamethasone (DEX) and protein synthesis inhibitor cycloheximide (CHX) on the expression of EGF-R gene in this cell line. DEX treatment and protein synthesis inhibition by CHX treatment cause a rapid 3 to 4 fold increase in the level of EGF-R mRNA and combined treatment of the above two agents have less than additive effect. It appears that mRNA for EGF-R accumulate within the cell during protein synthesis inhibition and upon removal of CHX, gets translated into EGF-R specific protein as judged by immuno-dot assay. We did not observe the phenomenon of ‘super induction’ nor much of an additive effect under condition of combined DEX and CHX treatment.
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  • 11
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    Molecular and cellular biochemistry 104 (1991), S. 35-43 
    ISSN: 1573-4919
    Keywords: α1-adrenergic receptors ; β-adrenergic receptors ; cardiac muscle ; cell culture ; gene expression ; protein kinase C
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The molecular mechanisms of cardiac myocyte growth are relevant to important problems in cardiovascular disease. A cell culture model has been developed to explore the role of adrenergic hormones in cardiac myocyte growth and gene expression. Activation of a cardiac myocyte α1-adrenergic receptor by catecholamines induces hypertrophic growth of neonatal rat cardiac myocytes and initiates selective increases in contractile protein gene transcription. These effects on growth and gene expression do not depend on contractile activity. The cardiac myocytes contain at least two subtypes of α1-adrenergic receptors and at least three isoforms of protein kinase C (PKC). A distinct α1 receptor subtype may mediate hypertrophy and gene transcription. Different isoforms of PKC are translocated to different intracellular sites on activation, and there is evidence that the β-PKC isoform may be an element in the signal transduction pathway from an α1 receptor at the surface to the cardiac myocyte nucleus. Growth regulation through a β-adrenergic receptor can also be demonstrated in the culture model. The growth response mediated through a β-adrenergic receptor differs in several respects from that transduced through an al adrenergic receptor.
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  • 12
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    Molecular and cellular biochemistry 103 (1991), S. 41-50 
    ISSN: 1573-4919
    Keywords: insulin-like growth factor-1 ; binding proteins ; diabetes ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Summary Multiple factors contribute to the growth retardation which is a characteristic feature of uncontrolled diabetes. In this report we have examined the effects of streptozotocin-induced (STZ) diabetes on expression of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-1 (IGFBP-1) in various tissues. As early as 7 days after STZ administration there was a modest reduction in IGF-I mRNA abundance. The reduction (10–30%) was of similar magnitude in each of the 7 tissues examined; liver, kidney, lung, diaphragm, quadraceps, heart and adipose tissue. However, the reduction achieved statistical significance only in the lung (p 〈 0.05) and diaphragm (p 〈 0.01). A further reduction in IGF-I mRNA abundance was seen in many tissues, 32 and 91 days after STZ administration. In contrast to the decrease in IGF-I mRNA, IGFBP-1 mRNA was significantly increased in the liver and kidney of diabetic rats. IGFBP-1 mRNA was detectable at only very low levels in other tissues but was increased in diabetic rats compared non-diabetic rats. In diabetic rats, a highly significant correlation (R = 0.75, p 〈 0.001) between hepatic IGFBP-1 mRNA and glucose was observed whereas there was no significant correlation between serum glucose and hepatic IGF-I mRNA abundance (R = 0.24, p = NS). Treatment of diabetic rats with insulin resulted in a small, non significant increase in hepatic and renal IGF-I mRNA and a significant decrease in renal IGFBP-1 mRNA abundance. The observations reported here are consistent with the hypothesis that diminished IGF-I expression and inhibition of available IGF-1 by increased levels of IGFBP-1 may explain the impaired growth seen in diabetic animals.
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  • 13
    ISSN: 1573-5028
    Keywords: blue light ; phytochrome ; gene expression ; signal transduction chain
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract When dark-grown aurea mutant tomato seedlings which lack more than 95% of the phytochrome present in isogenic wild-type seedlings are kept in white or blue light, four nuclear-encoded transcripts coding for plastidic proteins (the light-harvesting chlorophyll a/b-binding protein of photosystem I and II [cab-PSII], plastocyanin and subunit 2 of photosystem I) are present in comparable amounts. These transcript levels in red light are strongly reduced in aurea seedlings when compared with those of wild type. Thus, blue light is required for normal expression of these genes in the mutant, while red light alone is not sufficient. Red light-grown aurea seedlings are very sensitive to blue light, even 10 minutes of blue light every day suffices to cause a measurable increase in cab-PSII transcript level. The action of blue light on the expression of cab-PSII in the mutant is under phytochrome control. After 8 days of blue light, phytochrome is almost as effective in inducing cab-PSII mRNA as in the isogenic wild type, whereas after 8 days of red light, only a small phytochrome response was observed in the mutant. It is concluded that blue light sensitizes the mutant to the residual phytochrome which allows normal gene expression and survival of the mutant under daylight conditions.
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  • 14
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    Plant molecular biology 16 (1991), S. 225-234 
    ISSN: 1573-5028
    Keywords: β-tubulin ; gene expression ; developmental regulation ; light regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We examined the developmental expression of a diverged soybean β-tubulin gene (designated sb-1), which had been cloned and sequenced previously. A probe specific for the sb-1 gene was constructed from the 3′ transcribed untranslated sequence. As a control, a more general probe for β-tubulin genes and their transcripts was constructed from a highly conserved region of the third exon of another soybean β-tubulin gene, sb-2. Poly(A)+ RNA, extracted from various soybean tissues and organs, was probed alternatively with the sb-1 gene-specific probe and with the generic β-tubulin probe. Levels of β-tubulin transcripts recognized by the generic probe differed by a factor of approximately 3 in the different tissues and organs and varied with the state of organ development. Highest levels were found in young, unexpanded leaves and they decreased as leaf maturation occurred. In contrast, transcripts of sb-1 were nearly undetectable in young leaves, and they increased as leaf maturation occurred. Levels of sb-1 transcript were low in all organs of the light-grown plant examined, except the hypocotyl, where they were approximately 10-fold higher. However, the highest levels of sb-1 transcripts were observed in elongating hypocotyls of etiolated seedlings. Exposure of six-day-old etiolated seedlings to light for 12 hours halted further hypocotyl elongation and brought about a dramatic, nearly 100-fold, decrease in the steady-state level of sb-1 transcripts.
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  • 15
    ISSN: 1573-5028
    Keywords: gene expression ; photosystem II ; Solanum tuberosum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
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  • 16
    ISSN: 1573-5028
    Keywords: L-phenylalanine ammonia-lyase mRNA ; fungal elicitor ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An expression library containing cDNAs derived from transcripts from fungal elicitor-treated alfalfa cell suspension cultures was screened with an antiserum raised against phenylalanine ammonia-lyase (PAL) from alfalfa. A single immunoreactive clone was isolated which encoded a full-length PAL cDNA (APAL1) consisting of a 2175 bp open reading frame, 96 bp 5′-untranslated leader and 128 bp 3′-noncoding region. The deduced amino acid sequence was 86.5% similar to that of the PAL2 gene of bean, and encoded a polypeptide ofM r 78865. A second PAL cDNA species was isolated, whose 3′-untranslated region was 86% identical to that of APAL1. Southern blot analysis indicated that PAL is encoded by a small multigene family in alfalfa. PAL transcript levels were rapidly and massively induced, and preceded increased PAL extractable activity, on exposure of alfalfa suspension cells to elicitor from baker's yeast. PAL transcripts were most abundant in roots, stems and petioles during growth and development of alfalfa seedlings. These studies provide the basis for an examination of the developmental and environmental control of a key enzyme of phenylpropanoid synthesis in a plant species which is readily amenable to stable genetic transformation.
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  • 17
    ISSN: 1573-5028
    Keywords: Agrobacterium tumefaciens ; crown gall ; gene expression ; genomic position effects ; Solanum tuberosum L. ; T-cyt
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The promoter region of the Agrobacterium tumefaciens T-cyt gene was linked in a translational fusion to the coding DNA of the reporter gene uidA (for β-glucuronidase or GUS protein; EC 3.2.1.31) and to nos 3′ flanking DNA. The chimaeric gene was introduced by Agrobacterium transformation into potato (Solanum tuberosum L. cv. Désirée). In nine transgenic lines, the average GUS levels were highest in extracts from stems and roots of in vitro grown plants (ca. 11 000 GUS activity units per pmol MU per mg protein per min) but lower in leaves of the in vitro grown plants (ca. 7000 units). GUS activity was intermediate in stems and roots of plants grown in soil as well as in in vitro crown galls (ca. 3000 units). Activity was low in tubers, irrespective of whether these developed in vitro or in soil (both ca. 100 units), and lowest of all in leaves of soil-grown plants (ca. 10–15 units). However, in shoot cultures reestablished from soil-grown plants, GUS activity in the leaves increased to that determined in the original shoot cultures. Hence, plant culture conditions strongly influenced the expression of the T-cyt-uidA-nos gene. In particular, it was silenced in leaves of soil-grown plants. The results are compared with previous analyses of the promoter region of the wild-type T-cyt gene and with the growth properties of a large number of crown gall cell lines and crown-gall-derived plants, including over forty S. tuberosum cv. Désirée cell lines isolated in the present study that were transformed with the wild-type T-cyt gene and six promoter-mutated derivatives. A number of implications are discussed for crown gall formation and for control of expression of plant genes which contain Activator or G-box type 5′ expression control sequences.
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  • 18
    ISSN: 1573-5028
    Keywords: gene expression ; heterotrophic growth ; leucine zipper ; photosystem I ; photosystem II
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have cloned and sequenced thepsaA andpsaB genes from the unicellular cyanobacteriumSynechocystis sp. PCC 6803. These genes are arranged in tandem, are co-transcribed, and are highly homologous to thepsaA andpsaB genes previously characterized. RNA was isolated from light-grown cells, from cells put in total darkness with and without glucose, and from cells grown under light-activated heterotrophic growth (LAHG) conditions. Quantitation of hybridization to northern blots revealed only a slight decrease in the accumulation of thepsaA-psaB transcript in cells grown in complete darkness with glucose and in LAHG cells, relative to light-grown cells. Accumulation of thepsbA transcript steadily declines through dark incubation, with a steady-state level in LAHG cells 28% of that in light-grown cells. Transcripts frompsbD, psaD, andrbcLS accumulate in cells grown in complete darkness and in LAHG cells to approximately the same levels as in light-grown cells. Photosynthesis gene transcripts in cells grown in the dark without glucose were detected, but were highly degraded. Our data prove that transcripts from photosynthesis genes do accumulate in dark-grownSynechocystis 6803, which may allow for synthesis and assembly of photosystem (PS) I and PS II in the dark.
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  • 19
    ISSN: 1573-5028
    Keywords: hormones ; gene expression ; soybean ; water deficit ; tubulin ; cell wall proteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transfer of soybean seedlings to low-water-potential vermiculite (ψw = −0.3 MPa) results in a reversible decrease in hypocotyl growth and modulation of several polysomal mRNAs (Plant Physiol 92: 205–214). We report here the isolation of two cDNA clones (pGE16 and pGE95) which correspond to genes whose mRNA levels are increased, and one cDNA clone (pGE23) which corresponds to a gene whose mRNA level is decreased in the hypocotyl zone of cell elongation by water deficit. In well-watered seedlings mRNAs hybridizing to pGE16 and pGE95 are most abundant in mature regions of the seedling, but in water-deficient seedlings mRNA levels are reduced in mature regions and enhanced in elongating regions. RNA corresponding to soybean proline-rich protein 1 (sbPRP1) shows a similar tissue distribution and response to water deficit. In contrast, in well-watered seedlings, the gene corresponding to pGE23 was highly expressed in the hypocotyl and root growing zones. Transfer of seedlings to low-water-potential vermiculite caused a rapid decrease in mRNA hybridizing to pGE23. Sequence analysis revealted that pGE23 has high homology with β-tubulin. Water deficit also reduced the level of mRNA hybridizing to JCW1, an auxin-modulated gene, although with different kinetics. Furthermore, mRNA encoding actin, glycine-rich proteins (GRPs), and hydroxyproline-rich glycoproteins (HRGPs) were down-regulated in the hypocotyl zone of elongation of seedlings exposed to water deficit. No effect of water deficit was observed on the expression of chalcone synthase. Decreased expression of β-tubulin, actin, JCW1, HRGP and GRP and increased expression of sbPRP1, pGE95 and pGE16 in the hypocotyl zone of cell elongation could participate in the reversible growth inhibition observed in water-deficient soybean seedlings.
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  • 20
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    Plant molecular biology 16 (1991), S. 663-670 
    ISSN: 1573-5028
    Keywords: gene expression ; proline-rich protein ; wheat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA (WPRP1) encoding a wheat proline-rich protein has been isolated and sequenced. The amino acid composition shows 45% proline, with high levels of methionine, lysine and glutamic acid. The derived 378 residue amino acid sequence has a highly repetitive structure which is unlike those of other proline-rich proteins. The WPRP1 cDNA clone was used to determine the copy number and chromosomal location of the WPRP1 gene by restriction fragment length polymorphism analysis of wheat inbred lines. Although WPRP1 is encoded by a single-copy gene it is also a representative of a larger family of related sequences. RNA gel blot analysis showed that expression of WPRP1 is highest in rapidly growing tissue which together with its amino acid composition suggests a structural role for the encoded protein.
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  • 21
    ISSN: 1573-5028
    Keywords: conifer ; gene expression ; heterologous promoters ; inducible promoter activity ; particle acceleration ; transient assay
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Electrical discharge particle acceleration was used to test the transient expression of numerous inducible angiosperm promoters in a gymnospermPicea glauca (white spruce). Promoter expression was assayed in three different tissues capable ofin vitro regeneration, zygotic embryos, seedlings and embryogenic callus. The promoters tested include the light-inducibleArabidopsis and soybean ribulose-1,5-bisphosphate small subunit promoters and a maize phosphoenolpyruvate carboxylase promoter; a soybean heat-shock-inducible promoter, a soybean auxin inducible promoter and a maize alcohol dehydrogenase promoter. Promoters were cloned into a promoter-less expression vector to form a promoter-β-glucuronidase-nopaline synthase 3′ fusion. A similar construct was made using the cauliflower mosaic virus 35S (CaMV 35S) promoter as a control. All promoters were expressed in white spruce embryos, yet at levels lower than CaMV 35S. In addition, in the embryos the heat-shock and the alcohol dehydrogenase promoters showed inducible expression when given the proper induction stimulus. In seedlings, expression of all promoters was lower than in the embryos and expression was only inducible with the heat-shock promoter in the cotyledons. Of the tissues tested, the expression level of all promoters was lowest in embryogenic callus. Interestingly, the expression of the β-glucuronidase gene in embryogenic callus was restricted to the proembryonal head cells regardless of the promoter used. These results clearly demonstrate the use of particle bombardment to test the transient expression of heterologous promoters in organized tissue and the expression of angiosperm promoters in a gymnosperm.
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  • 22
    ISSN: 1573-5028
    Keywords: carnation ; Dianthus caryophyllus ; ethylene ; gene expression ; glutathione s-transferase ; senescence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Carnation flower petal senescence is associated with the expression of specific senescence-related mRNAs, several of which were previously cloned [5]. The cDNA clone pSR8 represents a transcript which accumulates specifically in senescing flower petals in response to ethylene. Here we report the structural characterization of this cDNA. A second cDNA clone was isolated based on shared sequence homology with pSR8. This clone, pSR8.4, exhibited an overlapping restriction endonuclease map with pSR8 and contained an additional 300 nucleotides. Primer extension analysis revealed the combined cDNAs to be near full-length and the transcript to accumulate in senescing petals. Analysis of the nucleotide sequence of SR8 cDNAs revealed an open reading frame of 220 amino acids sufficient to encode a 25 kDa polypeptide. Comparison of the deduced polypeptide sequence of pSR8 with other peptide sequences revealed significant similarity with glutathione s-transferases from a variety of organisms. The predicted polypeptide sequence shared 44%, 53% and 52% homology with GSTs from maize, Drosophila and man, respectively. We discuss our results in relation to the biochemistry of flower petal senescence and the possible role of glutathione s-transferase in this developmental process.
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  • 23
    ISSN: 1573-5028
    Keywords: ethylene ; fruit repening ; gene expression ; light ; Lycopersicon ; wounding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Regulation of three cDNA clones (pT52, pT53, and pT58) was analyzed in terms of wounding alone and wounding in conjunction with developmental and environmental cues (ripening, ethylene, and light) in tomato fruit tissue. The pT52-specific transcript level is induced by wounding in early-red and red stage fruit and by ethylene. The pT58-specific transcript level is also induced by wounding and ethylene in early-red stage fruit but is not induced by wounding in red fruit. The pT53-specific transcript level is repressed by wounding in early-red and red stage fruit. Like the pT52- and pT58-specific transcripts, the pT53-specific transcript is induced by ethylene. Furthermore, the level of the pT52-specific transcript is regulated by light. Analysis of unwounded tissue showed that the abundance of each cdNA-specific transcript changes during fruit ripening and that each of the transcripts is present in other plant organs as well. This analysis provides information about the interactions between developmental and environmental factors affecting these genes.
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  • 24
    ISSN: 1573-5028
    Keywords: Nicotiana tabacum ; plant transformation ; gene expression ; bacterial lysine decarboxylase ; protein transport ; chloroplasts ; cadaverine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A possible approach for altering alkaloid biosynthesis in plants is the expression of genes encoding key enzymes of a pathway such as lysine decarboxylase (ldc) in transgenic plants. Two strategies were followed here: one focused on expression of the gene in the cytoplasm, the other on subsequent targeting of the protein to the chloroplasts. Theldcgene fromHafnia alvei was therefore (a) placed under the control of the 1′ promoter of the bidirectional Tr promoter fromAgrobacterium tumefaciens Ti- plasmid, and (b) cloned behind therbcS promoter from potato fused to the coding region of therbcS transit peptide. Bothldc constructs, introduced intoNicotiana tabacum with the aid ofA. tumefaciens, were integrated into the plant genome and transcribed as shown by Southern and northern hybridization. However, LDC activity was only detectable in plants expressing mRNA under the control of therbcS promoter directing the LDC fusion protein into chloroplasts with the aid of the transit peptide domain. In plants expressing the processed bacterial enzyme cadaverine levels increased from nearly zero to 0.3–1% of dry mass.
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  • 25
    ISSN: 1573-5028
    Keywords: alcohol dehydrogenase ; anaerobiosis ; chromosome mapping ; gene expression ; Petunia hybrida ; plant development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A genomic clone for an alcohol dehydrogenase (Adh) gene has been isolated fromPetunia hybrida cv. V30 by screening aPetunia genomic library with a maizeAdh1 probe. A combination of RFLP and allozyme segregation data failed to demonstrate which of twoAdh loci, both of which map to chromosome 4, was the source of the cloned gene. The product of the cloned genes has been identified unequivocally by a transient expression assay inPetunia protoplasts. We have designated this genePetunia Adh1. The expression of this gene is tightly regulated in the developing anther, where its gene product is the predominant ADH isozyme. It is anaerobically inducible in roots, stems and leaves of seedlings. The induction of enzyme activity is correlated with induction ofAdh1 mRNA.
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  • 26
    ISSN: 1573-5028
    Keywords: tobacco ; 5-enolpyruvylshikimate-3-phosphate synthase ; cDNA clone ; gene expression ; gene amplification ; glyphosate ; cell culture ; tolerance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were obtained from a glyphosate-tolerant tobacco cell line. The cDNAs were 89% identical and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other plants. Tobacco EPSPS proteins were more similar to those from tomato and petunia than Arabidopsis. One cDNA clone, EPSPS-1, represented a gene that was amplified in glyphosate-tolerant cells, while the gene for EPSPS-2 was unaltered in these cells. Consequently, EPSPS-1 mRNA was more abundant in tolerant than unselected cells, whereas EPSPS-2 mRNA was at relatively constant levels in these cell lines. Exposure of unselected cells and tobacco leaves to glyphosate produced a transient increase in EPSPS mRNA. However, glyphosate-tolerant cells containing amplified copies of EPSPS genes did not show a similar response following exposure to glyphosate. A significant proportion of the EPSPS gene amplification was maintained when tolerant cells were grown in the absence of glyphosate for eight months. Plants regenerated from these cells also contained amplified EPSPS genes.
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  • 27
    ISSN: 1432-1351
    Keywords: Aplysia ; Motoneurons ; Immunocytochemistry ; Small cardioactive peptides ; Facilitation ; Depression ; Buccal ; Feeding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary We used physiological recordings, intracellular dye injections and immunocytochemistry to further identify and characterize neurons in the buccal ganglia of Aplysia calif ornica expressing Small Cardioactive Peptide-like immunoreactivity (SCP-LI). Neurons were identified based upon soma size and position, input from premotor cells B4 and B5, axonal projections, muscle innervation patterns, and neuromuscular synaptic properties. SCP-LI was observed in several large ventral neurons including B6, B7, B9, B10, and B11, groups of s1 and s2 cluster cells, at least one cell located at a branch point of buccal nerve n2, and the previously characterized neurons B1, B2 and B15. B6, B7, B9, B10 and B11 are motoneurons to intrinsic muscles of the buccal mass, each displaying a unique innervation pattern and neuromuscular plasticity. Combined, these motoneurons innervate all major intrinsic buccal muscles (I1/I3, I2, I4, I5, I6). Correspondingly, SCP-LI processes were observed on all of these muscles. Innervation of multiple nonhomologous buccal muscles by individual motoneurons having extremely plastic neuromuscular synapses, represents a unique form of neuromuscular organization which is prevalent in this system. Our results show numerous SCPergic buccal motoneurons with widespread ganglionic processes and buccal muscle innervation, and support extensive use of SCPs in the control of feeding musculature.
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  • 28
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    Cell & tissue research 263 (1991), S. 207-215 
    ISSN: 1432-0878
    Keywords: Somatolactin (SL) ; Pituitary gland, pars intermedia ; PAS-positive cells ; Immunocytochemistry ; Gadus morhua, Oncorhynchus mykiss, Poecillia latipinna (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary We report here on the cellular localization in the fish pituitary of somatolactin (SL), a putative new pituitary hormone related to growth hormone and prolactin, which has been recently identified in the piscine pituitary gland. Immunocytochemical staining, using anti-cod SL serum, revealed that in the cod pituitary gland, SL is produced by cells in the intermediate lobe, bordering the neural tissue. These cells, staining weakly with periodic-acid-Schiff (PAS), are distinct from the melanocyte stimulating hormone (MSH) cells which, as in all teleosts, are PAS-negative. SL-immunoreactivity was observed in the same location in all other teleost species examined: flounder, rainbow trout, killifish, molly, catfish and eel. In most fish the SL-immunoreactive cells are either strongly or weakly PAS-positive but in rainbow trout are chromophobic, indicating that the SL protein can probably exist in glycosylated and non-glycosylated forms. Thus, in demonstrating the cellular localization of SL, this study provides the first identification of the enigmatic, second cell-type of the fish pars intermedia.
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  • 29
    ISSN: 1432-0878
    Keywords: Neurofilaments ; Phosphorylation ; Axon ; Immunocytochemistry ; Golden syrian hamster, Mesocricetus auratus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The distribution of phosphorylated and nonphosphorylated neurofilament epitopes was determined immunocytochemically in adjacent 2 μm-thick sections of sciatic nerve, ventral root and spinal cord. Staining was scored as either intense, moderate or absent and the proportion of labeled axons was calculated for each category. Nearly all sciatic nerve and ventral root axons were immunoreactive with both antibodies against phosphorylated and non-phosphorylated neurofilaments and there were no significant differences in the number of intensely- or moderately-labeled axons. Within the spinal cord however, while the majority of large caliber axons was stained with both antibodies, there was a significant number of small caliber axons which stained only with antibodies against phosphorylated neurofilaments. These results show that phosphorylated and nonphosphorylated neurofilaments are extensively codistributed in CNS and PNS axons, and that in the CNS, staining intensity for non-phosphorylated epitopes is less in the smaller axons.
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  • 30
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    Cell & tissue research 263 (1991), S. 271-284 
    ISSN: 1432-0878
    Keywords: Serotonin ; Immunocytochemistry ; Avian brain ; Hypothalamus ; Japanese quail, Coturnix coturnix japonica (Aves)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The presence and topographical localization of the serotoninergic system in the brain of the Japanese quail (Coturnix coturnix japonica) have been studied by means of peroxidase-anti-peroxidase immunocytochemistry. The perimeter, diameter, area, and shape factor of immunoreactive cells have been recorded and analyzed morphometrically for intra- and interspecies comparison. The data reported here confirm and extend results previously obtained in the brain of other avian species. Serotonin-immunoreactive neurons of the quail are mainly located in the hypothalamic paraventricular organ and adjacent areas, and in the brainstem where they form three separate groups. The first of these groups consists of small-sized neurons located in the ventro-rostral mesencephalon. The second group is composed of medium-sized neurons located in the dorsal mesencephalo-pontine region. The third group is also formed by medium-sized neurons, and is located ventrally in the ponto-medullary region. In the quail brain, serotoninergic neurons are not restricted to nuclei located in the vicinity of the midsagittal plane, but show some lateralization, especially in the brainstem. The organization of the different groups of immunoreactive neurons based on this topographical distribution and morphometric analysis has been compared with descriptions of the serotoninergic system in other birds. Serotonin-immunoreactive nerve fibers are widely distributed throughout the brain, but appear to be particularly abundant in regions involved in the control of reproductive activities, such as the septal region, the medial preoptic nucleus, the nucleus intercollicularis, and the external zone of the median eminence. The data reported here have allowed the drawing of a map of serotoninimmunoreactive structure.
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  • 31
    ISSN: 1432-0878
    Keywords: Met-enkephalin-Arg6-Gly7-Leu8 ; Immunocytochemistry ; Neuropeptides ; Allatostatins ; Neurosecretory cells ; Corpus cardiacum/corpus allatum complex ; Diploptera punctata, Calliphora vomitoria (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Neuronal circuits in the brain and retrocerebral complex of the cockroach Diploptera punctata have been mapped immunocytochemically with antisera directed against the extended enkephalin, Met-enkephalin-Arg6-Gly7-Leu8 (Met-8). The pathways link median and lateral neurosecretory cells with the corpus cardiacum/corpus allatum complex. In females, nerve fibres penetrate the corpora allata and varicosities or terminals, immunoreactive to Met-8, surround the glandular cells. Males differ in having almost no Met-8 immunoreactivity in the corpora allata. The corpora cardiaca of both males and females are richly supplied with Met-8 immunoreactive material, in particular in the ‘cap’ regions immediately adjacent to the corpora allata. A similarity in the amino-acid sequences of Met-8 and the C-terminus of the recently characterised allatostatins of D. punctata suggests that the pathways identified with the Met-8 antisera may be the same as those by which the allatostatins are transported from the brain to the corpus allatum. In comparative studies on the blowfly Calliphora vomitoria, similar neuronal pathways have been identified except that no sexual dimophism with respect to amounts of immunoreactive material within the corpus allatum has been observed. These results suggest a possible homology in the neuropeptide regulation of the gland.
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  • 32
    ISSN: 1432-0878
    Keywords: Vitamin D-1,25(OH)2D3-like immunoreactivity ; 1,25(OH)2D3 receptor-like immunoreactivity ; Immunocytochemistry ; Calcium ions ; Orchestia cavimana (Crustacea)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary For the first time, immunoreactivity to 1,25(OH)2D3-like molecules and their receptors has been investigated in a calcium transporting epithelium of a crustacean, Orchestia, using vertebrate antisera on ultrathin cryosections of posterior caeca previously fixed in glutaraldehyde, then postfixed in osmium tetroxide. Both immunoreactivities were located mainly in the nuclei of epithelial cells. Quantitative differences in 1,25(OH)2D3-like immunoreactivity were noticed from one stage of the molt cycle to another. These results, together with other data, contribute to evidence that immunoreactive 1,25(OH)2D3-like molecules may be involved in the regulatory processes of calcium metabolism in this terrestrial crustacean and suggest an involvement of these substances in the regulation of calcium movements in the posterior caeca.
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  • 33
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    Cell & tissue research 264 (1991), S. 23-32 
    ISSN: 1432-0878
    Keywords: Male urethra ; Urethral epithelium ; Immunocytochemistry ; Ultrastructure ; Man
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The distal segment of the human male urethra, in particular the fossa navicularis, was studied with light- and electron microscopy as well as by means of histochemical and immunocytochemical methods. The fossa navicularis of the urethra contains a circumscribed zone of extremely thick, non-keratinized stratified squamous epithelium composed of cells containing a large amount of glycogen. These cells lack acid phosphatase activity and lysozyme-like immunoreactivity, both of which can be demonstrated to varying extents in the other zones of the distal male urethra. These glycogen-rich cells are considered to be the substrate for an endogenous flora of lactobacteria, whereas the acid-phosphatase activity and the lysozyme-like immunoreactivity indicate the presence of macrophages and the secretion of bactericidal agents at the epithelial surface. These observations suggest that the different zones with heterogeneous properties in the distal male urethra probably represent a defense system against the invasion of pathogenic microorganisms. Moreover, the glycogen-rich zone, which resembles the glycogen-rich epithelium of the vagina, is estrogen-dependent. This is demonstrated in cases of sex reversal in which after long-lasting estrogen treatment the glycogen-rich zone becomes extremely extended by displacement of the neighbouring epithelium.
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  • 34
    ISSN: 1432-0878
    Keywords: Enkephalins ; Tegmentum mesencephali ; Tectum opticum ; Immunocytochemistry ; Retrograde labeling ; Cobalt-lysine ; Salmo gairdneri, Salmo salar (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Immunocytochemistry using antibodies against Met-enkephalin and Leu-enkephalin has demonstrated a group of large enkephalin-immunoreactive neurons in the nucleus of the rostral mesencephalic tegmentum (mRMT) of two teleost fish, Salmo gairdneri and Salmo salar. Injections of cobalt-lysine in the medial optic tectum retrogradely labeled the above group of tegmental neurons. Tegmental neurons were labeled only ipsilaterally to the injection site. This indicates that enkephalinergic neurons in the nRMT project to the optic tectum, and that at least some of the enkephalinergic axons observed in the optic tectum belong to a tegmento-tectal pathway. Comparable enkephalinergic pathways have been described in reptiles and birds, where pretectal-mesencephalic nuclei contribute to the enkephalin-containing fibers that project to the optic tectum.
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  • 35
    ISSN: 1432-0878
    Keywords: Pancreas, endocrine ; Larval development ; Serial thin/semithin sections ; Immunocytochemistry ; Rana temporaria (Anura)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The pancreatic endocrine component was studied at different stages of development in the tadpoles of Rana temporaria. The material was embedded in Epon, and serial semithin and thin sections were made in order to correlate ultrastructural features and tinctorial traits of the endocrine cells. Serial semithin sections were also stained with the peroxidase-antiperoxidase immunocytochemical method and with silver impregnations for argyrophilia and argentaffinity. In early larvae (legless tadpoles), A and B cells are present. Both can be found within ducts and exocrine tissue or, more frequently, in cellular clusters among the ducts and acini. These primitive islets are solid structures, surrounded but not penetrated by capillaries. Mitoses were observed in A and B cells. In the following phase (tadpoles with hindlegs), D and pancreatic polypeptide-immunoreactive cells are also present, as well as numerous endocrine cells scattered among exocrine tissue. There is also a change in the vascular-insular pattern: capillaries not only surround but also penetrate the endocrine group. The structure of the endocrine pancreas in older tadpoles is similar. Tinctorial traits and ultrastructural features of endocrine cells are described, and the origin of primitive islets is discussed.
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  • 36
    ISSN: 1432-0878
    Keywords: Immunocytochemistry ; Neuropeptide Y ; Gonadotropin-releasing hormone ; Reproductive function ; Domestic chicken, Gallus domesticus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Nerve fibers and perikarya containing gonadotropin-releasing hormone (GnRH-like) immunoreactivity were investigated in the brain of the three-week-old chick, Gallus domesticus using the technique of immunocytochemistry. Six major groups of perikarya were found to include the olfactory bulb, olfactory tubercle/lobus parolfactorius, nucleus accumbens, septal preoptic hypothalamic region (three sub-nuclei), lateral anterior thalamic nucleus and in and about the oculomotor complex. The immunostaining was unusual in the latter group, suggesting that the neurons may contain a GnRH-II like material. Immunoreactive fibers for GnRH were found throughout the entire brain extending from the olfactory bulbs to the caudal brainstem. Two anatomical areas, not emphasized in the past literature, which had distinct GnRH-like immunoreactivity, included the lateral anterior thalamic nucleus and the preoptic recess. The former included a group of GnRH perikarya that is also known to be a retino-recipient area while the latter contained neuronal terminals some of which appeared to be contacting the cerebrospinal fluid of the preoptic recess. An attempt was made to list all anatomical structures that contained or were juxta-positioned to sites that displayed immunoreactive perikarya and fibers including circumventricular organs.
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  • 37
    ISSN: 1432-0878
    Keywords: Rhabdomeres ; Cytoskeleton ; Actin ; Immunocytochemistry ; Membrane shedding ; Leptotarsus spp. (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Rhabdomeres of tipulid flies lose membrane during turnover from a ‘shedding zone’ composed of microvillar tips. These distal domains lack intramicrovillar cytoskeletons and appear to be empty sacs of membrane. Recent concerns about the role of ninaC mechano-enzymes in the architecture of dipteran rhabodomeral microvilli and the dynamic role that they may play in the creation of shedding zones demand an examination of the distribution of actin in tipulid rhabdomeres. We compared rhabdomeres from tipulid retinae incubated before fixation for immunocytochemistry in a buffer without additives and a stabilising buffer that contained a cocktail of cysteine protease inhibitors; both were challenged by an anti-actin antibody for immunogold labelling after embedding in LR White Resin. Shedding zones thus processed collapse to structureless detritus. Stabilised and unstabilised shedding zones were immunonegative to anti-actin. To ensure that the negative results were not consequent upon conformational changes generated by the processing protocol, we examined microvilli of degenerating rhabdomeres of the Drosophila light-dependent retinal degeneration mutant rdgB KS222 (which separate and collapse without creating a shedding zone) and found the detritus they generate to be immunopositive to anti-actin. Stabilised and unstabilised regions of basal regions of tipulid rhabdomeres were equally immunopositive. We infer that (a) actin is absent from shedding zones; (b) actin is not degraded by microvillar cysteine proteases. The implications of these conclusions are discussed in relation to some functional models of arthropod photoreceptor microvilli.
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  • 38
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    Cell & tissue research 265 (1991), S. 485-492 
    ISSN: 1432-0878
    Keywords: ACTH ; Brain ; Hypothalamus ; Hypophysectomy ; Cortisol ; Metopirone ; Immunocytochemistry ; Pituitary ; Corticotropin-releasing factor ; Anguilla anguilla (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary An ACTH-like peptidergic system was demonstrated in the brain of three teleost species by immunocytochemistry. In order to investigate the origin of brain ACTH and factors modulating its synthesis, similar techniques were applied to the brain of eels (1) submitted to hypothysectomy in order to suppress pituitary ACTH and plasma cortisol, (2) injected with cortisol to inhibit pituitary ACTH synthesis and release, and (3) injected with metopirone to block cortisol synthesis and stimulate ACTH synthesis and release. Hypophysectomized eels showed a normal distribution of immunoreactive perikarya in the ventral hypothalamus and fibers in the brain, suggesting that brain ACTH does not arise from the pituitary. In cortisol-treated eels immunostaining was markedly reduced in brain perikarya and pituitary corticotropes, suggesting a reduced synthesis. In metopirone-injected eels, one third of the animals showed an increased immunostaining in perikarya and a dense network of immunoreactive fibers, suggesting that ACTH synthesis was increased. Brain ACTH was not affected in other animals. Pituitary corticotropes were rapidly degranulated. Responses of ACTH in the brain and pituitary occur independently when cortisol synthesis is inhibited. These responses are compared to those of the corticotropin-releasing factor system in the same eels.
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  • 39
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    Cell & tissue research 265 (1991), S. 527-534 
    ISSN: 1432-0878
    Keywords: Autonomic nervous system ganglia ; Enteric nervous system ; Intestine, large ; Tissue culture ; Immunocytochemistry ; Peptides ; Guinea-pig
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Ganglia of the myenteric plexus from the newborn guinea-pig, isolated by microdissection, were dissociated by a combination of enzymatic and mechanical methods. The neurones and glial cells in the resulting cell suspension were cultured for up to 21 days in vitro. The growth of the enteric ganglion cells in serum-free, hormone-supplemented (N1) medium and in serum-supplemented medium containing a mitotic inhibitor was compared over a period of 14 days in vitro. Enteric neurones were outnumbered by glia in both culture media, although glial cell proliferation was inhibited in both media compared with that in serum-supplemented medium without mitotic inhibitors. Glial cell numbers appeared to decline in serum-free medium after the first week in vitro. Neurites tended to be more varicose in the serum-free medium, and the morphology of the enteric glial cells also differed markedly in the two media. This is the first report of the dissociation and subsequent culture of myenteric ganglia that had previously been completely isolated from the remainder of the gut wall.
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  • 40
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    Cell & tissue research 265 (1991), S. 517-525 
    ISSN: 1432-0878
    Keywords: Spermiogenesis ; Spermatids ; DNA ; Immunocytochemistry ; Electron microscopy ; Bovine ; Mouse ; Rabbit
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary DNA distribution in mouse, rabbit and bull spermatids was analyzed by electron microscopy, after using a Feulgen-like HCl-osmium ammine procedure, and after immunocytochemistry with anti-DNA antibodies. In addition, nucleic acids were visualized with the intercalating dye ethidium bromide and phosphotungstic acid. The parts of DNA displaying a beta helix configuration (possibly A-T rich parts) were identified by epifluorescence microscopy after staining with Hoechst 33258. In all 3 species, young spermatid nuclei were seen to have large areas poor in DNA, as well as DNA-rich areas, which were mostly concentrated into a peripheral layer close to the acrosome and into one or several masses, displaying species-specific locations. These DNA-rich areas were stained with Hoechst 33258. Elongating spermatid nuclei contained homogeneously distributed DNA, and this was evident following both immunocytochemistry and nucleic acid histochemistry in all 3 species. However, the distribution appeared more heterogeneous after the Feulgen-like procedure, and was accompanied by a disappearance of Hoechst-fluorescence. In fully elongated spermatids, all nuclear areas stained with Hoechst 33258, while the 3 other techniques labeled either all or species-specific parts of the condensed chromatin. The reasons for these variable reactions are discussed in terms of technique specificities, DNA configuration and nucleoprotein moiety replacements.
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  • 41
    ISSN: 1432-0878
    Keywords: Retina ; Melatonin ; Rhythmic biosynthesis ; Immunocytochemistry ; Pike, Esox lucius (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The levels of melatonin and the activities of two enzymes of the melatonin biosynthetic pathway, serotonin N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were measured throughout the light-dark cycle in the retina of a teleost fish, the pike. HIOMT activity did not display significant variations, whereas NAT activity and melatonin content showed a daily rhythm, high levels occurring during the night. The profiles of the latter two rhythms did not closely match one another and differed from those previously described in the pineal organ of the same species. These results are discussed with respect to a possible paracrine role of retinal melatonin. Melatonin-like immunoreactivity was found in the photoreceptor cell layer and in the Müller cells of the inner nuclear layer. The intensity of the melatonin-like immunoreactivity varied throughout the 24 h light-dark cycle, in good correlation with the variations in the melatonin level as measured by radioimmunoassay.
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  • 42
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    Cell & tissue research 265 (1991), S. 611-614 
    ISSN: 1432-0878
    Keywords: Immunocytochemistry ; Met-enkephalin ; Merkel cells ; Dense-core granules ; Mouse (ICR)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The electron-microscopic immunogold method was applied to Merkel cells of adult mice to demonstrate the subcellular localization of met-enkephalin-like immunoreactivity. Post-embedding incubation with metenkephalin antisera showed that the gold particles were associated with the dense-core granules of the Merkel cells. The majority, but not all, of the dense-core granules were strongly labelled. Osmication caused a significant reduction in the number of gold particles on these granules. The nerve terminal associated with the Merkel cell did not show met-enkephalin-like immunoreactivity. To the best of our knowledge, this is the first report of the ultrastructural localization of a positive met-enkephalin immunoreactivity in the dense-core granules of Merkel cells in mice.
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  • 43
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    Cell & tissue research 266 (1991), S. 247-258 
    ISSN: 1432-0878
    Keywords: Monoclonal antibodies ; Immunocytochemistry ; Olfactory epithelium ; Respiratory epithelium ; Antigens ; (Rat Wistar) ; (Mouse BALB/c)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Two panels of monoclonal antibodies have been generated, each panel having a distinct specificity for antigens located in the ciliary zone of either the olfactory or respiratory epithelium of rats. Tissue specificity was confirmed in enzyme-linked immunosorbent assays on membrane fractions from various tissues. During ontogeny, the expression of olfactory-specific antigens preceeds that of respiratory-specific antigens; this observation correlates with differences in the genesis of the respective cilia type and confirms that different molecular entities are recognized. A spatial segregation of immunoreactivity in the chemosensory epithelium was observed for one of the olfactory-specific monoclonal antibodies; negative zones were located in the dorsal recess of the nasal cavity and on the tips of the turbinates. Olfactory-specific antibodies reacted with distinct polypeptide bands on Western blots from olfactory ciliary preparations.
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  • 44
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    Cell & tissue research 266 (1991), S. 359-364 
    ISSN: 1432-0878
    Keywords: Immunocytochemistry ; Neuropeptide ; Diuretic hormone ; Neurosecretion ; Manduca sexta, Eurema nicippe (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Separate antisera were raised to the N- and C-terminal half of the diuretic hormone from Manduca sexta. Antisera against the two halves of this peptide recognized the same cells in M. sexta, and preabsorption of the antisera with the peptides used as antigens abolished the immunoreactivity, confirming their specificity. The antisera reacted with two median neurosecretory cells on each side of the protocerebral groove in larvae, and with a group of about 80 small median neurosecretory cells in the adult, as well as their axons to, and their axon terminals in, the corpora cardiaca. During the early pupal stages, small cells, which are possibly derived from a common neuroblast, differentiate into immunoreactive neurosecretory cells, which explains the large increase in cell numbers in the adult. In the sleepy sulphur butterfly, Eurema nicippe, homologous median neurosecretory cells in the adult were immunoreactive with both antisera.
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  • 45
    ISSN: 1432-0878
    Keywords: Pigment-dispersing hormone ; Orthopteroid insects ; Immunocytochemistry ; Insect brain ; Periplaneta americana, Schistocerca gregaria, Teleogryllus commodus (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary In a comparative study, the anatomy of neurons immunoreactive with an antiserum against the crustacean β-pigment-dispersing hormone was investigated in the brain of several orthopteroid insects including locusts, crickets, a cockroach, and a phasmid. In all species studied, three groups of neurons with somata in the optic lobes show pigment-dispersing hormone-like immunoreactivity. Additionally, in most species, the tritocerebrum exhibits weak immunoreactive staining originating from ascending fibers, tritocerebral cells, or neurons in the inferior protocerebrum. Two of the three cell groups in the optic lobe have somata at the dorsal and ventral posterior edge of the lamina. These neurons have dense ramifications in the lamina with processes extending into the first optic chiasma and into distal layers of the medulla. Pigment-dispersing hormone-immunoreactive neurons of the third group have somata near the anterior proximal margin of the medulla. These neurons were reconstructed in Schistocerca gregaria, Locusta migratoria, Teleogryllus commodus, Periplaneta americana, and Extatosoma tiaratum. The neurons have wide and divergent arborizations in the medulla, in the lamina, and in several regions of the midbrain, including the superior and inferior lateral protocerebrum and areas between the pedunculi and α-lobes of the mushroom bodies. Species-specific differences were found in this third cell group with regard to the number of immunoreactive cells, midbrain arborizations, and contralateral projections, which are especially prominent in the cockroach and virtually absent in crickets. The unusual branching patterns and the special neurochemical phenotype suggest a particular physiological role of these neurons. Their possible function as circadian pacemakers is discussed.
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  • 46
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    Cell & tissue research 266 (1991), S. 385-389 
    ISSN: 1432-0878
    Keywords: Amniotic epithelium ; Cytoskeleton ; Filaments ; α-Actinin ; Ezrin ; Immunocytochemistry ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The amniotic epithelium constitutes a diffusion barrier controlling the passage of solutes and water between the aminotic cavity and maternal circulation. With the present immunocytochemical approach, we have shown that several major components of the cytoskeleton, i.e., actin, α-actinin, spectrin and ezrin, are preferentially associated with the apical and lateral cell surfaces of the human amniotic epithelium. Keratins are distributed throughout the entire cytoplasm, whereas vimentin mainly forms a perinuclear scaffold. These findings indicate a role of the various components of the cytoskeleton in the structural integrity and modulation of cell shape and junctional permeability.
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  • 47
    ISSN: 1432-0878
    Keywords: Proventriculus ; Endocrine secretory cells ; Secretory granules ; Peptide hormones ; Colocalization ; Immunocytochemistry ; Colloidal gold ; Chicken
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The endocrine cells of the chicken proventriculus were investigated immunocytochemically, using the peroxidase-antiperoxidase technique on paraffin and semithin sections for light microscopy, and immunogold staining in osmium-fixed material for electron microscopy. The fixation procedure also allowed a detailed ultrastructural investigation. Twenty-three antisera were tested and 7 immunoreactive cell-types were identified: D-cells containing somatostatin-like peptide; EG-cells immunoreactive to anti-glucagon, anti-GLP1 and antineurotensin; NT-cells labelled only with anti-neurotensin; BN-cells containing bombesin-like material; ENK-cells showing met-enkephalin immunoreactivity; EC-cells reactive to anti-serotonin; and APP-cells positive to anti-avian pancreatic polypeptide. In addition, enterochromaffin-like (ECL) cells, were also detected by electron microscopy. The presence of ENK-cells and the ultrastructure of these and NT-cells are described for the first time in chicken proventriculus, and glucagon, GLP1 and neurotensin are shown to be colocalized in the EG-cells.
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  • 48
    ISSN: 1432-0878
    Keywords: Neuroendocrine cells ; Intestine ; Chromogranin ; Serotonin ; Regulatory peptides ; Immunocytochemistry ; Podarcis hispanica (Reptilia, Lacertilia)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Numerous endocrine cells can be observed in the gut of the lizard Podarcis hispanica after application of the Grimelius silver nitrate technique. The argyrophilic endocrine cells are usually tall and thin in the small intestine but short, basal, and round in the large intestine. Eleven types of immunoreactive endocrine cells have been identified by immunocytochemical methods. Numerous serotonin-, caerulein/gastrin/cholecystokinin octapeptide-and peptide tyrosine-tyrosine-immunoreactive cells; a moderate number of pancreatic polypeptide-, neurotensin-, somatostatin-, glucagon-like peptide-1-and glucagon-immunoreactive cells, and few cholecystokinin N-terminal-and bombesin-immunoreactive cells were found in the epithelium of the small intestine. Coexistence of glucagon with GLP-1 or PP/PYY has been observed in some cells. In the large intestine a small number of serotonin-, peptide tyrosine-tyrosine-, pancreatic polypeptide-, neurotensin-, somatostatin-and glucagon-like peptide-1-immunoreactive cells were detected. Vasoactive intestinal peptide immunoreactivity was found in nerve fibers of the muscular layer. Substance P-immunoreactive nerve fibers were detected in lamina propria, submucosa and muscular layer. Chromogranin A-immunoreactive cells were observed throughout the intestine, although in lower numbers than argyrophilic cells.
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  • 49
    ISSN: 1432-0878
    Keywords: Gliobastoma multiforme ; Cell culture ; Glial fibrillary acidic protein ; Immunocytochemistry ; Cytogenetics ; Man
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Two human cell lines (GL15 and GL22) derived from glioblastoma multiforme were established and characterized by immunohistochemical and cytogenetic techniques. The expression of glial fibrillary acidic proteins and the karyotype were analyzed at different passages for both cell lines. The course of marker-pattern differed in the two cell lines. The main findings were a cell-density-dependent expression of glial fibrillary acidic protein in the cell line GL15 at all passages and a decreased expression of this protein over time in the cell line GL22. Both cell lines had hyperdiploid karyotypes and exhibited glioma-specific chromosomal abnormalities (gain of chromosome 7 and loss of chromosome 10). In the GL15 cell line no relevant chromosomal changes were produced during culturing, whereas in the GL22 cell line a hypodiploid clone appeared at the 42nd passage. The immunohistochemical and cytogenetic data resulting from this study confirm that the two cell lines established in our laboratory originated from astrocytic tumor cells.
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  • 50
    ISSN: 1432-0878
    Keywords: Pituitary adenylate cyclase-activating peptide (PACAP) ; Vasoactive intestinal peptide (VIP) ; Immunocytochemistry ; Respiratory tract ; Autonomic innervation ; Mammals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Pituitary adenylate cyclase-activating peptide (PACAP) is a vasoactive intestinal peptide (VIP)-like peptide recently isolated from ovine hypothalami. Nerve fibers displaying PACAP immunoreactivity were found in the respiratory tract of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. A moderate supply of PACAP-immunoreactive fibers was seen in the nasal mucosa of guinea pigs. Few to moderate numbers of PACAP-containing fibers occurred in the tracheo-bronchial wall of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. The fibers were distributed beneath the epithelium, around blood vessels and seromucous glands, and among bundles of smooth muscle. In the lungs, the immunoreactive fibers were observed close to small bronchioli. A few PACAP-immunoreactive nerve cell bodies were seen in the sphenopalatine and otic ganglia of guinea pigs. Simultaneous double immunostaining of the respiratory tract of sheep and ferrets revealed that all PACAP-containing nerve fibers stored VIP. We suggest that neuronal PACAP may take part in the regulation of smooth muscle tone and glandular secretion.
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  • 51
    ISSN: 1432-0878
    Keywords: Brain, vertebrate ; Phenylethanolamine-N-methyltransferase ; Adrenaline ; Immunocytochemistry ; HPLC ; Rat (Wistar, Sprague-Dawley, Long Evans, Zucker)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Immunocytochemistry was used to compare the immunoreactivity of adrenergic neurons to a well characterized specific immunoserum to phenylethanolamine-N-methyltransferase (PNMT) in different strains of rats commonly used in research studies. In adult animals, marked differences were found in the PNMT-immunoreactivity of neurons between Wistar rats and other strains, resulting in a lower PNMT-immunostaining intensity (i) within neuronal perikarya of the medulla oblongata, and (ii) more strikingly, within nerve fibers and terminals located in various brain regions. This low PNMT-immunoreactivity of nerve fibers was detected both in 14- and 35-day-old Wistar rats. On the other hand, the HPLC measurement of catecholamines, in particular of adrenaline in the hypothalamus and the medulla oblongata, did not show any difference between adult Wistar and Sprague-Dawley rats. These data suggest that the low PNMT-immunoreactivity observed in central adrenergic neurons of the Wistar rats is related to the poor recognition of the antigen by the PNMT-antibody used. Possibly, these nerve cells mainly display an isoform of the enzyme that is immunologically different from the PNMT contained within the adrenergic neurons of other rat strains.
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  • 52
    ISSN: 1432-0878
    Keywords: Pituitary gland ; Rathke's pouch ; Intermediate filaments ; Cytokeratins ; Development, ontogenetic ; Immunocytochemistry ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary An immunohistochemical study of the production of the intermediate filaments [vimentin, cytokeratin, and glial filament acidic protein (GFAP)] during development of the pituitary gland was made by use of fetal and adult human pituitary tissue. Among these intermediate filament proteins in the anterior and intermediate lobes of the pituitary, cytokeratin is the first to appear, followed by GFAP and vimentin. However, only cytokeratin is seen during the period of morphogenesis of the pituitary gland, with the type-II subfamily cytokeratin 8 being the earliest to appear. Among the simple-epithelial-type cytokeratins, cytokeratins 8 and 19 were observed within the pituitary primordium during morphogenesis. Cells immunoreactive for cytokeratins 8 and 19 showed a heterogeneous three-dimensional distribution pattern in Rathke's pouch. Both cytokeratins 8 and 19 tended to be strongly positive at sites in the pituitary primordium where cells had become more loosely arranged (i.e., areas far from the diencephalon) but were only weakly positive in areas in which the epithelial cells were densely packed (i.e., areas closely associated with the diencephalon). It is concluded that, during the period of morphogenesis, Rathke's pouch has the intermediate filaments characteristic of simple epithelium and shows different immunoreactivity for simple-epithelial-type cytokeratins from place to place according to the extent of cellular differentiation.
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  • 53
    ISSN: 1432-0878
    Keywords: Clotting factors ; Antimicrobial substances ; Hemocytes ; Immunocytochemistry ; Tachypleus tridentatus (Chelicerata)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The structure of hemocytes in the normal state and during blood coagulation, and the intracellular localization of three clotting factors and two antimicrobial factors were examined in the Japanese horseshoe crabTachypleus tridentatus. Two types of hemocytes were found in the circulating blood: non-granular and granular hemocytes. The latter contained numerous dense granules classed into two major types: L- and D-granules. The L-granules were larger (up to 1.5 μm in diameter) and less electron-dense than the D-granules (less than 0.6 μm in diameter). The L-granules contained three clotting factors and one antimicrobial factor, whereas the D-granules exclusively contained the other antimicrobial factor. After treatment with endotoxin, the L-granules were released more rapidly than the D-granules, although almost all granules were finally exocytosed. The granular hemocyte possessed a single Golgi complex; possible precursor granules of L-granules and D-granules contained tubular and condensed dense material, respectively. These data are discussed in relation to the self-defense mechanisms of the horseshoe crab.
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  • 54
    ISSN: 1432-0878
    Keywords: Gonadotropin ; Gonadotropin subunits ; Gonadotropes ; In situ hybridization ; Immunocytochemistry ; Pituitary gland, pars distalis ; Oogenesis ; Oncorhynchus mykiss (Teleostei)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Biosynthesis of salmon gonadotropins, GTH I and GTH II, during ovarian development, were examined by means of in situ hybridization histochemistry and indirect immunocytochemistry. In rainbow trout pituitary glands, expression of GTH Iβ- and IIβ-subunit genes appeared separately in distinct cells (GTH I- and GTH II-cells), whereas the GTH α-subunit gene was expressed in both cell-types. In the GTH I-cells, coordinated increases in GTh, α and Iβ messenger ribonucleic acids (mRNAs) occurred coincident with the onset of vitellogenesis, indicating active synthesis of GTH I during vitellogenesis. In contrast, in the GTH II-cells, both GTH α-and IIβ-mRNA signals markedly increased from a later stage of vitellogenesis and persisted throughout oocyte maturation and ovulation, supporting the idea that GTH II is actively synthesized as a maturational GTH. GTH α-mRNA levels in the GTH I-cells selectively decreased prior to final oocyte maturation, although Iβ-mRNA levels remained elevated, thus suggesting a decline of biosynthesis of GTH I after vitellogenesis. These findings clarify how the synthesis of GTH I and GTH II are coordinated in the piscine pituitary, and indicate that the expression of GTH subunit genes during gametogenesis is regulated differentially in a cell-specific manner, both temporally and spatially.
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  • 55
    ISSN: 1432-0878
    Keywords: Visual system ; Neuropeptides ; Serotonin ; Pigment-dispersing hormone ; Immunocytochemistry ; Neuromodulators ; Phormia terraenovae (Insecta) ; Leucophaea maderae (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The pigment-dispersing hormone (PDH) family of neuropeptides comprises a series of closely related octadecapeptides, isolated from different species of crustaceans and insects, which can be demonstrated immunocytochemically in neurons in the central nervous system and optic lobes of some representatives of these groups (Rao and Riehm 1989). In this investigation we have extended these immunocytochemical studies to include the blowfly Phormia terraenovae and the cockroach Leucophaea maderae. In the former species tissue extracts were also tested in a bioassay: extracts of blowfly brains exhibited PDH-like biological activity, causing melanophore pigment dispersion in destalked (eyestalkless) specimens of the fiddler crab Uca pugilator. Using standard immunocytochemical techniques, we could demonstrate a small number of pigment-dispersing hormone-immunoreactive (PDH-IR) neurons innervating optic lobe neuropil in the blowfly and the cockroach. In the blowfly the cell bodies of these neurons are located at the anterior base of the medulla. At least eight PDH-IR cell bodies of two size classes can be distinguished: 4 larger and 4 smaller. Branching immunoreactive fibers invade three layers in the medulla neuropil, and one stratum distal and one proximal to the lamina synaptic layer. A few fibers can also be seen invading the basal lobula and the lobula plate. The fibers distal to the lamina appear to be derived from two of the large PDH-IR cell bodies which also send processes into the medulla. These neurons share many features in their laminamedulla morphology with the serotonin immunoreactive neurons LBO-5HT described earlier (see Nässel 1988). It could be demonstrated by immunocytochemical double labeling that the serotonin and PDH immunoreactivities are located in two separate sets of neurons. In the cockroach optic lobe PDH-IR processes were found to invade the lamina synaptic region and form a diffuse distribution in the medulla. The numerous cell bodies of the lamina-medulla cells in the cockroach are located basal to the lamina in two clusters. Additional PDH-IR cell bodies could be found at the anterior base of the medulla. The distribution and morphology of serotonin-immunoreactive neurons in the cockroach lamina was found to be very similar to the PDH-IR ones. It is hence tempting to speculate that in both species the PDH-and serotonin-immunoreactive neurons are functionally coupled with common follower neurons. These neurons may be candidates for regulating large numbers of units in the visual system. In the flies photoreceptor properties may be regulated by action of the two set of neurons at sites peripheral to the lamina synaptic layer, possibly by paracrine release of messengers.
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  • 56
    ISSN: 1432-0878
    Keywords: Corticotropin-releasing factor (CRF) ; Immunocytochemistry ; Arginine vasotocin (AVT) ; Mesotocin (MST) ; Co-localization ; Natrix maura (Serpentes)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The anatomical distribution of neurons and nerve fibers containing corticotropin-releasing factor (CRF) has been studied in the brain of the snake, Natrix maura, by means of immunocytochemistry using an antiserum against rat CRF. To test the possible coexistence of CRF with the neurohypophysial peptides arginine vasotocin (AVT) and mesotocin (MST) adjacent sections were stained with antisera against the two latter peptides. CRF-immunoreactive (CRF-IR) neurons exist in the paraventricular nucleus (PVN). In some neurons of the PVN, coexistence of CRF with MST or of CRF with AVT has been shown. Numerous CRF-IR fibers run along the hypothalamo-hypophysial tract and end in the outer layer of the median eminence. In addition, some fibers reach the neural lobe of the hypophysis. CRF-IR perikarya have also been identified in the following locations: dorsal cortex, nucleus accumbens, amygdala, subfornical organ, lamina terminalis, nucleus of the paraventricular organ, nucleus of the oculomotor nerve, nucleus of the trigeminal nerve, and reticular formation. In addition to all these locations CRF-IR fibers were also observed in the lateral septum, supraoptic nucleus, habenula, lateral forebrain bundle, paraventricular organ, hypothalamic ventromedial nucleus, raphe and interpeduncular nuclei.
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  • 57
    ISSN: 1432-0878
    Keywords: Subcommissural organ ; Secretory pathway ; C1B8A8 monoclonal antibody ; Immunocytochemistry ; Immunoaffinity chromatography ; Bovine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The secretory pathway of the complex-type glycoprotein specific to the subcommissural organ (SCO) was examined using the monoclonal antibody (Mab) C1B8A8. Immunoreactive material was revealed in various compartments of the secretory ependymocyte, i.e., the endoplasmic reticulum, the Golgi area and the secretory vacuoles. In addition, immunoreactive material was also observed in the ventricular cavity. Evidence of a release both at the apical lining and at the basal process of the SCO ependymocytes suggests that the same protein could be secreted into the cerebrospinal fluid and the perivascular spaces. After immunoaffinity chromatography of soluble extracts of the SCO on Mab C1B8A8 immunoadsorbent columns, three glycopeptides were identified on Western blots; they were concanavalin A (Con A)-positive (88, 54 and 34 kDa) and wheat-germ agglutinin (WGA)-positive (54 and 34 kDa). The Con A-positive glycopeptide (88 kDa) is probably related to the high-mannose-type glycoprotein, the precursor form of the secreted compound, whereas the 54 kDa-glycopeptide that is both Con A- and WGA-positive could represent an intermediate form. The 34 kDa-glycopeptide that is strongly WGA-positive could be related to the monomeric form of the secreted compound. These three glycopeptides were not revealed in eluted fractions of soluble extracts of the ependyma that served as control.
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  • 58
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    Cell & tissue research 263 (1991), S. 71-79 
    ISSN: 1432-0878
    Keywords: Aromatase ; Immunocytochemistry ; Brain aromatase ; Preoptic area ; Hypothalamus ; Mouse
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The distribution of aromatase-immunoreactive cells was studied by immunocytochemistry in the mouse forebrain using a purified polyclonal antibody raised against human placental aromatase. Labeled perikarya were found in the dorso-lateral parts of the medial and tuberal hypothalamus. Positive cells filled an area extending between the subincertal nucleus in the dorsal part, the ventromedial hypothalamic nucleus in the ventral part, and the internal capsule and the magnocellular nucleus of the lateral hypothalamus in the lateral part. The same distribution was seen in the two strains of mice that were studied (Jackson and Swiss), and the number of immunoreactive perikarya did not seem to be affected by castration or testosterone treatment. No immunoreactivity could be detected in the medial regions of the preoptic area and hypothalamus; these were expected to contain the enzyme based on assays of aromatase activity performed in rats and on indirect autoradiographic evidence in mice. Our data raise questions concerning the distribution of aromatase in the brain and the mode of action of the centrally produced estrogens.
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  • 59
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    Cell & tissue research 263 (1991), S. 195-198 
    ISSN: 1432-0878
    Keywords: Pancreas, endocrine ; Islets of Langerhans ; Immunocytochemistry ; Endocrine cells four types ; Electron microscopy ; Sminthopsis crassicaudata (Marsupialia)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The endocrine pancreas of the Australian fattailed dunnart, Sminthopsis crassicaudata, was investigated by means of electron-microscopic immunocytochemistry using the protein A-gold technique on London resin (LR) white-embedded tissue. The primary antibodies used were raised against insulin, glucagon, somatostatin and pancreatic polypeptide. The morphology of the secretory granules differed in the four cell types. The insulin cells are pleomorphic, and the secretory granules composed of an electron-dense core surrounded by an electron-lucen halo. The glucago cells possess granules with an electron-dense core usually surrounded by a halo of less dense granular material. Somatostatin cells have large, less dense secretory granules. The pancreatic polypeptide cells show small, dense secretory granules. In order for an ultrastructural study to be considered reliable for the definite identification of endocrine cell types, it is essential that it be corroborated by immunocytochemical data at the light-or preferably electron-microscopic level. Recent developments in immuno-electron-microscopic techniques have contributed to a better knowledge of cells responsible for the secretion of a wide variety of hormones, as in this study.
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  • 60
    ISSN: 1432-0878
    Keywords: Pineal organ ; Epithalamus ; Pinealocytes ; Hydroxyindole O-methyltransferase (HIOMT) ; Immunocytochemistry ; HIOMT-mRNA ; In-situ hybridization ; Bovine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Hydroxyindole O-methyltransferase (HIOMT)-immunoreactive cells and melatonin synthesis were demonstrated in bovine epithalamus (including the pineal gland) with monoclonal antibodies and cRNA probes to HIOMT. The HIOMT-immunoreactive product was present in the cytoplasm of pinealocytes. All identifiable pinealocytes were clearly labeled in the pineal gland. The expression of the HIOMT gene was first identified in pinealocyte cytoplasm by in-situ hybridization (ISH). The distribution of the hybridization-positive cells in the pineal gland was compatible with that revealed by immunocytochemistry using the monoclonal antibody to HIOMT. In addition, HIOMT transcripts were found in the medial habenular nucleus, and the habenular and posterior commissure; they may correspond to S-antigen-immunoreactive cells demonstrated in the same regions of the hamster and the mouse. In these regions, the hybridization-positive cells did not exhibit HIOMT-immunoreactivity; thus, cells devoid of immunoreactivity may synthesize but rapidly transport the newly synthesized proteins. These results indicate (1) that the conversion of N-acetylserotonin into melatonin takes place in the cytoplasm of pinealocytes, (2) that some epithalamic cells in the habenular area may synthesize melatonin, and (3) that melatonin may act as a chemical messenger in centrally directed processes, as shown by using S-antigen immunocytochemistry.
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  • 61
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    Cell & tissue research 264 (1991), S. 321-328 
    ISSN: 1432-0878
    Keywords: Tooth pulp ; NGF receptor ; Calcitonin gene-related peptide ; Substance P ; Neuropeptide Y ; Immunocytochemistry ; Electron microscopy ; Cat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The distribution of nerve growth factor receptor (NGF receptor)-like immunoreactivity in pulps of developing primary and mature permanent cat canine teeth was examined, by use of a monoclonal antibody against NGF receptor detected by fluorescence immunohistochemistry and pre-embedding immunocytochemical light- and electron microscopy. Both primary and permanent pulps contained a vast number of NGF receptor-like immunoreactive nerves. Immunolabelling appeared to be localized both to axons and Schwann cells. In addition, many blood vessel walls in immature primary tooth pulps showed NGF receptor-like immunoreactivity, in contrast to permanent pulps where blood vessels rarely were NGF receptor-immunoreactive. Double-labelling immunofluorescence experiments revealed that in the permanent pulp a majority of the NGF receptor-positive nerves also showed calcitonin gene-related peptide (CGRP)-like immunoreactivity, and many showed substance P-like immunoreactivity. However, nerve fibers with neuropeptide Y-like immunoreactivity lacked NGF receptor-like immunoreactivity. In developing primary tooth pulps fewer NGF receptor-positive nerves were CGRP-like immunoreactive or substance P-like immunoreactive, as compared to the permanent pulp. Neuropeptide Y-like immunoreactive nerve fibers were not detected in the primary tooth pulp. The results suggest a role for nerve growth factor in both developing and mature sensory nerves of the tooth pulp.
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  • 62
    ISSN: 1432-0878
    Keywords: Serotonin ; Immunocytochemistry ; Olfactory interneurons ; Macrobrachium rosenbergii, Munida sarsi, Pacifastacus leniusculus (Crustacea)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary An antiserum raised against serotonin (5HT) was applied to the brains of representatives of four different infraorders of decapod crustaceans, and revealed two morphological classes of olfactory interneurons. They were classified by the position and size of their cell bodies, and by their connection pattern. One class consisted of giant olfactory interneurons and the other of globuli cells. They were regarded as input and intrinsic interneurons, respectively, because of their morphology. The two classes displayed a similar pattern in two of the infraorders, whereas only one class appeared in the other two infraorders.
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  • 63
    ISSN: 1432-0878
    Keywords: Inhibin ; LH ; FSH ; Pituitary gland, pars anterior ; Immunocytochemistry ; Maccaca fascicularis, Macaca mulatta (Primates)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Inhibin-like immunoreactivity was detected by immunocytochemistry in the pituitaries of untreated male crab-eating macaques (cynomolgus monkey) and rhesus monkeys, in rhesus monkeys actively immunized against FSH, and in one orchidectomized crab-cating macaque. Localizations were performed by the immunogold-silver staining with 5-nm colloidal gold-conjugated second or third antibodies and by the alkaline phosphatase-anti-alkaline-phosphatase technique. Two different inhibin-specific antisera, raised against the α-subunit or the entire inhibin molecule, provided identical staining patterns. Positive label was confined to the pars distalis of the pituitary and occurred exclusively in the cytoplasm of morphologically different cell types throughout the pars distalis in all pituitaries. Staining was most prominent in clusters of chromophobic cells. The presence of inhibin-like activity in the pituitary of an orchidectomized monkey with undetectable serum inhibin levels suggests that inhibin is produced within the pituitary gland. Co-localization studies for the β-subunits of the gonadotropic hormones revealed that on average 82% of the gonadotropes were bihormonal. Using the same protocol, co-localization of inhibin-like activity with gonadotropin-like immunoreactivity revealed only a small degree of common distribution (〈15%). Inhibinpositive cells were frequently in close proximity to gonadotropic cells and, thus, paracrine effects of inhibin on gonadotropin-synthesizing cells are conceivable.
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  • 64
    ISSN: 1432-0878
    Keywords: Neuropeptide Y (NPY) ; Immunocytochemistry ; Avian brain ; Japanese quail, Coturnix coturnix japonica
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary In the present study, we have demonstrated, by means of the biotin-avidin method, the widespread distribution of neuropeptide Y (NPY)-immunoreactive structures throughout the whole brain of the Japanese quail (Coturnix coturnix japonica). The prosencephalic region contained the highest concentration of both NPY-containing fibres and perikarya. Immunoreactive fibres were observed throughout, particularly within the paraolfactory lobe, the lateral septum, the nucleus taeniae, the preoptic area, the periventricular hypothalamic regions, the tuberal complex, and the ventrolateral thalamus. NPY-immunoreactive cells were represented by: a) small scattered perikarya in the telencephalic portion (i.e. archistriatal, neostriatal and hyperstriatal regions, hippocampus, piriform cortex); b) medium-sized cell bodies located around the nucleus rotundus, ventrolateral, and lateral anterior thalamic nuclei; c) small clustered cells within the periventricular and medial preoptic nuclei. The brainstem showed a less diffuse innervation, although a dense network of immunopositive fibres was observed within the optic tectum, the periaqueductal region, and the Edinger-Westphal, linearis caudalis and raphes nuclei. Two populations of large NPY-containing perikarya were detected: one located in the isthmic region, the other at the boundaries of the pons with the medulla. The wide distribution of NPY-immunoreactive structures within regions that have been demonstrated to play a role in the control of vegetative, endocrine and sensory activities suggests that, in birds, this neuropeptide is involved in the regulation of several aspects of cerebral functions.
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  • 65
    ISSN: 1432-0878
    Keywords: FMRFamide ; Brain, vertebrate ; Hypothalamus ; Pituitary gland, pars intermedia ; Nervus terminalis ; Immunocytochemistry ; Scyliorhinus torazame (Elasmobranchii)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The distribution of the molluscan cardioexcitatory tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH2) in the brain of the cloudy dogfish, Scyliorhinus torazame, was examined by immunocytochemistry. FMRFamide-like immunoreactivity was demonstrated to occur extensively in various regions of the dogfish brain, except for the corpus cerebelli. Immunoreactive neuronal perikarya were located in the ganglion of the nervus terminalis, the preoptic area, and the hypothalamic periventricular gray matter consisting of the nucleus medius hypothalamicus, the nucleus lateralis tuberis, and the nucleus lobi lateralis. some of the immunoreactive cells in the hypothalamus were identified as cerebrospinal fluid-contacting neurons. The bulk of the immunostained fibers in the nervus terminalis penetrated into the midventral portion of the telencephalon and ran dorsocaudally toward the basal telencephalon and hypothalamus, showing radial projections or ramifications. The labeled fibers were abundant in the midbasal part of the telencephalon and in the hypothalamus, where some fibers were found in loose networks around the cell bodies of the nucleus septi and hypothalamic periventricular nuclei. The fibers demonstrated in the hypothalamus terminated around the vascular wall of the primary capillary plexus of the median eminence or penetrated deeply into the pars intermedia of the hypophysis. These results suggest that, in the dogfish, an FMRFamide-like substance participates in the regulation of adenohypophysial function. This molecule may have a role as a neurotransmitter and/or neuromodulator in the central nervous system.
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  • 66
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    Protoplasma 163 (1991), S. 199-202 
    ISSN: 1615-6102
    Keywords: Actin ; Freeze substitution ; Fungi ; Hyphal tip ; Immunocytochemistry ; Magnaporthe grisea
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have successfully localized fungal actin for the first time using immuno-electron microscopy and hyphal tips of the rice blast pathogenMagnaporthe grisea. Following ultrarapid freezing, samples were processed in a novel substitution fluid of 10% acrolein in anhydrous ethanol and embedded in LR White resin. A monoclonal anti-actin antibody, previously shown to recognizeM. grisea actin, bound specifically to filasomes concentrated in the peripheral cytoplasm of subapical regions, and to the core-region of the Spitzenkörper.
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  • 67
    ISSN: 1615-6102
    Keywords: Chlamydomonas ; Cryofixation ; Freeze-substitution ; Immunocytochemistry ; Nuclear proteins ; Nucleus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We describe the preparation of monoclonal antibodies to nuclear antigens in the green alga,Chlamydomonas reinhardtii, and their localization at the light and electron microscope level. Supernatants from hybridomas were screened by the ELISA method and the four antibodies giving the strongest signal were subjected to further analysis. At the LM level immunogold silver staining was used on semi-thick resinless sections. We have examined at the EM level the distribution of these antigens by post-embedding immunocytochemical techniques on sections of conventionally fixed specimens compared to cryofixed and freeze-substituted ones. Enhanced ultrastructural preservation was observed in cells which were cryofixed, freeze-substituted and embedded at −35°C in Lowicryl K4M. Different preparative procedures involving cryofixation and substitution are described. Of the four antibodies three were localized under light and electron microscopy. All three were distributed in the interchromatin space. One of these antigens (QUL4D2, 54 kDa) is also found in the dense fibrillar component and fibrillar centers of the nucleolus.
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  • 68
    ISSN: 1615-6102
    Keywords: Polygalacturonan ; Pectin ; Methyl esterification ; Extracellular matrix ; Frankia ; Ceanothus ; Root nodule ; Immunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary During early stages of nodule development inCeanothus spp., theFrankia infection pathway is characterized by a distinctive host-derived extracellular matrix. In the present study, a major component of the host interface is shown to consist of pectic polysaccharides. The distribution of these pectic polysaccharides in developing nodules has been delineated in root and nodule tissue. The levels of polygalacturonic acid detected were extremely high in the root mucilage and in the intercellular infection matrix in the root cortex, as detected by indirect immunogold localization with an antibody, and with fluorescein-conjugated alginate and pectate probes. Polygalacturonans in the intercellular matrix and in nodule tissue were predominantly esterified. The non-esterified polygalacturonans were located in cell junctions. Within the infected nodule cortical cells, (poly)galacturonate content of the interfacial encapsulation surrounding theFrankia endosymbiont was very high, while the cell walls were not labeled above background, suggesting that the encapsulation is a specialized wall layer.
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  • 69
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    Protoplasma 162 (1991), S. 38-45 
    ISSN: 1615-6102
    Keywords: Green algae ; Immunocytochemistry ; Pyrenoid ; RuBisCo ; RuBisCo activase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary RuBisCo activase catalyzes the activation and maintains the activated state of the photosynthetic enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo, EC 4.1.1.39). We employed antisera prepared against the RuBisCo holoenzyme purified from tobacco and RuBisCo activase isolated from spinach to determine the localization of these proteins in leaves of C3-type higher plants and green algae. In leaves ofVicia faba, both RuBisCo activase and RuBisCo are distributed throughout the chloroplast stroma. In contrast, RuBisCo activase and RuBisCo are predominantly localized to the pyrenoid in the green algaeChlamydomonas reinhardtii andColeochaete scutata. The co-immunolocalization of RuBisCo activase and RuBisCo to the pyrenoid in these two green algal species suggests that pyrenoid-localized RuBisCo is catalytically competent. We conclude that the pyrenoid functions as a unique metabolic compartment of the chloroplast in which the reactions of the photosynthetic carbon reduction pathway are initiated.
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  • 70
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    Bulletin of experimental biology and medicine 111 (1991), S. 47-50 
    ISSN: 1573-8221
    Keywords: DNA-methylase ; multiple forms ; hydrophobic properties ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
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  • 71
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    Cellular and molecular neurobiology 11 (1991), S. 245-251 
    ISSN: 1573-6830
    Keywords: enkephalin ; mRNA ; depolarization ; gene expression ; aggregating cells ; neuronal cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary 1. Aggregating fetal rat brain cells express a significant amount of proenkephalin A (PENK) mRNA, and a selective radioimmunoassay shows that this mRNA is also translated into enkephalins. 2. Depolarization with potassium chloride (KC1) or veratridine increases the expression of PENK mRNA in a time-dependent fashion, with a maximal increase of sixfold. It is interesting, however, that depolarization of the same cultures with KC1 has no effect on the expression of prodynorphin mRNA. 3. An increase in PENK mRNA levels has been also observed in cultures treated with 8-Br-cAMP, phorbol 12-myristate-13-acetate (TPA), or dexamethasone. 4. However, incubation of the cultures with the opioid agonist etorphine or the antagonist naltrexone did not alter PENK gene expression, suggesting that there is not feedback control of opioids on PENK biosynthesis in these cells. 5. The increase in PENK mRNA in depolarized and in TPA-, dexamethasone-, or 8-Br-cAMP-treated cultures was not accompanied by a significant increase in the amount of free immunoreactive met-enkephalin. Fetal brain cell cultures are therefore a useful neuronal model system for studying the mechanism that regulated the expression of PENK mRNA.
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