ALBERT

Notes: The manganese-oxidizing factor of Pseudomonas putida strain GB-1 is associated with the outer membrane. One of the systems of protein transport across the outer membrane is the general secretory pathway (Gsp). The gsp genes are called xcp in Pseudomonas species. In a previous study, it was shown that mutation of the prepilin peptidase XcpA and of a homologue of the pseudopilin XcpT inhibited transport of the factor. In the present study, we describe the genomic region flanking the xcpT homologue (designated xcmT1). We show that xcmT1 is part of a two-gene operon that includes an xcpS homologue (designated xcmS). No other xcp-like genes are present in the regions flanking the xcmT1/xcmS cluster. We also characterized the site of transposon insertion of another transport mutant of P. putida GB-1. This insertion appeared to be located in a gene (designated xcmX) possibly encoding another pseudopilin-related protein. This xcmX is clustered with two other xcpT-related genes (designated xcmT2 and xcmT3) on one side and homologues of three csg genes (designated csmE, csmF and csmG) on the other side. The csg genes are involved in production of aggregative fibres in Escherichia coli and Salmonella typhimurium. A search for XcmX homologues revealed that the recently published genome of Ralstonia solanacearum and the unannotated genome of P. putida KT2440 contain comparable gene clusters with xcmX and xcp homologues that are different from the well-described ‘regular’xcp/gsp clusters. They do contain xcpR and xcpQ homologues but, for example, homologues of xcpP, Y and Z are lacking. The results suggest a novel Xcp-related system for the transport of manganese-oxidizing enzymes to the cell surface.

Notes: Haemophilus influenzae is an obligate commensal of the upper respiratory tract of humans that uses simple repeats (microsatellites) to alter gene expression. The mod gene of H. influenzae strain Rd has homology to DNA methyltransferases of type III restriction/modification systems and has 40 tetranucleotide (5′-AGTC) repeats within its open reading frame. This gene was found in 21 out of 23 genetically distinct H. influenzae strains, and in 13 of these strains the locus contained repeats. H. influenzae strains were constructed in which a lacZ reporter was fused to a chromosomal copy of mod downstream of the repeats. Phase variation occurred at a high frequency in strains with the wild-type number of repeats. Mutation rates were derived for similarly engineered strains, containing different numbers of repeats. Rates increased linearly with tract length over the range 17–38 repeat units. The majority of tract alterations were insertions or deletions of one repeat unit with a 2:1 bias towards contractions of the tract. These results demonstrate the number of repeats to be an important determinant of phase variation rate in H. influenzae for a gene containing a microsatellite.

Notes: The histological analysis of eastern Atlantic bluefin tuna Thunnus thynnus ovaries caught from February to September 1999–2000, made it possible to distinguish the presence of seven oocyte developmental stages and allowed the characterization of six time-dependent ovary maturity stages. The ovaries of mature (fork length, LF ≥ 110 cm) bluefin tuna were non-active from August (spent period) to March (quiescent period) when they contained only perinucleolar-stage oocytes. Ovary development started in April to early May (recrudescent period) with the appearance of oocytes at the lipid stage. Vitellogenesis appeared in mid-May (ripening period) and post-vitellogenesis occurred in late May to mid-June (pre-spawning period). In late June to early July, hydrated oocytes, a sign of imminent spawning, were found only in specimens caught in Balearic waters. Females ranging between 100 and 110 cm LF, captured during the recrudescent and ripening periods, had the largest oocytes at the lipid stage, most of which were degenerating. An extensive vitellogenic atresia was observed in the ovaries of five females caught during the spawning period in non-spawning areas.

Notes: 1 Prolonged bed rest or exposure to microgravity may cause several alterations in autonomic nervous system response (ANSR). 2 Hindlimb unloading (HU) rats were used as an animal model of simulated microgravity to investigate ANSR changes. The experiments were carried out to investigate the effects of simulated microgravity on the autonomic nervous response of the perfused mesenteric vascular bed (MVB), vas deferens and the colon and duodenum from 2-week HU rats. 3 In MVB preparations of HU rats, the frequency-dependent increases in perfusion pressure with perivascular nerve stimulation (PNS; 8–40 Hz) were inhibited, whereas the noradrenaline (NA) concentration-dependent (1–100 μm) perfusion pressure increases were potentiated. The latter most probably reflected up-regulation of α-adrenergic receptor function. Relaxant responses of NA-precontracted MVB to PNS (4–30 Hz) or isoprenaline were not different between control and HU preparations, while vasodilation induced by the endothelial agonist ACh was reduced. 4 Transmural stimulation (2–40 Hz) induced frequency-dependent twitches of the vas deferens which were reduced in vas deferens of HU rats, while the sensitivity to NA-induced contraction was significantly increased. 5 In the gastroenteric system of HU rat, direct contractile responses to carbachol or tachykinin as well as relaxant or contractile responses to nervous stimulation appeared unchanged both in the proximal colon rings and in duodenal longitudinal strips. 6 In conclusion, HU treatment affects peripheral tissues in which the main contractile mediators are the adrenergic ones such as resistance vessels and vas deferens, probably by reducing the release of neuromediator. This study validates NA signalling impairment as a widespread process in microgravity, which may most dramatically result in the clinical phenotype of orthostatic intolerance.

Notes: 1 Inflammatory bowel disease (IBD) is a condition that involves proinflammatory cytokines such as interleukins 1β and 6 (ILs). In this disease, it has been shown that an abnormal microcirculatory system is implicated. 2 Therefore, the effects of in vivo treatment for three days with interleukins 1β and 6 were investigated on rat isolated mesenteric vascular bed (MVB). 3 A significant concentration-dependent increase in vascular response to noradrenaline (NA) was found, with a significant difference in Emax between control (93.01 ± 16.78 mmHg) and treated preparations (137.91 ± 5.20 mmHg). Endothelin-1(ET-1) induced a significantly greater increase of perfusion pressure in treated rats in comparison with control rats at the highest concentration used (0.1 μm). 4 The concentration-dependent decrease of perfusion pressure induced by acetylcholine (ACh) in MVB precontracted with NA was significantly reduced in specimens from treated rats in comparison with control rats, with a significant difference in Emax between control and treated preparations. 5 Perivascular nerve stimulation (PNS) evoked contractions with no difference between treatments. Similarly, no difference in relaxant effect was found after PNS in specimens precontracted with NA, in the presence of guanethidine. 6 These findings indicate that the precocious inflammation acts only at postsynaptic level, facilitating vascular contraction. These data seem to support the hypothesis that vascular dysfunction caused by overproduction of ILs may contribute, among other immunological factors, to vasculitis in IBD that leads to intestinal ischaemia through vasoconstriction.

Notes: 1 The present study aimed to evaluate the role of κ-opioid receptors at two peripheral sites, the vas deferens and the proximal colon, in κ-opioid receptor knockout mice. We investigated the role of the κ-opioid receptor in the vas deferens twitch response and in the colonic ‘off-contraction’, a rebound contractile response which follows the inhibitory response to low frequencies stimulation (10, 20, 30 Hz) and which has been suggested to ‘locally’ reproduce the contractile component of the peristaltic reflex. 2 Transmural stimulation of the vas deferens at lower frequencies (10 Hz, 10 V, 1 ms pulse trains lasting 0.5 s) evoked a contractile response that was significantly higher in the preparations from knockout mice because of lack of κ-opioid receptors than in wild type mice. A selective κ-opioid receptor agonist, U-50,488H, induced a dose-dependent inhibition of the electrically stimulated contraction in vas deferens. The percentages of reduction of the twitch response were significantly lower in knockout mice than in wild type mice after treatment with U-50,488H. The reduction of twitch response caused by U-50,488H was not reversed by administration of nor-binaltorphimine (nor-BNI) (5 × 10−6 m), a selective κ-opioid receptor antagonist, in preparations from both knockout mice and wild type mice. U-50,488H has no effect on postsynaptic adrenergic receptors, as its administration did not affect the direct contractile response to noradrenaline. 3 Transmural stimulation (5 Hz, 20 V, 2 ms pulse trains lasting 30 s) induced inhibition of spontaneous activity of colonic strips during the period of stimulation, followed by an ‘off-contraction’ after the cessation of stimulation. The statistical evaluation of the ‘off-contraction’ responses between the two strains showed no significant difference. The off-contraction, measured in specimens from knockout mice, was inhibited concentration-dependently by U-50,488H (P 〈 0.01) and significantly less than from wild type mice. 4 The effect of U-50,488H was not reversed by administration of nor-BNI (5 × 10−6 m), either in preparations from knockout mice or from wild type mice. 5 Our data may suggest that κ-opioid receptors are involved in some peripheral responses to the nerve stimulation, as indicated by the effect of U-50,488H, a selective κ-opioid receptor agonist. However, the involvement of κ-opioid receptor was also present, although less apparent, in κ -opioid receptor knockout mice, suggesting either that this drug acts not only on κ-opioid receptors but also on other receptor sites, such as κ-like receptors. An alternative interpretation can be related to a sodium channel blocking action of U-50,488H, which could explain the inhibitory effects of twitch response still present but less evident in knockout strain and the lack of effect of the antagonist nor-BNI.

Notes: Radon-222 (Rn-222) is used as a transport tracer of forest canopy–atmosphere CO2 exchange in an old-growth, tropical rain forest site near km 67 of the Tapajós National Forest, Pará, Brazil. Initial results, from month-long periods at the end of the wet season (June–July) and the end of the dry season (November–December) in 2001, demonstrate the potential of new Rn measurement instruments and methods to quantify mass transport processes between forest canopies and the atmosphere. Gas exchange rates yield mean canopy air residence times ranging from minutes during turbulent daytime hours to greater than 12 h during calm nights. Rn is an effective tracer for net ecosystem exchange of CO2 (CO2 NEE) during calm, night-time hours when eddy covariance-based NEE measurements are less certain because of low atmospheric turbulence. Rn-derived night-time CO2 NEE (9.00±0.99 μmol m−2 s−1 in the wet season, 6.39±0.59 in the dry season) was significantly higher than raw uncorrected, eddy covariance-derived CO2 NEE (5.96±0.51 wet season, 5.57±0.53 dry season), but agrees with corrected eddy covariance results (8.65±1.07 wet season, 6.56±0.73 dry season) derived by filtering out lower NEE values obtained during calm periods using independent meteorological criteria. The Rn CO2 results suggest that uncorrected eddy covariance values underestimate night-time CO2 loss at this site. If generalizable to other sites, these observations indicate that previous reports of strong net CO2 uptake in Amazonian terra firme forest may be overestimated.

Notes: As part of the Large Scale Biosphere–Atmosphere Experiment in Amazônia (LBA), we have developed a bottom-up approach for estimating canopy-scale fluxes of isoprene. Estimating isoprene fluxes for a given forest ecosystem requires knowledge of foliar biomass, segregated by species, and the isoprene emission characteristics of the individual tree species comprising the forest. In this study, approximately 38% of 125 tree species examined at six sites in the Brazilian Amazon emitted isoprene. Given logistical difficulties and extremely high species diversity, it was possible to screen only a small percentage of tree species, and we propose a protocol for estimating the emission capacity of unmeasured taxa using a taxonomic approach, in which we assign to an unmeasured genus a value based on the percentage of genera within its plant family which have been shown to emit isoprene.Combining this information with data obtained from 14 tree censuses at four Neotropical forest sites, we have estimated the percentage of isoprene-emitting biomass at each site. The relative contribution of each genus of tree is estimated as the basal area of all trees of that genus divided by the total basal area of the plot. Using this technique, the percentage of isoprene-emitting biomass varied from 20% to 42% (mean=31%; SD=8%).Responses of isoprene emission to varying light and temperature, measured on a sun-adapted leaf of mango (Mangifera indica L.), suggest that existing algorithms developed for temperate species are adequate for tropical species as well. Incorporating these algorithms, estimates of isoprene-emitting biomass, isoprene emission capacity, and site foliar biomass into a canopy flux model, canopy-scale fluxes of isoprene were predicted and compared with the above-canopy fluxes measured at two sites. Our bottom-up approach overestimates fluxes by about 50%, but variations in measured fluxes between the two sites are largely explained by observed variation in the amount of isoprene-emitting biomass.

Notes: The podzolization process is studied through lipids in nine characteristic podzol horizons. Organic matter accumulates particularly with aluminium in the Bh horizon, while the hard, cemented Bs horizon below this is formed mainly by iron oxides. The low soil pH seems to have no great influence on the preservation of lipids as reflected by the absolute amounts present and the presence of bacterial lipid markers throughout the profile. Independent of soil pH, lipids accumulate in organically enriched horizons. Albeit, high molecular weight organic compounds accumulate to a relatively greater extent than lipids in these horizons. A lipid signal related to the aerial parts, i.e. leaves and flowers, of Calluna is observed only in the O horizon. This ‘n-alkane, steroid and triterpenoids’ signal is quickly lost in the underlying Ah horizon due to (bacterial) oxidation. The other total lipid extracts obtained are dominated by root-derived compounds. In subsoil horizons rich in organic matter, i.e. the Ahb and Bh horizons, root-derived friedooleanan and steroid compounds dominate the total lipid signal. Degraded horizons, poor in organic matter, i.e. the E2, Bhs, Bs and B/C horizons, are dominated by C22 and C24ω-hydroxy acids, long-chain (〉 C20) n-alkanoic acids with a strong even-over-odd predominance and C22 and C24n-alkanols. Steroid and root-derived triterpenoids with a friedooleanan structure have been removed from these horizons through degradation. Based on total organic carbon content and lipid composition, the formation of an E1 horizon has started, but is not yet complete. In the Ahb horizon, a contribution from buried vegetation to the total lipid signal is still present, although degradation and an input from roots have significantly altered the original signal. Overall, lipid data indicate that degradation (microbial oxidation) is an important process that should be taken into account, in addition to leaching, when describing podzolization processes in soils.

Notes: The mineralization of nitrogen from soil organic matter is important when one tries to optimize nitrogen fertilization and assess risks of N losses to the environment, but its measurement is laborious and expensive. We have explored the possibilities for monitoring N mineralization directly using time domain reflectometry (TDR). Net N and S mineralization were monitored over a 101-day period in two layers (0–30 and 30–60 cm) of a loamy sand soil during aerobic incubation in a laboratory experiment. At the same time electrical conductivity of the bulk soil, σa, was measured by TDR. A series of calibration measurements with different amounts of KNO3 at different soil moisture contents was made with the topsoil to calculate the electrical conductivity, σw, of the soil solution from σa and θ. The actual σw was determined from the conductivity of 1:2 soil:water extracts (σ1:2) with a mass balance approach using measured NO3– concentrations, after correction for ions present prior to the addition of KNO3. The average N mineralization rate in the topsoil was small (0.12 mg N kg−1 day−1), and, as expected, very small in the subsoil (0.023 mg N kg−1 day−1). In the top layer NO3– concentrations calculated from σa determined by TDR slightly underestimated measured concentrations in the first 4 weeks, and in the second half of the incubation there was a significant overestimation of measured NO3–. Using the sum of both measured NO3– and SO42– reduced the overestimation. In the subsoil calculated NO3– concentrations strongly and consistently overestimated measured concentrations, although both followed the same trend. As S mineralization in the subsoil was very small, and initial SO42– concentrations were largely taken into account in the calibration relations, SO42– concentrations could not explain the overestimation. The very small NO3– and SO42– concentrations in the B layer, at the lower limit of the concentrations used in the calibrations, are a possible explanation for the discrepancies. A separate calibration for the subsoil could also be required to improve estimates of NO3– concentrations.