Alpha- and beta-Proteobacteria control the consumption and release of amino acids on lake snow aggregates

We analyzed the composition of aggregate (lake snow)-associated bacterial communities in Lake Constance from 1994 until 1996 between a depth of 25 m and the sediment surface at 110 m by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes of various specificity. In addition, we experimentally examined the turnover of dissolved amino acids and carbohydrates together with the microbial colonization of aggregates formed in rolling tanks in the lab. Generally, between 40 and more than 80% of the microbes enumerated by DAPI staining (4',6'-diamidino-2-phenylindole) were detected as Bacteria by the probe EUB338. At a depth of 25 m, 10.5% +/- 7.9% and 14.2% +/- 10.2% of the DAPI cell counts were detected by probes specific for alpha- and beta-Proteobacteria. These proportions increased to 12.0% +/- 3.3% and 54.0% +/- 5.9% at a depth of 50 m but decreased again at the sediment surface at 110 m to 2.7% +/- 1.4% and 41.1% +/- 8.4%, indicating a clear dominance of beta-Proteobacteria at depths of 50 and 110 m, where aggregates have an age of 3 to 5 and 8 to 11 days, respectively. From 50 m to the sediment surface, cells detected by a Cytophaga/Flavobacteria-specific probe (CF319a) comprised increasing proportions up to 18% of the DAPI cell counts. gamma-Proteobacteria always comprised minor proportions of the aggregate-associated bacterial community. Using only two probes highly specific for clusters of bacteria closely related to Sphingomonas species and Brevundimonas diminuta, we identified between 16 and 60% of the alpha-Proteobacteria. In addition, with three probes highly specific for close relatives of the beta-Proteobacteria Duganella zoogloeoides (formerly Zoogloea ramigera), Acidovorax facilis, and Hydrogenophaga palleroni, bacteria common in activated sludge, 42 to 70% of the beta-Proteobacteria were identified. In the early phase (<20 h) of 11 of the 15 experimental incubations of aggregates, dissolved amino acids were consumed by the aggregate-associated bacteria from the surrounding water. This stage was followed by a period of 1 to 3 days during which dissolved amino acids were released into the surrounding water, paralleled by an increasing dominance of beta-Proteobacteria. Hence, our results show that lake snow aggregates are inhabited by a community dominated by a limited number of alpha- and beta-Proteobacteria, which undergo a distinct succession. They successively decompose the amino acids bound in the aggregates and release substantial amounts into the surrounding water during aging and sinking.     

Conformational intermediates and fusion activity of influenza virus hemagglutinin

Three strains of influenza virus (H1, H2, and H3) exhibited similar characteristics in the ability of their hemagglutinin (HA) to induce membrane fusion, but the HAs differed in their susceptibility to inactivation. The extent of inactivation depended on the pH of preincubation and was lowest for A/Japan (H2 subtype), in agreement with previous studies (A. Puri, F. Booy, R. W. Doms, J. M. White, and R. Blumenthal, J. Virol. 64:3824-3832, 1990). While significant inactivation of X31 (H3 subtype) was observed at 37 degrees C at pH values corresponding to the maximum of fusion (about pH 5.0), no inactivation was seen at preincubation pH values 0.2 to 0.4 pH units higher. Surprisingly, low-pH preincubation under those conditions enhanced the fusion rates and extents of A/Japan as well as those of X31. For A/PR 8/34 (H1 subtype), neither a shift of the pH (to >5.0) nor a decrease of the temperature to 20 degrees C was sufficient to prevent inactivation. We provide evidence that the activated HA is a conformational intermediate distinct from the native structure and from the final structure associated with the conformational change of HA, which is implicated by the high-resolution structure of the soluble trimeric fragment TBHA2 (P. A. Bullough, F. M. Hughson, J. J. Skehel, and D. C. Wiley, Nature 371:37-43, 1994).     

A comparison of the dose-response behavior of canine airways and parenchyma

We compared the histamine responsiveness of canine airways and parenchymal tissues in six anesthetized paralyzed open-chest mongrel dogs, partitioning total lung resistance (RL) into airway resistance (Raw) and tissue viscance (Vti). Pressure was measured during tidal breathing (frequency was 0.3 Hz) at the trachea and in three alveolar regions by use of alveolar capsules. Measurements were taken before and after the delivery of increasing concentrations of aerosolized histamine (0.1-30 mg/ml). We found that Vti accounted for 78 +/- 8% of RL under base-line conditions; this proportion remained relatively constant throughout the histamine concentration-response curve. There was a significant correlation between percent change in Vti and percent change in Raw at all levels of histamine-induced constriction (P less than 0.001). Moreover, the sensitivity of the tissues and airways (defined as the concentration of histamine required to double resistance) was remarkably similar. We conclude that, at this frequency of ventilation, Vti accounts for the major portion of RL both under base-line conditions and after histamine-induced constriction. Although increases in RL cannot be attributed solely to events occurring in the airways, the close correlation between changes in Raw and Vti and the similar sensitivities of the two support the use of indexes reflecting changes in airway caliber as an indicator of overall lung histamine responsiveness.     

Photosynthesis in aquatic adventitious roots of the halophytic stem-succulent Tecticornia pergranulata (formerly Halosarcia pergranulata)

In flood-tolerant species, a common response to inundation is growth of adventitious roots into the water column. The capacity for these roots to become photosynthetically active has received scant attention. The experiments presented here show the aquatic adventitious roots of the flood-tolerant, halophytic stem-succulent, Tecticornia pergranulata (subfamily Salicornioideae, Chenopodiaceae) are photosynthetic and quantify for the first time the photosynthetic capacity of aquatic roots for a terrestrial species. Fluorescence microscopy was used to determine the presence of chloroplasts within cells of aquatic roots. Net O₂ production by excised aquatic roots, when underwater, was measured with varying light and CO₂ regimes; the apparent maximum capacity ( P _max) for underwater net photosynthesis in aquatic roots was 0.45  µ mol O₂ m⁻² s⁻¹. The photosynthetic potential of these roots was supported by the immunolocalization of PsbA, the major protein of photosystem II, and ribulose-1-5-bisphosphate carboxylase/oxygenase (Rubisco) in root protein extracts. Chlorophyllous aquatic roots of T. pergranulata are photosynthetically active, and such activity is a previously unrecognized source of O₂, and potentially carbohydrates, in flooded and submerged plants.     

Structural diversity and organization of three gene families for Kunitz-type enzyme inhibitors from potato tubers ( Solanum tuberosum L.)

In the potato, Kunitz-type enzyme inhibitors are abundant and highly polymorphic small proteins found in tubers. DNA sequence analysis of 1596 unselected ESTs (expressed sequence tags) from mature tubers of the cultivars Provita and Saturna resulted in the identification of 55 different DNA sequences with high sequence similarity to Kunitz-type enzyme inhibitors. The frequency of Kunitz-type inhibitor ESTs in Provita was four times higher than in Saturna tubers, and none of the Provita ESTs was identical to any of the Saturna ESTs. A phenogram constructed from the deduced amino acid sequences of the inhibitors revealed three major homology groups-A, B and C. Group A inhibitors were all derived from Provita ESTs. Inhibitor groups A and B were more similar to each other than to group C inhibitors, and for most members within-group similarity was at least 90%. Non-conservative amino acid substitutions and insertion/deletion polymorphisms suggest functional differentiation between members of the gene family. A minimum of 21 genes for Kunitz-type enzyme inhibitors (six for group A, nine for group B and six for group C) was estimated to exist in the potato genome. Genetic mapping and the identification of BAC (bacterial artificial chromosome) clones containing more than one member of the gene family indicated that most inhibitor genes of groups A, B and C are organized in a cluster that maps to a single region on potato chromosome III.     

Hepatic overexpression of sterol carrier protein-2 inhibits VLDL production and reciprocally enhances biliary lipid secretion

We examined in vivo a role for sterol carrier protein-2 (SCP-2) in the regulation of lipid secretion across the hepatic sinusoidal and canalicular membranes. Recombinant adenovirus Ad.rSCP2 was used to overexpress SCP-2 in livers of mice. We determined plasma, hepatic, and biliary lipid concentrations; hepatic fatty acid (FA) and cholesterol synthesis; hepatic and biliary phosphatidylcholine (PC) molecular species; and VLDL triglyceride production. In Ad.rSCP2 mice, there was marked inhibition of hepatic fatty acids and cholesterol synthesis to <62% of control mice. Hepatic triglyceride contents were decreased, while cholesterol and phospholipids concentrations were elevated in Ad.rSCP2 mice. Hepatic VLDL triglyceride production fell in Ad.rSCP2 mice to 39% of control values. As expected, biliary cholesterol, phospholipids, bile acids outputs, and biliary PC hydrophobic index were significantly increased in Ad.rSCP2 mice. These studies indicate that SCP-2 overexpression in the liver markedly inhibits lipid synthesis as well as VLDL production, and alters hepatic lipid contents. In contrast, SCP-2 increased biliary lipid secretion and the proportion of hydrophobic PC molecular species in bile. These effects suggest a key regulatory role for SCP-2 in hepatic lipid metabolism and the existence of a reciprocal relationship between the fluxes of lipids across the sinusoidal and canalicular membranes.     

Growth, photosynthesis and ozone uptake of young beech (Fagus sylvatica L.) in response to different ozone exposures

Beech seedlings (Fagus sylvatica L.) were exposed to episodes of O₃ in environmentally controlled growth chambers during one growing season. Three treatments were applied: charcoal-filtered air, charcoal-filtered air with the addition of 40 ppb O₃ for seven episodes of 5 days' duration (9000-1700 hours), and charcoal-filtered air with the addition of 100 ppb O₃ for seven episodes of 5 days' duration (9000-1700 hours). The accumulated exposure over a threshold of 40 ppb in the last treatment reached 13,911 ppb h. Throughout the growing season we measured growth as well as photosynthetic properties and related effects to external and calculated internal doses of O₃, using stomatal conductance (g_s) data. Growth, measured as diameter increment and biomass, was not significantly affected by the O₃ treatments. In the 100-ppb treatment, light-saturated CO₂ assimilation rates and chlorophyll content were significantly reduced, and the chlorophyll fluorescence parameter F_v/F_m was significantly reduced at times of high uptake rates and coincided with strong reductions of assimilation rates. O₃ uptake was lowered in the 100-ppb treatment due to reduced g_s. There was serious visible damage by the end of the exposure period in the 100-ppb treatment, while the treatment with 40 ppb O₃ did not seem to cause any significant changes.     

Relation of phosphatidylinositol metabolism to glycolytic pathway in skeletal muscle membranes

Skeletal muscle triads are possessing the whole set of enzymes of the phosphatidylinositol (PI)-linked signal generating pathway, PI-kinase, PI(4)P-kinase, and PI(4,5)P₂-phospholipase C (PLC). The activities of these enzymes are comparable to those found in other cell types for which a functional role of the PI-pathway in intracellular signal transduction has been established. For skeletal muscle an unequivocal function and an initiating signal for Ins(1,4,5)P₃-liberation is still unknown. However, the observed Ca-dependency of PLC activity suggests that here Ins(1,4,5)P₃ production is a consequence rather than a cause of increasing cytosolic Ca²⁺. Recently, the glycolytic enzyme aldolase, whose activity can be modulated by inositol polyphosphates, has been localized in the triadic structure. The enzyme which has a high affinity to Ins(1,4)P₂, Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄, seems to be compartmentalized to the junctional foot structure from which it is released upon binding of these molecules. This phenomenon could reflect a capability for regulation of the glycolytic flux even for aldolase, especially if a non steady-state situation in the junctional gap is considered. Meanwhile we have accumulated evidence for the operation of a partial glycolytic sequence in the junctional region established by the enzymes aldolase, glyceraldehyde-3-P (GAP) dehydrogenase and phosphoglycerate kinase. This system is able to produce ATP upon oxidation of GAP and could be, because of the inositol polyphosphate-sensing abilities of aldolase, a target for the membrane associated PI-pathway. The ATP production is however transient which indicates the coupling to an ATP hydrolyzing reaction. Thus, it appears that the ATP produced by the membrane associated system is effectively utilized by an ATP consuming membrane localized system like PI-metabolism or protein kinases. There are indications that exogeneously added ATP does not equilibrate with the ATP synthesized in the junctional region which suggests an effective structural or kinetical compartmentalization of this system. Therefore it is hypothesized that the ATP synthesized by the membrane associated glycolytic sequence is utilized in membrane localized reactions.     

Plant genome archaeology: evidence for conserved ancestral chromosome segments in dicotyledonous plant species

We have developed genetic maps, based on expressed sequence tags (ESTs) that are homologous to Arabidopsis genes, in four dicotyledonous crop plant species from different families. A comparison of these maps with the physical map of Arabidopsis reveals common genome segments that appear to have been conserved throughout the evolution of the dicots. In the four crop species analysed these segments comprise between 16 and 33% of the Arabidopsis genome. Our findings extend the synteny patterns previously observed only within plant families, and indicate that structural and functional information from the model species will be, at least in part, applicable in crop plants with large genomes.