Inhibition of dehydration-induced fusion between liposomal membranes by carbohydrates as measured by fluorescence energy transfer

The relative abilities of a number of naturally occurring carbohydrates to inhibit dehydration-induced fusion between palmitoyloleoylphosphatidylcholine:phosphatidylserine (85:15) large unilamellar vesicles have been studied. Fusion events were quantified using a fluorescence resonance energy transfer technique. Trehalose was most effective at inhibiting fusion (0.4 g/trehalose/g lipid showed 30% probe intermixing), followed by maltose (60% intermixing), fructose (60%), sucrose (70%), glucose (80%), cellobiose, glycerol, raffinose, and myo-inositol (90%). The relative abilities of these carbohydrates to inhibit fusion correlate directly with their abilities to interact with phospholipids, maintain bilayer fluidity, and preserve biological membranes. The results are discussed in relation to the crystalline structure of the carbohydrates and their possible influence on level of interaction with phosphate head groups.     

Influence of Nutrient Concentrations on MPN Quantification and Enrichment of Nitrate-Reducing Fe(II)-Oxidizing and Fe(III)-Reducing Bacteria from Littoral Freshwater Lake Sediments

The application of culture-dependent studies to quantify Fe-metabolizing microorganisms from the environment is a necessity, as there are so far no universal functional marker genes for application in culture-independent studies. Media composition can vary between studies, therefore, we determined the effects of three different growth media on the quantification (MPNs) and identity (via cloning and sequencing of dominant DGGE bands) of nitrate-reducing Fe(II)-oxidizers and lactate- or acetate-oxidizing Fe(III)-reducers from a lacustrine sediment: low sulphate freshwater medium (FWM), sterile filtered bicarbonate-buffered lake water (BLW) and a mixture of both (MIX). We consistently found fewer cells in the BLW than in the FWM and the MIX. The DGGE banding patterns of the microbial communities enriched in different media types clustered together according to the e^? donor and acceptor couples and not according to the medium used. Thus, although the medium composition significantly influenced the quantification and thereby conclusions on the abundance and potential significance of the targeted group within the ecosystem, biodiversity assessments through enrichment cultures were less influenced by the medium, but instead were affected by the type and concentration of the e^? donor/acceptor.     

The occurrence of the freshwater isopod Heterias (Fritzianira) exul in the Lakes Region of Chile,with notes on the genus Heterias (Asellota: Janiridae)

Heterais exul, previously known from Brazil and Argentina, was collected from a number of lakes and streams in the Lakes Region of Chile (between Valdivia and Puerto Montt). It was found in a wide variety of habitats: on leaf litter, detritus, algae, rotting wood, emergent vegetation, and clean sandy bottoms. Heterias, Pseudasellus, and Fritzianira are placed in synonymy, but Fritzianira is maintained as a subgenus of Heterias because of its lack of an antenna) scale. The 3 Australian species of Heterias and H. exul are believed to have evolved from a common freshwater Gondwanaland ancestor.     

Successive histochemical differentiation steps during postnatal development of the collecting duct in rabbit kidney

Immunohistochemical experiments with monoclonal antibodies (mabs) on the kidney of neonatal rabbits revealed that the primary expression of collecting duct typic structures does not occur in a continuous and parallel, but in a subsequent developmental process. Only mabs RCT-30 A, and CD 4-V revealed immunoreactivity at the ontogenetically oldest parts of the collecting duct, the ampullae, while the other used markers (CD 1-3, CD 5-V, RCT-30 and RMCX) did not. In contrast, all of the tested antibodies showed positive reactions at the medullary and cortical collecting duct of the neonatal kidney as well as of the adult kidney. Additional incubations with wheat-germ agglutinin (WGA) a marker of terminal-differentiated collecting duct cells demonstrated weak-labelled ampulla cells beside intensively labelled ampullary neck and medullary collecting duct cells. With peanut agglutinin (PNA) labelling a 3 step transition could be illuminated: weak-labelled ampulla cells were found beside continuously bright labelled ampullary neck cells and finally a punctuate pattern downwards to the papilla. If the ampullary neck is the zone of proliferation, our findings of WGA- and PNA-co-labelling in this zone indicate, that in early developmental stages characteristic structures of different mature cells, probably principal and intercalated cells, are co-expressed within one single cell type. Thus intercalated cells might derive from principal cells.     

Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids.

Coenzyme A (CoA) transferase from Clostridium acetobutylicum ATCC 824 was purified 81-fold to homogeneity. This enzyme was stable in the presence of 0.5 M ammonium sulfate and 20% (vol/vol) glycerol, whereas activity was rapidly lost in the absence of these stabilizers. The kinetic binding mechanism was Ping Pong Bi Bi, and the Km values at pH 7.5 and 30 degrees C for acetate, propionate, and butyrate were, respectively, 1,200, 1,000, and 660 mM, while the Km value for acetoacetyl-CoA ranged from about 7 to 56 microM, depending on the acid substrate. The Km values for butyrate and acetate were high relative to the intracellular concentrations of these species; consequently, in vivo enzyme activity is expected to be sensitive to changes in those concentrations. In addition to the carboxylic acids listed above, this CoA transferase was able to convert valerate, isobutyrate, and crotonate; however, the conversion of formate, n-caproate, and isovalerate was not detected. The acetate and butyrate conversion reactions in vitro were inhibited by physiological levels of acetone and butanol, and this may be another factor in the in vivo regulation of enzyme activity. The optimum pH of acetate conversion was broad, with at least 80% of maximal activity from pH 5.9 to greater than 7.8. The purified enzyme was a heterotetramer with subunit molecular weights of about 23,000 and 25,000.     

Isolation and Characterization of Mutants of Clostridium acetobutylicum ATCC 824 Deficient in Acetoacetyl-Coenzyme A:Acetate/Butyrate:Coenzyme A-Transferase (EC 2.8.3.9) and in Other Solvent Pathway Enzymes

Mutants of Clostridium acetobutylicum ATCC 824 exhibiting resistance to 2-bromobutyrate or rifampin were isolated after nitrosoguanidine treatment. Mutants were screened for solvent production by using an automated alcohol test system. Isolates were analyzed for levels of butanol, ethanol, acetone, butyrate, acetate, and acetoin in stationary-phase batch cultures. The specific activities of NADH- and NADPH-dependent butanol dehydrogenase and butyraldehyde dehydrogenase as well as those of acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (butyrate-acetoacetate coenzyme A-transferase [EC 2.8.3.9]) (CoA-transferase), butyrate kinase, and phosphotransbutyrylase were measured at the onset of stationary phase. Rifampin-resistant strain D10 and 2-bromobutyrate mutant R were found to be deficient in only CoA-transferase, while several other mutants exhibited reduced butyraldehyde dehydrogenase and butanol dehydrogenase activities as well. The colony morphology of 2-bromobutyrate mutant R was similar to that of the parent on RCM medium; however, it had about 1/10 the level of CoA-transferase and increased levels of butanol dehydrogenase and butyraldehyde dehydrogenase. A nonsporulating, spontaneously derived degenerated strain exhibited reduced levels of butyraldehyde dehydrogenase, butanol, dehydrogenase, and CoA-transferase compared with those of the original strain. When C. acetobutylicum ATCC 824 was grown on medium containing low levels of 2-bromobutyrate, an altered colony morphology was observed. Not all strains resistant to 2-bromobutyrate (12 mM) were non-solvent-producing strains.     

Taurine and glycine conjugation and sulfation of lithocholate in primary hepatocyte cultures

Rat primary liver cells were used to study taurine and glycine conjugation and sulfation of lithocholate. After addition of [14C]lithocholate to the tissue culture medium, synthesis and excretion of amidated and/or sulfated products were investigated for up to 24 h. After incubation for 1 h, more than 83% of the labeled bile salt was amidated but not sulfated and between 5 and 11% was sulfated, with more than 80% of the sulfated bile salts being also amidated. After 24 h, the proportion of sulfated lithocholate had increased to about 23% and more than 99% of the lithocholate sulfate was additionally conjugated with glycine or taurine. Both sulfates and non-sulfates were preferably amidated with taurine. We conclude that in primary rat hepatocytes, (1) lithocholate is rapidly and almost completely conjugated with glycine or taurine (amidated), whereas sulfation of lithocholate (and its amidates) proceeds slowly and even after 24 h represents only a small proportion of the total lithocholate metabolites, and (2) sulfated and unsulfated bile salts are both preferably amidated with taurine.     

Commissural ring nerve: A female-specific neurosecretory tract supplied by bifurcating median neurons in the cockroach Periplaneta americana (L.) and the cricket Teleogryllus commodus (Walker)

Summary In the American cockroach, Periplaneta americana, and the Australian field cricket, Teleogryllus commodus, the two nerves supplying the bases of the cerci are joined by a branch that crosses behind the last abdominal ganglion. This commissural ring nerve is restricted to females, and it contains many axons filled with granular and agranular vesicles. The axons stem from somata located within the ganglion. There are one (Periplaneta) or two (Teleogryllus) groups of median neurons with bilaterally symmetrical bifurcations, and a group of postero-ventral neurons on each side. In T. commodus, these neurons are distinct from others associated with the cerci. In the two species, the ring nerve neurons contribute to a neuropile near the root of each cereal nerve. The bifurcating median neurons arborize on both sides before entering the ring nerve, while the postero-ventral ones branch more extensively ipsilateral to their somata. The possibilities are discussed that the bifurcating neurons may be homologous to dorsal unpaired median neurons, and that the ring nerve may be a neurohemal area.     

Comparative analysis of the lipopolysaccharides of a rough and a smooth strain of Pseudomonas syringae pv. phaseolicola

The lipopolysaccharides (LPS) of a rough (R) and a smooth (S) strain of Pseudomonas syringae pv. phaseolicola were analysed. The S-LPS revealed markedly more rhamnose and fucose, but less glucose, than the R-LPS. The presence of 3-O-methyl-rhamnose (acofriose) in the S-LPS was confirmed by cochromatography with authentic acofriose. SDS polyacrylamide gel electrophoresis of the S-LPS demonstrated a cluster of regularly spaced high molecular weight fractions, which was almost lacking in the R-LPS. The main fatty acids of the lipid A of both LPS species were 3-OH-10:0,3-OH-12:0,2-OH-12:0, and 12:0. Two N-linked diesters were demonstrated: 3-O(12:0)-12:0 and 3-O(2-OH-12:0)-12:0. S-LPS was subjected to mild hydrolysis and the degraded polysaccharide separated into three fractions by gel permeation chromatography on a Fractogel TSK HW-50 column. Fraction I, representing nearly only the O-specific side chain, consisted of rhamnose and fucose in a molar ratio of 4:1, with 4% of the rhamnose being 3-O-methylated (acofriose). Fraction II, representing mostly core material, was composed of glucose, rhamnose, heptose, glucosamine, galactosamine, alanine, and a still unidentified amino compound, in an approximate molar ratio of 3:1:1:1:1:1:1, and KDO. Fraction III consisted of released monomers and salts. The LPS was highly phosphorylated (3.28% phosphorus in the core fraction). The thus characterized composition of the LPS O-chain seems to be unique for the pathovar phaseolicola of P. syringae, although many similarities exist to other pathovars as well as to other bacterial species.Abbreviations LPS lipopolysacchairdes - GC/MS combined gas liquid chromatography-mass spectrometry - HVE high voltage electrophoresis - KDO 2-keto-3-deoxyoctonic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate P.s. pv. phaseolicola is termed P. phaseolicola in the text