Structure of the core oligosaccharide of a rough-type lipopolysaccharide of Pseudomonas syringae pv. phaseolicola.

The core structure of the lipopolysaccharide (LPS) isolated from a rough strain of the phytopathogenic bacterium Pseudomonas syringae pv. phaseolicola, GSPB 711, was investigated by sugar and methylation analyses, Fourier transform ion-cyclotron resonance ESI MS, and one- and two-dimensional 1H-, 13C- and 31P-NMR spectroscopy. Strong alkaline deacylation of the LPS resulted in two core-lipid A backbone undecasaccharide pentakisphosphates in the ratio approximately 2.5 : 1, which corresponded to outer core glycoforms 1 and 2 terminated with either L-rhamnose or 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), respectively. Mild acid degradation of the LPS gave the major glycoform 1 core octasaccharide and a minor truncated glycoform 2 core heptasaccharide, which resulted from the cleavage of the terminal Kdo residues. The inner core of P. syringae is distinguished by a high degree of phosphorylation of L-glycero-D-manno-heptose residues with phosphate, diphosphate and ethanolamine diphosphate groups. The glycoform 1 core is structurally similar but not identical to one of the core glycoforms of the human pathogenic bacterium Pseudomonas aeruginosa. The outer core composition and structure may be useful as a chemotaxonomic marker for the P. syringae group of bacteria, whereas a more conserved inner core structure appears to be representative for the whole genus Pseudomonas.     

Structural studies of the capsular polysaccharide and lipopolysaccharide O-antigen of Aeromonas salmonicida strain 80204-1 produced under in vitro and in vivo growth conditions.

Aeromonas salmonicida is a pathogenic aquatic bacterium and the causal agent of furunculosis in salmon. In the course of this study, it was found that when grown in vitro on tryptic soy agar, A. salmonicida strain 80204-1 produced a capsular polysaccharide with the identical structure to that of the lipopolysaccharide O-chain polysaccharide. A combination of 1D and 2D NMR methods, including a series of 1D analogues of 3D experiments, together with capillary electrophoresis-electrospray MS (CE-ES-MS), compositional and methylation analyses and specific modifications was used to determine the structure of these polysaccharides. Both polymers were shown to be composed of linear trisaccharide repeating units consisting of 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA), 3-[(N-acetyl-L-alanyl)amido]-3,6-dideoxy-D-glucose[3-[(N-acetyl-L-alanyl)amido]-3-deoxy-D-quinovose, Qui3NAlaNAc] and 2-acetamido-2,6-dideoxy-D-glucose (2-acetamido-2-deoxy-D-quinovose, QuiNAc) and having the following structure: [-->3)-alpha-D-GalpNAcA-(1-->3)-beta-D-QuipNAc-(1-->4)-beta-D-Quip3NAlaNAc-(1-]n, where GalNAcA is partly presented as an amide and AlaNAc represents N-acetyl-L-alanyl group. CE-ES-MS analysis of CPS and O-chain polysaccharide confirmed that 40% of GalNAcA was present in the amide form. Direct CE-ES-MS/MS analysis of in vivo cultured cells confirmed the formation of a novel polysaccharide, a structure also formed in vitro, which was previously undetectable in bacterial cells grown within implants in fish, and in which GalNAcA was fully amidated.     

Mesozooplankton interactions with the shelf around the sub-Antarctic Prince Edward Islands archipelago

Mesozooplankton surveys were conducted in April/May for fourconsecutive years (1996–1999) in the vicinity of the PrinceEdward Islands (PEIs), Southern Ocean. The PEIs are locatedin the Polar Frontal Zone, directly in the path of the east-flowingAntarctic Circumpolar Current. Zooplankton were collected byoblique tows using a Bongo net fitted with 300 µm mesh.The abundance, biomass and average size of the mesozooplanktonin the upstream (USR), inter-island (IIR) and downstream (DSR)regions indicated that some groups and species were significantlyaffected by their interaction with the shallow shelf watersof the PEIs. Total mesozooplankton abundance and biomass weretypically highest in the DSR, but no consistent pattern wasevident in the USR and IIR. Copepods, euphausiids and fish weregenerally of a low average size in the IIR. This small sizewas largely attributed to the reduced abundance, or completeabsence, of mesopelagic species from the shelf region. Of totalbiomass, the mesopelagic species Euphausia longirostris, Euphausiasimilis, Pleuromamma abdominalis, Paraeuchaeta biloba and Oncaeaantarctica together contributed an average of 16% to the USR,2% to the IIR and 15% to the DSR. Conversely, epipelagic speciesshowed no consistent pattern of abundance and biomass distributionbetween regions. The low incidence of mesopelagic species overthe island shelf was attributed mainly to reduced advectionof deep water into the shelf region (average depth = 200 m),rather than predation, particularly during the through-flowmode between the islands. This resulted in substantial regionaldifferences in euphausiid community structure. The epipelagicspecies Euphausia vallentini and Thysanoessa vicina completelydominated the IIR, comprising on average 89% of total euphausiidbiomass in this region. However, predation may be importantduring the water-trapping mode between the islands. Advectionof zooplankton into the IIR appeared to be affected by the proximityof the Subantarctic Front (SAF). In 1996, when the SAF was farnorth of the PEIs, reduced current velocities resulted in somedegree of water retention over the shelf and an increased predationimpact. Conversely, when the SAF was close to the PEIs in 1999,more large plankton were transported over the island shelf.High current velocities and productivity associated with theSAF appear to increase the biomass and size of allochthonouszooplankton/nekton advected into the IIR, and consequently mayhave increased the availability of prey to land-based predators.The long-term southward movement of the SAF recently observedin the vicinity of the PEIs may therefore have important implicationsfor the ecosystem of these islands.     

Osmotic water permeability of cell membranes from Mesembryanthemum crystallinum leaves: effects of age and salinity

The osmotic water permeability ( P _os) of cell membranes isolated from leaves of 40-, 50- and 60-day-old Mesembryanthemum crystallinum plants was estimated by measuring light-scattering kinetics using stopped-flow spectrophotometry. The measurements were performed on the plasma membrane (PM), purified tonoplast (TP), and TP-enriched vesicles. The PM and TP-enriched vesicles were obtained by partitioning the microsomal fraction in an aqueous polymer two-phase system, whereas the purified TP vesicles were prepared by microsomal vesicle flotation on a sucrose cushion. The P _os of isolated membranes declined with plant age. The kinetic experiments showed that there was no difference between the P _os of the PM and TP isolated from plants of all ages. A 24-h exposure of plants to 400 m M NaCl caused a decline in the P _os as well. These findings suggest that, during M. crystallinum transition to CAM, which was induced by plant ageing or salinity, plant osmoregulatory responses included changes in the P _os of the leaf-cell membranes. These variations in the P _os are discussed in the context of adaptive mechanisms responsible for the maintenance of the water balance in the common ice plant.     

Regions of homology in small colicinogenic plasmids

Restriction maps have been constructed for the colicinogenic plasmids (ColA, ColD, and ColK. Their regions of homology with the ColE1 plasmid and its deletion derivative pAO3 carrying the region responsible for autonomous replication of ColE1 plasmid were determined by means of blotting hybridization and heteroduplex analysis. The plasmids ColA, ColD, and ColK were shown to contain DNA fragments homologous to the region of ColE1 involved in the regulation of replication.     

Hydrazides of N-blocked amino acids as sole substrates with unique difunctional behavior under papain catalysis

Hydrazides of five N-acylamino acids have been used alone as substrates for papain catalysis to yield N¹,N²-diacylhydrazines. With the exception of N-(benzyloxycarbonyl)(Z)- -alanine hydrazide, they were very effective as both acylating agents of the enzyme and nucleophiles in attacking the enzyme-substrate intermediate. Although Z- -alanine hydrazide was a minimal acylating agent, it was a satisfactory nucleophile. The most favorable reaction involved Z- -alanine hydrazide in producing N¹,N²-bis(Z- -alanyl)hydrazine. When Z- -alanine hydrazide was the substrate, this same chiral diacylhydrazine was formed along with meso N¹-(Z- -alanyl)-N²-(Z- -alanyl)hydrazine. For the acylation step, the enzyme displayed powerful, essentially stereospecific, bias toward the enantiomer. Once the thioester intermediate was formed, little preference was detected for attack by the enantiomers as nucleophiles. The most direct procedure for synthesis of substrates was conversion of Z-amino acids to their esters by means of dry HCl in an absolute alcohol. Treatment with hydrazine produced the hydrazides in excellent yield.     

Synthesis of the tetrasaccharide repeating-unit of the O-specific polysaccharide from Salmonella senftenberg

The oligosaccharide β-d-Man-(1 → 4)-α-l-Rha (1 → 3)-d-Gal-(6 ← 1)-α-d-Glc, which is the repeating unit of the O-specific polysaccharide chain of the lipopolysaccharide from Salmonella senftenberg, was obtained by glycosylation of benzyl 2,4-di-O-benzyl-6-O-(2,3,4-tri-O-benzyl-6-O-p-nitrobenzoyl-α-d-glucopyranosyl)-β-d-galactopyranoside or benzyl 2-O-acetyl-6-O-(2,3,4-tri-O-benzyl-6-O-p-nitrobenzoyl-α-d-glucopyranosyl)-β-d-galactopyranoside with 3-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-d-mannopyranosyl)-β-l-rhamnopyranose 1,2-(methyl orthoacetate) followed by removal of protecting groups.     

Stereochemical aspects of forcing special substrate hydrazides to behave either as electrophiles or as nucleophiles during catalysis by crude papain

Restriction of hydrazides of N-blocked amino acids mainly to electrophilic action, in acylating crude papain, has been achieved by means of a large amount of aniline, with formation of insoluble anilides of N-acylamino acids. Similarly, nucleophilic behavior, on the part of a hydrazide, has been promoted by introducing a large proportion of an N-acylamino acid to produce an insoluble N¹,N²-diacylhydrazine. Achiral, chiral and racemic hydrazides and their corresponding N-acylamino acids were utilized in the study. Among the more informative combinations of reactants were Z-dl-alanine hydrazide with aniline and then with Z-glycine. A stereospecific response in the former situation produced Z-l-alanine anilide. In the latter case, a stereoselective interaction produced Z-Gly-NHNH-lAla-Z more rapidly than Z-Gly-NHNH-d-Ala-Z. The final incubation period yielded an optically pure D product. Differences in stereochemical control have been delineated in terms of different spatial aspects for interactions at the S and S′ subsites of sulfhydryl proteolytic enzymes. A racemic reactant encountered firm stereospecificity as an electrophile at the S subsite but only modest stereoselectivity as a nucleophile at the S′ subsite. The ready availability of crude papain allows an effective procedure for the synthesis of substantial quantities of diacylhydrazines.     

Isolation of calcium pump system and purification of calcium ion-dependent ATPase from heart muscle.

The procedure for the isolation of the highly active fraction of sarcoplasmic reticulum from pigeon and dog hearts is described. The method is based on the partial loading of heart microsomes with calcium and oxalate ions and the precipitation of loaded vesicles in sucrose and potassium chloride concentration gradients. Preparations obtained possess high activity of Ca2+-dependent ATPase and are also able to accumulate up to 10 mumol Ca2+ per mg protein. Purification of sarcoplasmic reticulum membranes is accompanied by a decrease in concentration of cytochrome a+a3 and an increase in the content of [32P]phosphoenzyme. The basic components in "calcium-oxalate preparation" from hearts are proteins with molecular weights of about 100000 (Ca2+-dependent ATPase) and 55000 Calcium-oxalate preparation from pigeon hearts was used for subsequent purification of Ca2+-dependent ATPase. Specific activity of purified enzyme from pigeon hearts is 12-16 mumol Pi/min per mg protein. Enzyme activity of purified Ca2+-dependent ATPase is inhibited by EGTA and is not sensitive to azide, 2,4-dinitrophenol and ouabain. The data obtained demonstrate the similarity of calcium pump systems and Ca2+-dependent ATPases isolated from heart and skeletal muscles.