Strain specificity of outer membrane proteins in Escherichia coli.

Outer membrane proteins of various strains of Escherichia coli were compared using three different systems of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The outer membranes of E. coli K-12, E. coli B, and E. coli J-5 had distinctive protein compositions. As regards proteins which interact with peptidoglycan, E. coli K-12 contained O-8 and O-9, while E. coli B possessed one protein which migrated to the position of O-9. Although E. coli J-5 possessed two such proteins, O-8' and O-9', their positions on polyacrylamide gel were different from those of O-8 and O-9. Protein O-7, which migrates slightly more slowly than O-8, was found specifically in E. coli K-12. Proteins O-10 and O-11 were found in all strains tested, although the relative amounts were different depending on the strain. Strains of E. coli K-12 and E. coli J-5 gave three major bands, O-2a, O-2b, and O-3, in the region of high molecular weight. These proteins were repressed by iron in the cultivation media. Strains of E. coli B, on the other hand, gave only O-2b and O-3. E. coli J-5 gave two other major bands in this region, but the amounts were not controlled by iron in the cultivation media.     

Studies on Sulfur-containing Carbonates and Their Pesticidal Activities

Several S-substituted-benzyl O-substituted-benzyl dithiocarbonates were synthesized and their acaricidal activities with Tetranychus urticae Koch were examined. Some dithiocarbonates, such as S-benzyl O-4-chlorobenzyl dithiocarbonate, S-benzyl O-4-bromobenzyl dithiocarbonate, S-benzyl O-3-methoxylbenzyl dithiocarbonate, S-3-methylbenzyl O-4-chlorobenzyl dithiocarbonate and S-3-methoxybenzyl O-4-chlorobenzyl dithiocarbonate, showed high ovicidal activities.

Various S-benzyl O-substituted-phenyl dithiocarbonates together with some related compounds were prepared and their biological activities were evaluated. Among them, S-benzyl O-phenyl-, S-benzyl O-2-acetylphenyl- and S-benzyl O-4-methylthiophenyl-dithiocarbonates showed high ovicidal activity to the two spotted spider mite, Tetranychus urticae Koch. S-Benzyl O-2-alkylphenyl-, S-benzyl O-2-allylphenyl- and S-benzyl O-2-dimethyl-aminomethylphenyl-dithiocarbonates showed herbicidal activities to barnyardgrass and radish. S-Benzyl O-4-alkylphenyl dithiocarbonate was herbicidal only to radish. Some structure-activity relationships are discussed.

Several thionocarbonates were synthesized and their antifugal activities were examined. O-Benzyl O-4-nitrophenyl thionocarbonate, O-benzyl O-3-nitrophenyl thionocarbonate and O-benzyl O-4-acetylphenyl thionocarbonate showed excellent activities to Pellicularia sasakii. O-Benzyl O-2- and 4-acetylphenyl thionocarbonate showed the highest activities to Pyricularia oryzae.     

Influence of molecular size and osmolarity of sugars and dextrans on the synthesis of outer membrane proteins O-8 and O-9 of Escherichia coli K-12.

Supplementation of the growth medium with high concentrations of sugars or low-molecular-weight dextrans results in a drastic change in the ratio of outer membrane proteins O-8 and O-9, due to induction of O-8 synthesis and suppression of O-9 synthesis. Sugars and dextrans of molecular weights greater than 600 to 700 switched the synthesis of O-9 to that of O-8 more effectively than those of lower molecular weight, although the effect was almost the same within each of the two groups irrespective of the differences in molecular weight within the group. Proteins O-8 or O-9, or both, are responsible for the formation of pores that allow the passive diffusion of hydrophilic molecules whose molecular weights are smaller than about 600 (T. Nakae, Biochem. Biophys. Res. Commun. 71:877-884, 1976). The results indicate that substances that cannot pass through the outer membrane switch the synthesis of O-9 to that of O-8 more effectively than those that can penetrate this membrane with the aid of O-8, O-9, or both. It is suggested that the osmotic pressure exerted on the outer membrane plays an important role in the regulation of synthesis of the two proteins.     

Effects of fatty acids on superoxide radical generation in leukocytes.

Acetylated ferricytochrome c was employed for the detection of superoxide radicals (O-2) generated both in intact cells and in subcellular fractions of leukocytes. Certain saturated fatty acids, myristate in particular, induced the production of O-2 in leukocytes, suggesting a correlation between the formation of O-2 and the hydrophobic interaction of fatty acids with the leukocyte plasma membrane. As compared with O-2 radical generation from phagocytizing leukocytes a greater stimulation of O-2 formation was observed in cells in which myristate was added. The enhanced activity which generated O-2 in the cell-free system was located in a particulate fraction but not in the cytosol. The rate of O-2 generation in the particulate fraction was higher in the presence of NADPH than in the presence of NADH. The effects of reagents such as KCN, 2,4-dichlorophenol and aminotriazole on the O-2 generation in this fraction are examined and the nature of the O-2 generating system is discussed.     

Lipopolysaccharide and mouse virulence of Salmonella: O antigen is important after intraperitoneal but not intravenous challenge

Abstract The quality of the O-antigenic polysaccharide part of the cell wall lipopolysaccharide (LPS) is a virulence determinant in Salmonella strains: isogenic derivatives with antigen O-4,12 have been shown to be more virulent than those with O-6,7 when given intraperitoneally (i.p.) to mice. The O-6,7 LPS activates complement by the alternative pathway more efficiently than does O-4,12. We show here that the O-6,7 (but not O-4,12) bacteria were rapidly killed in the peritoneal cavity of the mice, resulting in approx. 100-fold reduced numbers of bacteria reaching the liver; the subsequent rate of growth of the bacteria was not affected. After intravenous challenge, both O-6,7 and O-4,12 sister strains survived equally well in the liver and spleen and were of approximately equal virulence. We suggest that the rapid activation of complement by the O-6,7 LPS leads to the killing of these bacteria by the peritoneal cells and thereby to reduced virulence.     

Pathogenicity for several laboratory animals of toxoplasma oocysts originated from naturally infected cats.

The pathogenicity of four strains, O-1, O-2, O-3, and O-4, of Toxoplasma isolated in the form of oocyst from the feces of naturally infected cats was examined for such laboratory animals as mice, rats, rabbits, guinea pigs, and dogs, in comparison with that of the Beverley strain. Suspensions of seven graded doses of oocysts of each strain ranging from 1.0 X 10(-1) to 1.0 X 10(5) were inoculated orally into seven groups of five mice each. The O-1, O-2, and O-3 strains were as pathogenic for mice as the Beverley strain, but the O-4 strain was not so pathogenic as any other strain. Rats, rabbits, guinea pigs, and dogs were inoculated orally with around 1.0 X 10(5) oocysts. The four O strains were not so severly pathogenic for rats and rabbits as to cause death. The O-2 and O-3 strains showed strong pathogenicity for guinea pigs, almost all of which, when inoculated with them, died after manifesting severe clinical symptoms. The pathogenicity of the O-1 and O-2 strains showed essentially the same tendency for dogs as for any other animal. In inoculation with oocysts, as well as with proliferative forms or cysts, the same pathogenicity was not observed in different strains, even if the same species of host animals was used. On the contrary, the same pathogenicity was not always found even in one strain when a different species of animals was used.     

植物病原细菌中超氧阴离子释放及其释放位点的研究

实验发现很多植物病原细菌具有自身释放超氧阴离子的现象 ,其释放规律与菌株的致病性可能存在一定的关系。并且植物病原细菌超氧阴离子的释放是多位点的 ,在细胞膜、细胞壁及无菌滤液中用化学方法及电子自旋共振法 (Electronspinresonance ,ESR)都能够检测到超氧阴离子的释放。无论是自然生理状态下 ,还是SOD酶活性被抑制后 ,都显示各组分中无菌滤液的超氧阴离子浓度最高 ,可能是植物病原细菌超氧阴离子释放的主要位点。     

In vitro analysis of the origin and maintenance of O-2Aadult progenitor cells

We have been studying the differing characteristics of oligodendrocyte- type-2 astrocyte (O-2A) progenitors isolated from optic nerves of perinatal and adult rats. These two cell types display striking differences in their in vitro phenotypes. In addition, the O- 2Aperinatal progenitor population appears to have a limited life-span in vivo, while O-2Aadult progenitors appear to be maintained throughout life. O-2Aperinatal progenitors seem to have largely disappeared from the optic nerve by 1 mo after birth, and are not detectable in cultures derived from optic nerves of adult rats. In contrast, O-2Aadult progenitors can first be isolated from optic nerves of 7-d-old rats and are still present in optic nerves of 1-yr-old rats. These observations raise two questions: (a) From what source do O-2Aadult progenitors originate; and (b) how is the O-2Aadult progenitor population maintained in the nerve throughout life? We now provide in vitro evidence indicating that O-2Aadult progenitors are derived directly from a subpopulation of O-2Aperinatal progenitors. We also provide evidence indicating that O-2Aadult progenitors are capable of prolonged self renewal in vitro. In addition, our data suggests that the in vitro generation of oligodendrocytes from O-2Aadult progenitors occurs primarily through asymmetric division and differentiation, in contrast with the self-extinguishing pattern of symmetric division and differentiation displayed by O-2Aperinatal progenitors in vitro. We suggest that O-2Aadult progenitors express at least some properties of stem cells and thus may be able to support the generation of both differentiated progeny cells as well as their own continued replenishment throughout adult life.     

Differential O-3/O-4 regioselectivity in the glycosylation of alpha and beta anomers of 6-O-substituted N-dimethylmaleoyl-protected D-glucosamine acceptors

An assessment of the relative O-3/O-4 reactivities of both methyl alpha- and beta-d-glycosides of N-dimethylmaleoyl (DMM) d-glucosamine acceptors protected at O-6 with benzoyl (Bz), benzyl (Bn), and tert-butyldiphenylsilyl (TBDPS) groups is presented using per-O-benzoylated beta-d-galactofuranosyl and per-O-acetylated alpha-d-galactopyranosyl trichloroacetimidates as glycosyl donors. Using the former donor, the alpha anomer of the 6-O-benzoylated compound gave exclusive substitution at O-3, whereas the other two compounds with alpha-configuration kept this site as preferential. The beta anomer of the 6-O-benzoylated compound gave the same amounts of reaction products on O-3 and O-4, whereas the other beta analogs carried a more reactive O-4. The same reactions were carried out using as donor the less-reactive per-O-acetylated alpha-d-galactopyranosyl trichloroacetimidate. Although the same trend was found to occur, the O-4 was always relatively more reactive with the pyranosyl donor than with the furanosyl donor, when keeping the remaining factors constant. Furthermore, the beta anomers of the acceptor gave almost exclusive substitution at O-4. These observations confirm and extend the utility of these 'matching' donor and acceptor reactivities.     

Arrangement of proteins O-8 and O-9 in outer membrane of Escherichia coli K-12. Existence of homotrimers and heterotrimers.

1. The molecular arrangement of major outer membrane proteins O-8 and O-9 that exist as trimers has been studied by means of cross-linking with dimethylsuberimidate. 2. The cross-linked samples were examined on a urea/sodium dodecyl sulfate/polyacrylamide gel which was developed to separate cross-linked trimer and dimer of O-8 from those of O-9. 3. Cells simultaneously synthesizing both O-8 and O-9 formed heterotrimers (trimers containing both proteins) as well as homotrimers. 4. Quantitative analyses revealed that there was no discrimination between O-8 and O-9 in the assembly process to form trimers. 5. When cells were grown sequentially under two different sets of conditions so that the cells synthesized either one of the two proteins in the first stage and the other in the second stage of growth, no heterotrimers were formed. This result indicates that subunit exchange did not take place between trimers which had been incorporated into the outer membrane.     

O-acetylated oligosaccharides from pectins of potato tuber cell walls.

Acetylated trigalacturonides and rhamnogalacturonan I (RG-I)-derived oligosaccharides were isolated from a Driselase digest of potato tuber cell walls by ion-exchange and size-exclusion chromatography. The oligosaccharides were structurally characterized by fast atom bombardment-mass spectroscopy, nuclear magnetic resonance spectroscopy, and glycosyl-linkage composition analysis. One trigalacturonide contained a single acetyl group at O-3 of the reducing galacturonic acid residue. A second trigalacturonide contained two acetyl substituents, which were located on O-3 or O-4 of the nonreducing galacturonic acid residue and O-3 of the reducing galacturonic acid residue. RG-I backbone-derived oligomers had acetyl groups at O-2 of the galacturonic acid residues. Some of these galacturonic acid residues were O-acetylated at both O-2 and O-3 positions. Rhamnosyl residues of RG-I oligomers were not acetylated.     

Irradiation in vitro discriminates between different O-2A progenitor cell subpopulations in the perinatal central nervous system of rats

The effects of X irradiation on oligodendrocyte-type-2-astrocyte (O-2A) progenitor cells derived from different regions of the perinatal central nervous system (CNS) of rats were investigated in vitro. The O-2A progenitor cells can differentiate into either oligodendrocytes or type-2 astrocytes. The depletion of these cells could lead to demyelination, seen as a delayed reaction after irradiation of the CNS in vivo. To quantify cell survival, O-2A progenitor cells were grown on monolayers of type-1 astrocytes. Monolayers of type-1 astrocytes stimulate O-2A progenitor cells to divide. O-2A progenitor cells were irradiated in vitro and clonogenic cell survival was measured. The O-2A progenitor cells derived from perinatal optic nerve were quite radiosensitive in contrast to O-2A progenitor cells derived from perinatal spinal cord and perinatal corpus callosum. Furthermore, O-2A progenitor cells derived from the optic nerve formed smaller colonies, with most colonies showing early differentiation into oligodendrocytes. In contrast, more than half of the colonies derived from corpus callosum did not show any differentiation after 2 weeks in vitro and kept growing. These differences support the view that perinatal O-2A progenitor cells derived from the optic nerve are committed progenitor cells while the O-2A progenitor cells derived from the perinatal corpus callosum and the perinatal spinal cord have more stem cell properties.     

Structure of the pyrroloquinoline quinone radical in quinoprotein ethanol dehydrogenase

Quinoprotein alcohol dehydrogenases use the pyrroloquinoline quinone (PQQ) cofactor to catalyze the oxidation of alcohols. The catalytic cycle is thought to involve a hydride transfer from the alcohol to the oxidized PQQ, resulting in the generation of aldehyde and reduced PQQ. Reoxidation of the cofactor by cytochrome proceeds in two sequential steps via the PQQ radical. We have used a combination of electron nuclear double resonance and density functional theory to show that the PQQ radical is not protonated at either O-4 or O-5, a result that is at variance with the general presumption of a singly protonated radical. The quantum mechanical calculations also show that reduced PQQ is unlikely to be protonated at O-5; rather, it is either singly protonated at O-4 or not protonated at either O-4 or O-5, a result that also challenges the common assumption of a reduced PQQ protonated at both O-4 and O-5. The reaction cycle of PQQ-dependent alcohol dehydrogenases is revised in light of these findings.     

Lipase-catalysed preparation of acetates of 4-nitrophenyl beta-D-xylopyranoside and their use in kinetic studies of acetyl migration

Di-O-acetates and mono-O-acetates of 4-nitrophenyl beta-D-xylopyranoside were prepared by use of lipase PS-30. Polarity of organic solvents and reaction time affected the regioselectivity of the di-O-acetylation as well as the yields of monoacetates. The kinetics of acetyl groups migration in these derivatives was studied in aqueous media using HPLC. Migration of the acetyl group strongly depended on pH. The highest rate of acetyl migration was observed from O-2 to O-3 in both 2,4-di-O-acetate and 2-O-acetate. On the contrary, acetyl exchange between O-3 and O-4 in both directions was slower than between O-2 and O-3. The 2,3-di-O-acetate and 4-O-acetate showed to be the most stable towards acetyl migration. The 3,4-di-O-acetate and 4-O-acetate were dominant in the corresponding equilibration mixtures.     

Coexistence of perinatal and adult forms of a glial progenitor cell during development of the rat optic nerve

We have studied the developmental appearance of the O-2A(adult) progenitor cell, a specific type of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell that we have identified previously in cultures prepared from the optic nerves of adult rats. O-2A(adult) progenitors differ from their counterparts in perinatal animals (O-2A perinatal progenitor cells) in antigenic phenotype, morphology, cell cycle time, rate of migration, time course of differentiation into oligodendrocytes or type-2 astrocytes and sensitivity to the lytic effects of complement in vitro. In the present study, we have found that O-2A(adult) progenitor-like cells first appear in the developing optic nerve approximately 7 days after birth and that by 1 month after birth these cells appear to be the dominant progenitor population in the nerve. However, the perinatal-to-adult transition in progenitor populations is a gradual one and O-2A(adult) and O-2A perinatal progenitors coexist in the optic nerve for 3 weeks or more. In addition, cells derived from optic nerves of P21 rats express characteristic features of O-2adult and O-2A perinatal progenitors for extended periods of growth in the same tissue culture dish. Our results thus indicate that the properties that distinguish these two types of O-2A progenitors from each other are expressed in apparently identical environments. Thus, these cells must either respond to different signals present in the environment, or must respond with markedly different behaviours to the binding of identical signalling molecules.     

Coordination chemistry of vitamin C. Part I. Interaction of L-ascorbic acid with alkaline earth metal ions in the crystalline solid and aqueous solution

The interaction of L-ascorbic acid with alkaline earth metal ions has been investigated in aqueous solution at pH 6-7. The solid salts of the type Mg(L-ascorbate)2.4H2O, Ca(L-ascorbate)2.2H2O, Sr(L-ascorbate)2.2H2O and Ba(L-ascorbate)2.2H2O were isolated and characterized by means of 13C NMR and FT-IR spectroscopy. Spectroscopic and other evidence suggested that in aqueous solution, the binding of the alkaline earth metal ions is through the O-3 atom of the ascorbate anion, while in the solid state the binding of the Mg(II) is different from those of the other alkaline earth metal ion salts. The Mg(II) ion binds to the O-3, O-1 atom of the two ascorbate anions and to two H2O molecules, while the eight-coordination around the Ca(II), Sr(II), and Ba(II) ions would be completed by the coordination of three acid anions, through O-5, O-6 of the first, O-3, O-5, O-6 of the second and O-1 of the third anion as well as to two H2O molecules. The structural properties of the alkaline earth metal-ascorbate salts are different in the solid and aqueous solution.     

Determining the crystal structure of cellulose III(I) by modeling

Recently, a one-chain monoclinic unit cell for cellulose III(I) having P2(1) symmetry and a single glucose in the asymmetric unit was proposed, based on high-resolution diffraction patterns. The new work challenged a two-chain structure that was published 25 years earlier, although it did not provide new three-dimensional coordinates. Our goals were to solve the structure by modeling, find whether modeling would reject the previously determined two-chain unit cell, and compare the model with the anticipated experimental structure. Combinations of three rotamers of the O-2, O-3, and O-6 hydroxyl groups produced 27 'up' and 27 'down' starting structures. Clusters ('minicrystals') of 13 cellotetraose chains terminated by methyl groups for each of the 54 starting structures were optimized with MM3(96). Hydroxyl groups on 16 of these 54 structures reoriented to give very similar hydrogen-bonding schemes in the interiors, along with the lowest energies. Hydrogen bonds included the usual intramolecular O-3H...O-5' linkage, with O-6' also accepting from O-3H. Interchain hydrogen bonds form an infinite, cooperative O-6H...O-2H...O-6 network. Direct comparison of total minicrystal energies for the one- and two-chain unit cell was inappropriate because the two-chain cell's alternate chains are shifted 0.9 A along the z-axis. To get comparable energy values, models were built with both cellotetraose and cellohexaose chains. The differences in their energies represent the energies for the central layers of cellobiose units. The one-chain cell models had much lower energy. The eight best 'up' one-chain models agree reasonably well with the structure newly determined by experiment.     

Increased superoxide radicals generation from alveolar macrophages in immature guinea-pigs

To study the effect of maturation on abilities of superoxide radicals (O-2) generation in the airways, we compared stimuli-induced O-2 generation by alveolar macrophages in immature (aged 10+/-2 days) and adult (aged 90+/-2 days) guinea-pigs. The production of O-2 was assayed by chemiluminescence method, using a Cypridina luciferin analog as a highly sensitive and specific probe for O-2. Whereas no significant difference in cell components of bronchoalveolar lavage fluid was observed between immature and adult animals, O-2 generation induced by stimulation of alveolar macrophages was greater in immature than in adult animals, with significant differences observed after platelet-activating factor (100 nM) or phorbol myristate acetate (0.5 micro g/ml). The results suggest that alveolar macrophages from immature animals are far more potent O-2 generators than the same cells of adult animals.     

Full NMR assignment and revised structure for the capsular polysaccharide from Streptococcus pneumoniae type 15B

The capsular polysaccharide from Streptococcus pneumoniae Type 15B is a component of the 23-valent polysaccharide vaccine against pneumococcal disease. We report full NMR assignments for the native and de-O-acetylated polysaccharide, and confirm that the phosphorylated substituent is glycerol-2-phosphate rather than phosphocholine, located on O-3 of the side chain beta-Galp residue. The polysaccharide is O-acetylated on the terminal alpha-Gal residue, distributed between O-2, O-3, O-4 and O-6 in a ratio of 6:12:12:55, with approximately 15% of the repeat units not O-acetylated.