Multiantennary group-specific polysaccharide of group B Streptococcus

The group-specific antigen of group B Streptococcus is composed of four different oligosaccharide units of Mw 766 (III), 1277 (II), 1462 (IV), and 1788 (I). The major constituent sugars of the oligosaccharides are alpha-L-rhamnopyranose, alpha-D-galactopyranose, 2-acetamido-2-deoxy-beta-D-glucopyranosyl, and D-glucitol except that III does not contain alpha-D-galactopyranosyl or 2-acetamido-2-deoxy-beta-D-glucopyranosyl residues and IV contains no D-glucitol but has one additional beta-L-rhamnopyranosyl residue. The structures of II and III have been previously elucidated [Michon, F., Katzenellenbogen, E., Kasper, D. L., & Jennings, H. J. (1987) Biochemistry 26, 476-486]. In the group B antigen all the oligosaccharides are linked by one type of phosphodiester bond from O6 of the D-glucitol residue of one oligosaccharide to O6 of the alpha-D-galactopyranosyl residue of the next to form a complex and highly branched multiantennary structure. However, despite the heterogeneous nature of its component oligosaccharides, some order has been identified in the biosynthesis of the group B antigen from chemical and enzymatic sequence studies. Because III lacks an alpha-D-galactopyranosyl residue but has a D-glucitol residue, it is situated at the reducing terminus of all the branches of the group B antigen where it is always adjacent to a II moiety. Conversely, IV has an alpha-D-galactopyranosyl residue but has no D-glucitol and is therefore located at the reducing terminus of the group B antigen where it probably functions as a linker molecule between the group B polysaccharide and the cell wall peptidoglycan of the group B streptococcal organisms. Oligosaccharide I contains two alpha-D-galactopyranosyl residues and one D-glucitol residue and thus constitutes the branch point in the group B antigen, whereas II contains one of each of the above residues and therefore is situated in linear interchain positions. The group B antigen is highly branched and probably has a unique multiantennary structure.     

Structural analysis of the major urinary oligosaccharides in feline alpha-mannosidosis.

Two homologous series of urinary oligosaccharides were identified by h.p.l.c. and fast-atom-bombardment mass spectrometry in feline alpha-mannosidosis. The predominant series has the composition Man2-8GlcNAc2 and a minor series the composition Man2-9GlcNAc. The structure of the most abundant oligosaccharide, which accounts for over 80% of the urinary oligosaccharide, was shown to be alpha-D-Manp(1----3)[alpha-D-Manp-(1----6)]beta-D-Manp -(1----4)-beta-D-GlcpNA c-(1----4)-D-GlcNAc by gas chromatography and mass spectrometry. Such a structure is consistent with the incomplete catabolism of complex N-linked glycans due to a deficiency of alpha-D-mannosidase in tissue lacking an endohexosaminidase activity.     

A convenient six-point blood sampling schedule for determining whole body cholesterol kinetics in humans

Parameters of total body cholesterol metabolism in humans can be determined by using a three-pool model to analyze the turnover of plasma cholesterol following the injection of radiolabeled cholesterol. In the past this required a rigorous schedule of approximately 36 blood samples over a 10-month period. We have developed a convenient sampling schedule involving only six large samples, each analyzed in sextuplicate. Such a reduction in the frequency of samples is possible only when considerable confidence in the model is available. In general, the simplified sampling strategy depends upon considerable prior experience with the model, only moderate biological error, and estimatable subject to subject variation in model parameters. Because the timing of the samples is critical and because the optimal times will differ for different subjects, the six-point strategy involves using the first three samples (drawn at days 1, 7, and 24 or, for hypercholesterolemic subjects, at days 1, 8, and 28) in conjunction with results from previous studies to set the time for the next sample; the process is reiterated for the last two points. In this study, we have compared parameter estimates obtained by the new six-point schedule with those obtained simultaneously (in the same, single turnover study) by the old 36-point schedule in the same 26 subjects. Both schedules gave comparable values. In particular, the coefficients of variation between values obtained by the two methods for each of the four parameters for which we have developed predictive equations were quite low: PR 1.5%, M1 4.1%, M3min 13%, Mtot min 4.3%. The simplified six-point schedule makes it feasible to study long-term cholesterol turnover in substantial numbers of patients.     

Host-Pathogen Interactions : XXIX. Oligogalacturonides Released from Sodium Polypectate by Endopolygalacturonic Acid Lyase Are Elicitors of Phytoalexins in Soybean

Recent studies have demonstrated that an apparently homogeneous preparation of an α-1,4-d-endopolygalacturonic acid lyase (EC 4.2.2.2) isolated from the phytopathogenic bacterium Erwinia carotovora induced phytoalexin accumulation in cotyledons of soybean (Glycine max [L.] Merr. cv Wayne) and that this pectin-degrading enzyme released heat-stable elicitors of phytoalexins from soybean cell walls, citrus pectin, and sodium polypectate (KR Davis et al. 1984 Plant Physiol 74: 52-60). The present paper reports the purification, by anion-exchange chromatography on QAE-Sephadex columns followed by gel-permeation chromatography on a Bio-Gel P-6 column, of the two fractions with highest specific elicitor activity present in a crude elicitor-preparation obtained by lyase treatment of sodium polypectate. Structural analysis of the fraction with highest specific elicitor activity indicated that the major, if not only, component was a decasaccharide of α-1,4-d-galactosyluronic acid that contained the expected product of lyase cleavage, 4-deoxy-β-l-5-threohexopyranos-4-enyluronic acid (4,5-unsaturated galactosyluronic acid), at the nonreducing terminus. This modified decagalacturonide fraction exhibited half-maximum and maximum elicitor activity at 1 microgram/cotyledon (6 micromolar) and 5 micrograms/cotyledon (32 micromolar) galactosyluronic acid equivalents, respectively. Reducing 90 to 95% of the carboxyl groups of the galactosyluronic acid residues abolished the elicitor activity of the decagalacturonide fraction. The second most elicitor-active fraction contained mostly undeca-α-1,4-d-galactosyluronic acid that contained 4,5-unsaturated galactosyluronic acid at the nonreducing termini. This fraction exhibited half-maximum and maximum elicitor activity at approximately 3 micrograms/cotyledon (17 micromolar) and 6 micrograms/cotyledon (34 micromolar) galactosyluronic acid equivalents, respectively. These results confirm and extend previous observations that oligogalacturonides derived from the pectic polysaccharides of plant cell walls can serve as regulatory molecules that induce phytoalexin accumulation in soybean. These results are consistent with the hypothesis that oligogalacturonides play a role in disease resistance in plants.     

Lack of relationship in humans of the parameters of body cholesterol metabolism with plasma levels of subfractions of HDL or LDL, or with apoE isoform phenotype

The factors involved in regulating parameters of whole body cholesterol metabolism in humans have been explored in a series of investigations. Several physiological variables have been identified (weight, excess weight, plasma cholesterol, and age) that can predict 53-76% of the variation in production rate (PR) and in the sizes of the rapidly exchanging pool of body cholesterol (M1) and of the minimum estimates of the slowly exchanging pool of body cholesterol (M3min) and of total body cholesterol (Mtotmin). Surprisingly, measurements of the plasma levels of HDL cholesterol and of the major HDL apolipoproteins (apoA-I, A-II, and E) did not provide additional information useful in predicting parameters of whole body cholesterol metabolism. A study was therefore conducted to investigate possible relationships of the plasma levels of subfractions of lipoproteins, determined by analytic ultracentrifugation, and of apoprotein E phenotype, with the parameters of whole body cholesterol metabolism. Ultracentrifugal analysis of plasma lipoprotein subfractions was performed at the Donner Laboratory in 49 subjects; all of these subjects were currently undergoing whole body cholesterol turnover studies or had previously had such studies and were in a similar metabolic state as judged by plasma lipid and lipoprotein values. Apoprotein E phenotyping was carried out in 71 subjects. Differences in model parameters were sought among subjects with various apoprotein E phenotypes. Ultracentrifugal LDL subfractions Sof 0-2 (the region of LPa), Sof 0-7 (smaller LDL), Sof 7-12 (larger LDL), Sof 12-20 (IDL), and ultracentrifugal HDL subfractions Fo1.20 0-1.5 (smaller HDL3), Fo1.20 2-9 (larger HDL3 plus HDL2), and Fo1.20 5-9 (larger HDL2 or HDL2b) were examined for correlations with each other and with parameters of whole body cholesterol metabolism.     

Characterization of a structurally complex heptasaccharide isolated from the pectic polysaccharide rhamnogalacturonan II

A heptasaccharide was released from the plant cell-wall, pectic polysaccharide rhamnogalacturonan II by selective acid hydrolysis of the glycosidic linkages of apiosyl residues. The heptasaccharide was purified to homogeneity by gel filtration and anion-exchange chromatography. Some of the heptasaccharide molecules were found to be mono- and some di-O-acetylated, but the location of the acetic ester groups was not determined. The heptasaccharide was found to have the following structure, where AceA = an aceryl (3-C-carboxy-5-deoxy-l-xylosyl) residue, and Api = an apiose residue.

     

Swainsonine affects the processing of glycoproteins in vivo

Rats, sheep and guinea pigs treated with swainsonine excrete 'high mannose' oligosaccharides in urine. The major rat and guinea pig oligosaccharide is (Man)5GlcNAc, whereas sheep excrete a mixture of oligosaccharides of composition (Man)2-5GlcNAc2 and (Man)3-5GlcNAc. The presence of these oligosaccharides suggests that Golgi alpha-D-mannosidase II as well as lysosomal alpha-D-mannosidase is inhibited by swainsonine resulting in storage of abnormally processed asparagine-linked glycans from glycoproteins. Altered glycoprotein processing appears to have little effect on the health of the intoxicated animal, but the accompanying lysosomal storage produces a disease state.     

Determination of molsidomine in plasma by high-performance liquid column chromatography

A high-performance liquid chromatographic method is described for the analysis of the anti-anginal compound 5-ethoxycarbonyl-3-morpholinosydnonimine (Molsidomine) in human and dog plasma. The drug was extracted from plasma into chloroform and the analysis was carried out on a reversed-phase column, the column effluent being monitored by UV absorption at 312 nm. The method is sensitive (2 ± 0.3 ng/ml) and specific. The method was applied to a study in which human volunteers received an aqueous solution of the drug and then, on a separate occasion, a tablet formulation. Peak plasma levels of 20–30 ng/ml (tablet) and 10–19 ng/ml (aqueous solution) were obtained following a 2-mg oral dose.     

Extended performance using a high field magnet mass spectrometer

The development of a high field magnet for high mass electron impact, chemical ionization, field desorption and fast atom bombardment mass spectrometric studies is described. Its utility is illustrated with examples from structural studies of vitamin B12 biosynthetic intermediates, oligosaccharides, glycopeptides and the bleomycin antibiotics. The technique has also greatly assisted sequence studies of protein derived peptides.     

Delayed metamorphosis in Galleria mellonella: Interaction of chilling and space deprivation

A 2 × 2 factorial experiment was performed with each of 18 age classes within the last instar of Galleria mellonella. The two experimental factors applied to each age class were chilling and space deprivation; combinations of each factor were tested at two levels (present or absent). The 3-parameter Weibull function was used to calculate the proportion of animals undergoing larval-pupal ecdysis. Significant differences among time-trend statistics were related to interaction between chilling and space deprivation, as well as to age class. In general, chilling without space deprivation produced a small delay relative to the unchilled, not space-deprived condition. With space deprivation, however, chilling accelerated pupation compared to space deprivation alone. The order in which the chilled, space-deprived and space-deprived groups pupated expresses the essence of the interaction. Contrary to an additive effect, delayed development resulting from space deprivation appeared to be mitigated by chilling. Salivary gland stiffening was observed in chilled, space-deprived animals. With space deprivation, stiffening was associated with faster development compared to that of unchilled animals. These observations are discussed in the context of extant hypotheses of endocrine mechanisms responsible for the separate effects of chilling and space deprivation.