The role of enteric bacteria in the anerobic metabolism of 5-aminosalicylate

The primary (and inactive) enteric metabolite of 5-aminosalicylate is N-acetyl-5-amino-salicylate. Previous studies have demonstrated acetylation of this anti-inflammatory agent by intestinal and bacterial homogenates. To assess the contribution of anerobic bacteria to the N-acetylation in vivo, we have measured the production of N-acetyl-5-aminosalicylate in anerobic microculture. Our results indicate that enteric bacteria play a minor role in N-acetylation, but may contribute to the production of other metabolites of pharmacologic and toxicological interest.     

Glycosylated equine prolactin and its carbohydrate moiety

Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were purified to homogeneity from side fractions obtained during isolation of LH/FSH from horse pituitaries. Both PRL forms were isolated together in high yield by the isolation procedure used for glycosylated porcine PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, NaCl and isoelectric precipitation, and gel filtration. Purification of G-ePRL required additional Con A chromatography. The N-terminal amino acid sequencing for 32 cycles of G-ePRL and ePRL resulted in sequences identical to the known primary structure of ePRL. Based on MALDI mass spectrometry analysis and SDS-PAGE mobilities,G-ePRL and ePRL had estimated molecular weights of 25,000 and 23,000 Da, respectively. G-ePRL displayed only 60% of the immunoreactivity of ePRL in homologous radioimmunoassay. Using the Nb2 lymphoma cell bioassay, ePRL was found to have about l/30th the mitogenic activity of bovine PRL; G-ePRL was approximately l/10th as active as ePRL. Glycosylation of G-ePRL at Asn³¹ was confirmed by isolation and sequence analysis of an enzymatically derived G-ePRL glycopeptide spanning residues 29–37. Monosaccharide compositions of intact G-ePRL and this glycopeptide were very similar (Man₃, GlcNAc₂, GalNAc₁, Fuc_0.6, Gal_0.2, NeuAc_0.15) and resembled that of G-pPRL. The glycopeptide contained one sulfate residue as determined by ion chromatography after acid hydrolysis, indicating the presence of a sulfated monosaccharide. Comparative carbohydrate analysis of G-ePRL and other G-PRL preparations suggests that the functionally significant Asn³¹ carbohydrate unit is a fucosylated complex mono- and/or biantennary oligosaccharide terminating with a sulfated GalNAc residue and two or three Man residues.     

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of ¹²⁵I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (K_d) as the controls. The binding of ¹²⁵I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized ¹²⁵I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside.     

In vivo crystal formation in Escherichia coli of an over-expressed soluble form of penicillin-binding protein 5

Accumulation of either native membrane-bound or soluble variants of PBP5 over-expressed in the cytoplasm was investigated by electron microscopy of ultra-thin sections. One of the soluble forms of PBP5 (PBP5s353) formed well-ordered crystals inside the cells. Cells sectioned perpendicular to their long axis showed a diamond-shaped crystal whereas cells cut parallel to their long axis contained a long, narrow crystal. In both sectioning directions an ordered ultrastructure was visible as shown by optical diffraction. Computer processing was used to enhance the crystal images. From this the unit cell parameters were calculated as a = 7.6 nm, b = 4 nm, c = 4.2 nm, gamma = 75 degrees. The calculated unit-cell volume of 120 nm3 is large enough to contain one protein molecule.     

Clonal descent and microevolution of Neisseria meningitidis during 30 years of epidemic spread

Serogroup A meningococci of subgroups III, IV-1 and IV-2 are probably descended from a common ancestor that existed in the nineteenth century. The 10.5 kb sequences spanning five distinct chromosomal loci, encoding cell-surface antigens, a secreted protease or housekeeping genes and intergenic regions, were almost identical in strains of those subgroups isolated in 1966, 1966 and 1917 respectively. During the subsequent two to three decades, all of these loci varied as a result of mutation, translocation or import of DNA from unrelated neisseriae. Thus, microevolution occurs frequently in naturally transformable bacteria. Many variants were isolated only once or within a single geographical location and disappeared thereafter. Other variants achieved genetic fixation within months or a few years. The speed with which sequence variation is either eliminated or fixed may reflect sequential bottlenecks associated with epidemic spread and contrasts with the results of phylogenetic analyses from bacteria that do not cause epidemics.