Search

Toxicity in serum has been reported in cases of recurrent spontaneous abortions and endometriosis. The null hypothesis was that serum toxicity was not involved in failed pregnancies after in vitro fertilization procedures. The objective was to expose donor sperm to pregnant versus nonpregnant patient sera and analyze for sperm DNA damaging effects using a novel comparative genomic hybridization method. Luteal phase sera (N = 21 cases) were drawn one week after embryo transfer. Colloid-washed donor sperm were incubated (48 h, 37°C, 5% CO2 in air) in 0% or 50% sera. Single-stranded DNA (ssDNA) of control sperm were stained in Hoechst 33342 and hybridized to Sybr Gold-stained ssDNA of sera-treated sperm. Image analyses were performed and fluorescent intensities analyzed. Nonpregnant patient sera (57% of cases) were associated with DNA fragmentation (64.4 ± 8.8 pixels; mean ± S.E.M.) when compared with pregnant patient sera (106.3 ± 8.4 pixels). There were no differences in the sera of biochemical (108.2 ± 15.3) versus clinical pregnancy cases (105.3 ± 11.4). The results suggest that nonpregnant patient sera contained factor(s) that cause DNA fragmentation leading to pregnancy losses.

The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) consists of interacting cytoplasmic and membrane proteins that catalyze the phosphorylation and translocation of sugar substrates across the cell membrane. One PTS protein, II-BGlc, is the membrane receptor specific for glucose and methyl D-glucopyranosides; the protein has been purified to homogeneity from Salmonella typhimurium [Erni, B., Trachsel, H., Postma, P. & Rosenbusch, J. (1982) J. Biol. Chem. 257, 13726-13730]. In the present experiments, the Escherichia coli ptsG locus, which encodes II-BGlc, was isolated from a transducing phage library and subcloned into plasmid vectors. The resulting plasmids complement the following phenotypic defects of ptsG mutants: growth on glucose, uptake and phosphorylation of methyl alpha-D-glucoside, and repression of the utilization of non-PTS sugars by methyl alpha-glucoside. The transformed cells overproduce II-BGlc 4- to 10-fold, and a Mr 43,000 polypeptide was synthesized from the plasmids in an in vitro transcription/translation system. The E. coli and S. typhimurium II-BGlc proteins differ in their physical properties, and a modified, three-step purification procedure was developed for isolating the E. coli protein.

Chitin catabolism by the marine bacterium Vibrio furnissii involves chemotaxis to and transport of N-acetyl-D-glucosamine (GlcNAc) and D-glucose. We report the properties of the respective permeases that complemented E. coli Glc- Man- mutants. Although the V. furnissii Glc-specific permease (55,941 Da) shares 38% identity with E. coli IIGlc (ptsG), it is 67% identical to MalX of the E. coli maltose operon (Reidl, J., and Boos, W. (1991) J. Bacteriol. 173, 4862-4876). An adjacent open reading frame encodes a protein with 52% identity to E. coli MalY. Glc phosphorylation requires only V. furnissii MalX and the accessory phosphoenolpyruvate:glycose phosphotransferase system proteins. The V. furnissii equivalent of IIGlc was not found in the 25,000 transformants screened. The GlcNAc/Glc-specific permease (52,894 Da) shares 47% identity with the N-terminal, hydrophobic domain of E. coli IINag, but is unique among IINag proteins in that it lacks the C-terminal domain and thus requires IIIGlc for sugar fermentation in vivo and phosphorylation in vitro. While there are similarities between the phosphoenolpyruvate:glycose phosphotransferase system of V. furnissii and enteric bacteria, the differences may be important for survival of V. furnissii in the marine environment.

We have previously reported that the chitin catabolic cascade in Vibrio furnissii involves multiple signal transducing systems, and that mono- and disaccharide chemoreceptors/transporters are essential components of some of these systems. This and the accompanying papers (Bouma, C. L., and Roseman, S. (1996) J. Biol. Chem 271, 33457-33467; Keyhani, N. O., Wang, L.-X., Lee, Y. C., and Roseman, S. (1996) J. Biol. Chem. 271, 33409-33413) describe some of the sugar transporters. A 13-kilobase pair fragment of V. furnissii DNA was found to impart a Glc+, Man+ phenotype to Escherichia coli ptsG ptsM mutants, and encodes the mannose transporter, ptsM, of the phosphoenolpyruvate:glycose phosphotransferase system. Unlike the E. coli mannose permease, V. furnissii IIMan is inactive with GlcNAc and Fru, and is encoded by four genes rather than three. The gene order is manXYZW, where the product of manY corresponds to IIPMan, manZ to the mannose receptor IIBMan, and manX and manW to the single E. coli gene, manX (which encodes IIIMan, viz. IIAMan). Thus, in V. furnissii, the E. coli manX equivalent comprises two genes, which are separated in the genome by two other genes of the ptsM complex. Two additional open reading frames were detected in the V. furnissii DNA fragment. One encodes a GlcNAc-6-P deacetylase, and the other is similar to aldolase.