In-vitro antibiotic susceptibility and molecular analysis of anaerobic bacteria isolated in Cape Town, South Africa.

One hundred and twenty-three anaerobic isolates from Cape Town, South Africa, were tested in vitro against cefoxitin, chloramphenicol, metronidazole, co-amoxiclav, benzylpenicillin and clindamycin. Forty-five Gram-positive organisms were tested against RP59500 (a streptogramin). Resistance in the Gram-positive isolates was as follows: of the Clostridium perfringens isolates, 4% were resistant to benzylpenicillin and 4% to clindamycin; of the Peptostreptococcus anaerobius isolates, 10% were resistant to benzylpenicillin, 10% to cefoxitin and 10% to metronidazole; of the isolates of Peptostreptococcus spp., 12% were resistant to benzylpenicillin, 6% to metronidazole, 6% to chloramphenicol and 12% to RP59500. Of the Gram-negative organisms, those in the Bacteroides fragilis group were resistant to benzylpenicillin (83%), cefoxitin (5%), clindamycin (5%) and co-amoxiclav (2%). One clindamycin-resistant B. fragilis isolate carried a plasmid homologous to the ermF erythromycin resistance determinant.

The value of including broth cultures as part of a routine culture protocol.

Three hundred seventeen clinical specimens from both superficially and deeply infected sites were prospectively examined to assess the true value of including liquid media as part of the routine culture procedure. All broth cultures were subcultured after overnight incubation onto plate media. The isolates obtained from the broth cultures were then compared with the isolates obtained on primary solid media. The isolates obtained from the broth cultures only were evaluated for clinical relevance by review of the patients' records. Twenty-two clinically relevant isolates were obtained from the broth cultures only, but the isolation of these additional organisms altered patient management for only two patients. It would appear from these results that the additional expense and time involved in culturing clinical specimens in fluid media is unwarranted.

Osteoprotegerin: a novel secreted protein involved in the regulation of bone density.

A novel secreted glycoprotein that regulates bone resorption has been identified. The protein, termed Osteoprotegerin (OPG), is a novel member of the TNF receptor superfamily. In vivo, hepatic expression of OPG in transgenic mice results in a profound yet nonlethal osteopetrosis, coincident with a decrease in later stages of osteoclast differentiation. These same effects are observed upon administration of recombinant OPG into normal mice. In vitro, osteoclast differentiation from precursor cells is blocked in a dose-dependent manner by recombinant OPG. Furthermore, OPG blocks ovariectomy-associated bone loss in rats. These data show that OPG can act as a soluble factor in the regulation of bone mass and imply a utility for OPG in the treatment of osteoporosis associated with increased osteoclast activity.

Identification of the residues involved in homodimer formation of recombinant human erythropoietin.

Recombinant human erythropoietin (rHuEPO) is biologically functional when in a monomeric state; upon extensive heating, rHuEPO forms a dimer. The nature of this dimeric linkage was investigated after isolation of the dimer by gel filtration. The dimer fraction was subjected to tryptic digestion, and the peptides were separated by reversed-phase HPLC. SDS-PAGE, N-terminal sequencing, capillary electrophoresis and mass spectrometry (both liquid-chromatographic electrospray and matrix-assisted laser desorption ionization) were employed to compare the tryptic peptides from heat-treated rHuEPO and untreated rHuEPO. Results demonstrated that elevated heat broke the intramolecular disulfide bond between Cys-7 and Cys-161 and an intermolecular disulfide bond then formed from these residues, producing a covalently linked rHuEPO homodimer. Dimer formation was also mathematically modeled and shown to fit a simple equilibrium.

Microheterogeneity of erythropoietin carbohydrate structure.

The microheterogeneity of the carbohydrate structures on recombinant human erythropoietin (rHuEPO) expressed in Chinese hamster ovary cells has been evaluated by electrospray ionization (ESI) mass spectrometry (MS) of glycopeptide fragments. The microheterogeneity is largely associated with the presence or absence of terminal N-acetylneuraminic acid (Neu5Ac) residues, varying amounts of O-acetylation of the Neu5Ac residues, and the presence or absence of N-acetyllactosamine extensions. The N-linked carbohydrate structures were structurally diverse; 52 different N-linked oligosaccharide structures were identified. Consistent structural assignments could be made from data obtained using different proteolytic digests, ESI solvent systems (aqueous/methanol systems with acetic or formic acid), and on-line or off-line LC/MS analysis. All glycosylation sites exhibited some level of O-acetylation of Neu5Ac residues. Interestingly, glycosylation site asparagine-83 exhibits mono-O-acetyl and di-O-acetyl Neu5Ac residues, while the other sites, asparagine-24, asparagine-38, and serine-126, exhibit mainly mono-O-acetyl Neu5Ac derivatization. This difference in O-acetylation may be site specific or due to sample handling of labile structures. However, mild base treatment of rHuEPO with NaOH on ice removed the O-acetyl groups associated with a given carbohydrate structure, without adversely affecting the underlying oligosaccharide structure, resulting in a simplified mass spectra. Nuclear magnetic resonance spectroscopy of Neu5Ac residues released by neuraminidase treatment of total rHuEPO indicated that Neu5,9Ac2 residues were present. Additional resonances were also observed that were consistent with other Neu5Ac O-acetyl linkages; these O-acetyl resonances could be removed by mild base hydrolysis of rHuEPO.

Structural requirements for addition of O-linked carbohydrate to recombinant erythropoietin.

To define the structural requirements for addition of O-linked glycosylation in vivo, recombinant erythropoietin (rEPO) variants were constructed. Thirty-three independent Ser or Thr substitutions were constructed and examined to see which were subject to O-linked carbohydrate addition. Variants with Thr mutations at positions 123 and 125, but not elsewhere, contained additional carbohydrate, which suggests that several positions around the existing O-linked glycosylation site (Ser126), but not elsewhere, contain the necessary information for O-linked carbohydrate addition. Two forms of the Thr125 variant were identified. One form was glycosylated only at residue 125, and a second form was glycosylated at both Thr125 and Ser126, the normal O-glycosylation site. We have also found that glycosylation is less efficient when rEPO is improperly folded and that prolines at -1 and +1 relative to the O-glycosylation site enhance glycosylation.

Peptide mapping and evaluation of glycopeptide microheterogeneity derived from endoproteinase digestion of erythropoietin by affinity high-performance capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) has been employed to characterize the peptide map of recombinant human erythropoietin (rHuEPO) expressed from Chinese hamster ovary (CHO) cells. The methodology employs an ion pairing agent, 100 mM heptanesulfonic acid in 40 mM sodium phosphate buffer, pH 2.5, to increase peptide resolution, to decrease analyte wall interactions, and to evaluate glycopeptide microheterogeneity. The total tryptic map is segregated into two regions, nonglycosylated and glycosylated peptides. Reproducibility of the peptide map is excellent; the map results in baseline separation of 16 tryptic peptides and one doublet peak composed of two peptides (resolution 0.22). The map furthermore allows for the evaluation of the microheterogeneity associated with the three rHuEPO glycopeptides. At least 12 glycopeptide forms were separated in the initial peptide map. Peptides were identified by Edman sequencing, and the glycopeptides were further subjected to Dionex anion-exchange chromatography. To simplify the level of complexity associated with the glycopeptides, much of the characterization employed asialoglycopeptides and employed several endoproteolytic diagnosis. The relative percent distribution for each purified asialoglycopeptide was calculated to define the level of complexity and to tentatively assign a known structure to the HPCE peak. The level of structural complexity of the asialoglycopeptides appears to increase from the simplest O-linked form to the more complex N83, N38, and N24 glycosylation positions, respectively. HPCE evaluation of glycopeptide microheterogeneity appears to be simpler, faster, and just as sensitive as other more frequently employed methods for glycopeptide characterizations.

Human pepsinogen C (progastricsin). Isolation of cDNA clones, localization to chromosome 6, and sequence homology with pepsinogen A.

The entire pepsinogen C (PGC) coding sequence was determined by analysis of a series of five overlapping cDNA clones identified in a library constructed from human gastric mucosa poly(A+) RNA. A partial cDNA clone was initially identified using a 256-fold degenerate oligonucleotide probe for amino acid residues 4-12 of pepsin C, and subsequently 4 additional clones were identified upon rescreening with a probe complementary to the 5' region of the original cDNA clone. Northern analysis of gastric mucosa poly(A+) RNA with a PGC cDNA probe revealed an mRNA 1.5-kilobase species that was indistinguishable from that detected with a human pepsinogen A (PGA) cDNA probe. In contrast, the PGC and PGA cDNA probes detected distinct genomic restriction fragments indicating there was no detectable cross-hybridization under high stringency conditions. The PGC gene was localized to human chromosome 6 by analysis of a panel of human x mouse somatic cell hybrids. The regions containing the active site aspartyl groups of PGC are conserved in relationship to several other aspartic proteinases. We propose that the absence of detectable immunologic cross-reactivity between the two groups of human pepsinogens, A and C, results from divergent evolution of sequences located on the surface of the zymogens in contrast to the strongly conserved active site regions located within the binding cleft of the enzymes that are inaccessible for antigenic recognition.

Clorazepate use may prevent alcohol withdrawal convulsions.

Clorazepate dipotassium was administered orally for the five-day prophylactic treatment of potential, incipient and overt withdrawal signs and symptoms in 226 patients on admission to an inpatient alcohol treatment unit. Conservative estimates based on these patients' histories and on literature reports predicted that between 7 and 40 (3% to 18%) of these persons would be expected to have a withdrawal convulsion. No patients experienced convulsions. This complete absence of seizures suggests that clorazepate is effective in counteracting convulsive and other manifestations of the alcohol withdrawal syndrome.

Modulation of serotonin receptors by specific phosphatidylcholines.

Treatment of mouse cortical brain membranes with dioleoylphosphatidylcholine produced a large (50%) decrease in serotonin binding sites. The time course for this effect paralleled an increase in oleic acid in membrane phosphatidycholine and an increase in membrane fluidity. "Active Lipid" produced a similar decrease in serotonin binding sites, while fluidizing the membranes even more strongly. Distearoylphosphatidylcholine had no effect on serotonin binding sites or membrane fluidity by itself, but was capable of counteracting both the reduction in binding sites and membrane fluidity produced by "Active Lipid". The data indicate that specific phosphatidylcholines can have profound effects on serotonin receptors, but a clear picture of the relative importance of membrane fluidity per se versus more specific phospholipid effects will require further investigation.

Relationship between repression-sensitization and word frequency estimates for pleasant and unpleasant words.

College students (45 females and 12 males) estimated the frequency of pleasant and unpleasant English words. 28 subjects estimated how often they, personally, had seen the words (Personal condition), and 29 subjects estimated how often a "language expert" had encountered the words (Kucera-Francis condition). Byrne's Repression-Sensitization scores were obtained for all subjects. In both conditions repressors judged pleasant words to be more frequent, and sensitizers judged unpleasant words to be more frequent (p less than .01). The results may clarify some contradictions in the literature on frequency estimation and may help to explain the relationship between repression-sensitization and perceptual defense/vigilance.