Differentiation of two strains of Mycoplasma gallisepticum with monoclonal antibodies and flow cytometry

Identification of infecting Mycoplasma spp. is difficult and not routine for strain. This paper describes a procedure for the rapid identification of the strain of M. gallisepticum. Monoclonal antibodies were prepared against M. gallisepticum F and M. gallisepticum S6. Aliquots of 24-hour broth cultures of these organisms were incubated briefly with either of the monoclonal antibodies. A second incubation was made with anti-mouse immunoglobulin conjugated to fluorescein isothiocyanate. Fluorescent intensity associated with the organisms was measured with a flow cytometer. The criterion for identification was a comparative increase in fluorescent intensity when the strain and monoclonal antibody were homologous. The procedure correctly differentiated the F and S6 strains of M. gallisepticum in a blind study.     

Capitated risk-bearing managed care systems could improve end-of-life care

A surface protein antigen (PAc) of Streptococcus mutans, in particular the A-region of this PAc molecule, has been noted as a possible target in research for an effective dental caries vaccine. To identify the antigenic peptide binding to major histocompatibility complex (MHC) class II (HLA-DR) molecules in the A-region, we prepared a panel of overlapping synthetic peptides in the second unit of the A-region, and established that a simple enzyme-linked immunosorbent assay (ELISA) binding assay could be achieved by incubating the DR-crude. Binding to DR molecules of these peptides from nine donors was investigated by using the ELISA binding assay. It was revealed that the PAc(316-334) peptide bound more strongly to the HLA-DR molecule in seven out of nine subjects. In particular, DR8 (DRB1*0802), DR5 (DRB1*1101) and DR6 (DRB1*1402 and *1405), which bound strongly to PAc(316-334) peptide, were identified. Moreover, we synthesized glycine-substituted peptide analogues of the peptide and examined the binding motif of the binding region. As a result, the multiple binding motif in DR8, DR5 and DR6 was found in L-RV-K-A. It is suggested that a peptide vaccine for dental caries that is more effective for humans, with fewer adverse side-effects, could be designed by combining the multiple binding motif with the B-cell epitope to produce only the inhibiting antibody against dental caries. The peptide could therefore be useful for peptide vaccine development in the general human population.     

Coenzyme A disulfide reductase, the primary low molecular weight disulfide reductase from Staphylococcus aureus. Purification and characterization of the native enzyme

The human pathogen Staphylococcus aureus does not utilize the glutathione thiol/disulfide redox system employed by eukaryotes and many bacteria. Instead, this organism produces CoA as its major low molecular weight thiol. We report the identification and purification of the disulfide reductase component of this thiol/disulfide redox system. Coenzyme A disulfide reductase (CoADR) catalyzes the specific reduction of CoA disulfide by NADPH. CoADR has a pH optimum of 7.5-8.0 and is a dimer of identical subunits of Mr 49,000 each. The visible absorbance spectrum is indicative of a flavoprotein with a lambdamax = 452 nm. The liberated flavin from thermally denatured enzyme was identified as flavin adenine dinucleotide. Steady-state kinetic analysis revealed that CoADR catalyzes the reduction of CoA disulfide by NADPH at pH 7.8 with a Km for NADPH of 2 muM and for CoA disulfide of 11 muM. In addition to CoA disulfide CoADR reduces 4,4'-diphosphopantethine but has no measurable ability to reduce oxidized glutathione, cystine, pantethine, or H2O2. CoADR demonstrates a sequential kinetic mechanism and employs a single active site cysteine residue that forms a stable mixed disulfide with CoA during catalysis. These data suggest that S. aureus employs a thiol/disulfide redox system based on CoA/CoA-disulfide and CoADR, an unorthodox new member of the pyridine nucleotide-disulfide reductase superfamily.     

Use of a gonadotropin-releasing hormone agonist in the evaluation of postmenopausal virilization due to ovarian hyperthecosis. A case report

BACKGROUND: Hyperthecosis in a postmenopausal woman is a very rare cause of virilization, and only five cases have been reported previously. CASE: A woman presented with a nine-year history of increasing hirsutism and a mild virilization beginning in the perimenopausal period. Initial androgen metabolite concentrations suggested attenuated late-onset adrenal hyperplasia, but a trial of dexamethasone treatment was ineffective. Subsequent use of leuprolide acetate resulted in a biochemical and clinical improvement in the signs and symptoms. CONCLUSION: This case is unique because gonadotropin-releasing hormone agonist administration was utilized as both a diagnostic and therapeutic modality.     

Depression of lipogenesis in swine adipose tissue by specific dietary fatty acids

The objective of this study was to document the influence of specific dietary fatty acids on rates of lipid synthesis and sensitivity to insulin in porcine adipose tissue. Weanling pigs were assigned to one of six groups, and each group was fed diets containing 10 g/100 g of added cornstarch or 10 g/100 g of added fatty acid. The fatty acid-enriched diets contained either a combination of 14:1 plus 16:1 (14:1/16:1 diet), 16:0, 18:0, 18:1, or 18:2 (n-6). With the exception of the cornstarch diet, all diets contained approximately 35% 14:0. Subcutaneous adipose tissue samples were collected at slaughter from the area overlying the first cranial vertebra. Fresh samples were incubated for 2 h in 20 mM glucose and 0, 10, 100 or 1,000 microU/mL of porcine insulin. The smallest adipocytes were observed in adipose tissue from pigs fed the 16:0 or 18:2 diets. Glucose incorporation into lipids was greater (P < .05) in adipose tissue from cornstarch-fed pigs than in adipose tissue from the other treatment groups. Lipogenesis was 67, 53, 35, 32, and 20% lower (P < .05) in adipose tissue from 16:0-, 14:1/16:1-, 18:0-, 18:2-, and 18:1-fed pigs, respectively, than in adipose tissue from the cornstarch-fed pigs. Insulin increased lipogenesis by 19% (P < .05) in adipose tissue from the cornstarch-fed pigs and by 15 to 40% (P < .05) in adipose tissue from the 14:1/16:1-fed pigs. Insulin did not stimulate lipogenesis (P > .4) in adipose tissue from pigs fed the 16:0, 18:0, or 18:1 diets. The data suggest that fatty acid chain length and unsaturation are determinants in the effects of dietary fat and insulin on de novo lipogenesis.     

Detection of polyomaviral DNA in clinical samples from immunocompromised patients: correlation with clinical disease

Characterization of the region between HLA-B and the TNF loci in the human MHC revealed the presence of duplicated loci, named CL1 and CL2, that included repeat sequences. Development and use of a PCR typing methodology that amplified both CL microsatellites simultaneously indicated that PCR product patterns analysed on native agarose gels were allelic (Abraham et al., 1992). The purpose of the current study was to determine the molecular explanation for the unique patterns achieved. Sequence analysis of the CL1 locus from 32 chromosomes representing 10 ancestral haplotypes indicated that six alleles were present. The CL microsatellites also provided an opportunity to study the evolutionary relationships between MHC haplotypes from different racial groups. Sequence comparison of closely related ancestral haplotypes from different racial groups suggested that the CL1 microsatellite has not changed in the period since divergence.     

The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Asclepios

The impact of demographic, lifestyle, and seminal factors on the sperm chromatin structure assay (SCSA) parameters was evaluated in a population of 277 healthy Danish men. This cohort was established within the framework of a European Concerted Action on occupational hazards to male reproductive capability in order to examine the possible reproductive effects of exposure to styrene or pesticides. The SCSA measures the susceptibility of sperm DNA to in-situ acid-induced denaturation, by multiparameter flow cytometric analysis after staining with the DNA-specific fluorescent dye acridine orange. The green versus red bivariate cytogram patterns were quite variable among donors, showing a wide heterogeneity of sperm DNA denaturability. Nevertheless, in those cases where we had the possibility to measure two semen samples from the same donor, the cytogram pattern remained stable over time (0.64 < r < 0.78). Analysis of variance demonstrated that the SCSA results can be influenced by the age of the donor (P < 0.0001), smoking habits (P < 0.05), the presence of leukocytes and immature germ forms in the ejaculate (P < 0.0001), and the duration of sexual abstinence (P < 0.0001). Furthermore, the relationship between the SCSA data and sperm concentration, morphology, and vitality was weak (-0.22 < r < -0.46). Therefore, the SCSA provides independent and complementary measurements of semen quality and is thus a useful tool for epidemiological studies, but the effects of some confounders should be accounted for in the survey design and analysis.     

Increased flight surgeon role in military aeromedical evacuation

BACKGROUND: Physicians were involved in the development of aeromedical evacuation (medevac) and flight surgeons flew as crewmembers on the first U.S. military medevac flights. However, since World War II flight surgeons have not been routinely assigned to operational medevac units. The aeromedical literature addressing the role of physicians in medevac is controversial. Recent contingencies involving the U.S. Air Force (USAF) have required the augmentation of medevac units with flight surgeons. RECENT CHANGES IN THE EUROPEAN THEATER: Beginning in 1992, the United States Air Forces Europe (USAFE) assigned three flight surgeons to the medevac squadron. Between 2 February 1993 and 24 March 1994 USAFE moved 241 patients on 29 missions out of the former Yugoslavia--most of these missions had a flight surgeon on the crew. Because advance medical information on the status of these patients is often nonexistent, the presence of a physician on the crew proved life-saving in some instances. In peacetime operations, there has been a recent trend in the European theater for the USAF to move more unstable patients. OBSERVED BENEFITS OF PHYSICIANS IN MEDEVAC: Dedicated medevac flight surgeons have proven to have the specific experience and training to perform effectively in the role of in-flight medical attendant. In addition, they are effective in negotiating with referring physicians about the urgency of movement, required equipment, the need for medical attendants, etc. These flight surgeons also provide medical coverage of transiting patients in the Aeromedical Staging Flight (ASF), thus providing needed continuity in the medevac system. CONCLUSION: Dedicated medevac flight surgeons fill a unique and valuable role in medevac systems. Agencies with medevac units should consider assigning flight surgeons to these units.