Molecular cloning of MER-2, a human chromosome-11-encoded red blood cell antigen,using linkage of cotransfected markers

We report the molecular cloning of a human gene MER-2located on chromosome 11 that encodes a cell surface antigen which is polymorphic on red blood cells. An essential element of the cloning strategy was cotransfection-induced linkage of pSV2-neo, which encodes resistance to the antibiotic G418, to the human MER-2gene. An important feature of the pSV2-neo construct is that the same gene (the transposon, Tn5) that encodes G418 resistance in eukaryotic cells confers neomycin resistance in bacteria. Chinese hamster ovary (CHO) cells were cotransfected with pSV2-neo and genomic DNA from a CHO ×human cell hybrid containing a single human chromosome (chromosome 11). Transfectants expressing both the human MER-2gene and G418 resistance were isolated by selection in the antibiotic G418, followed by indirect immunofluorescence using the monoclonal antibody 1D12, which recognizes the MER-2 antigen, manual enrichment, and single-cell cloning. Genomic DNA from a primary transfectant positive for MER-2expression and G418 resistance was used to construct a cosmid library and cosmid clones able to grow in neomycin were isolated. Of 150,000 cosmid clones screened, 90 were resistant to neomycin and of these, 11 contained human repetitive sequences. Five neomycin-resistant cosmid clones containing human repetitive DNA were able to transfect CHO cells for G418 resistance and MER-2expression.     

Frequent carriage of Panton-Valentine leucocidin genes by Staphylococcus aureus isolates from surgically drained abscesses

Between 1 February and 15 April 2002, 95 patients were admitted to Gaston Bourret Territorial Hospital (New Caledonia, France) for drainage of community-acquired soft tissue abscesses. Staphylococcus aureus was detected in 68 cases (72%). Two-thirds of the patients with S. aureus infection had furuncles, which were located on the limbs in 82% of cases. The median interval between symptom onset and hospital admission was 5.7 days. Three-quarters of the patients were Melanesians living in tribes. Fifty-four S. aureus isolates were screened for toxin genes. Panton-Valentine leucocidin (PVL) genes were detected in 48 isolates (89%), the exfoliative toxin A gene was detected in 1 isolate, and no toxin genes were detected in 4 isolates. S. aureus nasal carriage was detected in 39.7% of patients with S. aureus infections. Two infecting S. aureus strains and two nasal carriage strains were resistant to methicillin. Comparative pulsed-field gel electrophoresis, performed in 16 cases, showed that five of six patients with PVL-positive nasal carriage strains were infected by the same strains. In contrast, 8 of 10 patients with PVL-negative nasal carriage strains were infected by PVL-positive strains. PVL genes thus appear to be a major virulence factor in both primary and secondary S. aureus skin infections.     

Notch 2 and Notch 1/3 segregate to neuronal and glial lineages of the developing olfactory epithelium.

The murine olfactory epithelium (OE) generates olfactory receptor neurons (ORNs) throughout development and into adulthood, but only a few of the factors regulating olfactory neuro- and glio-genesis have been delineated. Notch receptors maintain CNS neuronal progenitors and drive glial differentiation, and the Notch effectors Hes 1 and 5 are expressed in the OE, but the Notch receptors that stimulate Hes gene activation in defined lineages during OE development have not been determined. Here, we first use RT-PCR to reveal which Notch receptors and ligands are expressed in the developing and adult OE. This is followed by immunofluorescent detection, combined with lineage-specific markers to define the stage-specific developmental expression of different Notch family members. We show that throughout development, Notch 1 and 3 are expressed in cells retained within the lamina propria, where Notch 3 is expressed in olfactory ensheathing cells (OECs). In contrast, Notch 2 is expressed in apical embryonic and early postnatal OE neuronal progenitors. In postnatal and adult OE, Notch 1 is expressed predominantly in Bowman's glands, and Notch 2 in sustentacular cells. Notch 2 and Notch 1/3 may, therefore, have different roles in the commitment and differentiation of neuronal and glial lineages of the OE during development, and the maintenance of non-neuronal phenotypes postnatally.     

Early atherogenesis in the White Carneau pigeon. III. Lipid accumulation in nascent foam cells.

The role of lysosomes in aortic atherogenesis in White Carneau pigeons was examined by means of acid phosphatase cytochemistry. Foam cells were the major constituent of nascent atherosclerotic lesions in pigeons fed a 0.5% cholesterol diet for either 5 or 10 weeks. Seventy-four percent of foam cell lipid from animals at 5 weeks was in cytoplasmic droplets. The remaining lipid appeared in secondary lysosomes. After 10 weeks of cholesterol feeding, lysosomal lipid accounted for 73% of the lipid volume. The lipid accumulation correlated with increases in both size and number of lysosomes. An average of 2.4 lysosomes per 10(4) cu mu of cytoplasm was observed at 5 weeks. This value doubled by 10 weeks. The average lysosome diameter also increased between 5 and 10 weeks from 2.2 mu to 5.75 mu. Concomitantly, the complexity of lysosomes increased from simple, spherical organelles at 5 weeks to complex, multichambered organelles at 10 weeks. In contrast, lipid storage within cytoplasmic lipid droplets did not change either in size or in number. These observations suggest that by 5 weeks lipid storage within cytoplasmic droplets was maximized, and continued increases in lipid stores occurred predominantly through lysosomal loading.     

Prediction of sulfamethoxazole-trimethoprim synergistic action against members of the family Enterobacteriaceae with a two-plate agar dilution breakpoint MIC system.

Synergy between sulfamethoxazole (SMZ) and trimethoprim (TMP) was predicted by a two-plate agar dilution breakpoint MIC system. Comparison of the results of this new system with those of the disk diffusion system (P.M. Waterworth, Postgrad. Med. J. Suppl. 45:21-27, 1969) after tests with 1,518 Enterobacteriaceae isolates showed an overall correlation of 99.8%, a sensitivity of 99.7%, and a specificity of 100%. The method involves spot inoculation of 10(3) organisms onto each of two plates, one containing 160 micrograms of SMZ per ml and the other 8 micrograms of TMP per ml (in Oxoid IsoSensitest medium with 3% agar supplemented with 7% saponin-lysed horse blood), and then incubation overnight at 37 degrees C in air. All but three organisms for which SMZ-TMP was found to be synergistic by disk testing were inhibited on both plates. Three isolates of Proteus mirabilis, which failed to correlate with disk testing by this new system, all showed SMZ MICs of 1,000 micrograms/ml. The SMZ-TMP combination was falsely predicted to be nonsynergistic against these three organisms. There were no false synergy predictions by the breakpoint MIC system. Laboratories should report susceptibility to the SMZ-TMP combination only when there is synergy between the constituents. This simple, reliable agar dilution technique enables laboratories to accurately report synergy between SMZ and TMP.     

Typing of strains from a single-source outbreak of Pseudomonas pickettii.

Plasmid profiles, genome restriction fragment polymorphisms, carbohydrate oxidation-fermentation reactions, methylumbelliferyl substrate hydrolysis patterns, antimicrobial susceptibilities, and results obtained with the Biolog GN biochemical substrate kit were used to type 19 common-source, but mixed-biotype, outbreak strains and one epidemiologically distinct strain of Pseudomonas pickettii. Biotyping with conventional and methylumbelliferyl substrates failed to distinguish between strains. Plasmid profile testing was found to be inconsistent and not reproducible. The Biolog GN kit allowed greater strain differentiation than restriction fragment polymorphism did (12 biotypes versus 5 biotypes); antimicrobial susceptibility testing yielded 4 biotypes, and oxidation-fermentation tests gave 3 biotypes. Oxidation-fermentation results were consistent with restriction fragment polymorphs in all but 1 of the 20 strains tested. For ease of typing, comprehensive typeability, and reproducibility, oxidation-fermentation tests should be performed initially and followed if necessary by restriction fragment polymorph analysis for the elucidation of P. pickettii infection outbreaks.     

Mapping of the influenza virus genome II. Identification of the P1, P2, and P3 genes

Polyacrylamide gel electrophoresis of the RNAs of influenza A viruses was performed under conditions in which each of the eight RNA segments of influenza A/PR/8/34 (H0N1) virus migrates differently from the equivalent segments of influenza A/Hong Kong/8/68 (H3N2) virus. As reported previously [Palese, P. and Schulman, J. L. Proc. Nat. Acad. Sci. USA73, 2142–2146 (1976)], analysis of RNA patterns of recombinant viruses derived from these two parents permitted the identification of the fourth RNA segment (the slowest-moving RNA segment is counted as #1) of each virus as the gene coding for hemagglutinin, and the fifth segment of Hong Kong virus and the sixth segment of PR8 virus as the genes for the respective neuraminidases. Three more genes have been identified by correlating migration patterns of RNAs with protein patterns of recombinant viruses on gradient polyacrylamide gels. The slowest-moving band (RNA 1) is the gene coding for the third largest influenza virus protein (P3). RNA segment 2 codes for the largest protein (Pl), and the P2 protein is coded for by RNA segment 3.     

Immune response in urinary tract infection determined by radioimmunoassay and immunofluorescence: serum antibody levels against infecting bacterium and Enterobacteriaceae common antigen.

A solid-phase radioimmunoassay (RIA) procedure was compared with the indirect fluorescent antibody (IFA) test in a serological study of 76 female adults with urinary tract infections. Relative serum antibody activity was determined against patients' homologous infecting enterobacteria by RIA and IFA and against heterologous enterobacterial common antigen (Escherichia coli O14) by RIA. There was marked correlation between results of the IFA and RIA methods using the homologous system; 22 of 51 patients (43%) with pyelonephritis had significantly elevated serum antibody activity by both IFA (titers greater than or equal to 512) and RIA (binding ratio greater than or equal to 2.0) when compared with normal serum controls; three had significant antibody activity detectable by RIA only. Eighteen (72%) of 25 patients with pyelonephritis had RIA binding ratios of greater than or equal to 2.0 against their homologous bacterial isolates and the enterobacterial common antigen; an additional 6 patients had binding ratios of greater than or equal to 2.0 against the antigen only. All 25 patients with cystitis had low serum antibody levels by IFA and RIA when tested against their own isolate as well as enterobacterial common antigen. The RIA procedure was objective, quantitative, and less tedious to perform than IFA.     

THE INTERACTION OF RESPONSES IN THE BRAIN TO SEMANTIC STIMULI

Long time-constant EEG recording during paired stimuli has led to the discovery of the contingent negative variation or expectancy wave (Walter, 1964). This effect is produced when a conditional stimulus signals that an imperative stimulus demanding action, decision, or attention will follow at a short, constant time interval. Symbolic and meaningful stimuli were presented to subjects tachistoscopically, and the evoked responses in the brain were electronically averaged. The cerebral evoked responses to such psychological stimuli are more complex than to flashes. A slow negative DC potential shift (CNV) was seen during the interval between an auditory ready signal and the visual exposure if recognition of the stimulus was required, or if it was interesting. Following the visual exposure, a slow positive DC shift occurred. The method has been developed to study the brain responses to psychological stimuli. The amplitude of the responses relates to the information content and subjective factors rather than to the physical strength of the stimulus.