Genetic Linkage and Cotransfer of a Novel, vanB-Containing Transposon (Tn5382) and a Low-Affinity Penicillin-Binding Protein 5 Gene in a Clinical Vancomycin-Resistant Enterococcus faecium Isolate

Mechanisms for the intercellular transfer of VanB-type vancomycin resistance determinants and for the almost universal association of these determinants with those for high-level ampicillin resistance remain poorly defined. We report the discovery of Tn5382, a ca. 27-kb putative transposon encoding VanB-type glycopeptide resistance in Enterococcus faecium. Open reading frames internal to the right end of Tn5382 and downstream of the vanX_B dipeptidase gene exhibit significant homology to genes encoding the excisase and integrase of conjugative transposon Tn916. The ends of Tn5382 are also homologous to the ends of Tn916, especially in regions bound by the integrase enzyme. PCR amplification experiments indicate that Tn5382 excises to form a circular intermediate in E. faecium. Integration of Tn5382 in the chromosome of E. faecium C68 has occurred 113 bp downstream of the stop codon for the pbp5 gene, which encodes high-level ampicillin resistance in this clinical isolate. Transfer of vancomycin, ampicillin, and tetracycline resistance from C68 to an E. faecium recipient strain occurs at low frequency in vitro and is associated with acquisition of a 130- to 160-kb segment of DNA that contains Tn5382, the pbp5 gene, and its putative repressor gene, psr. The interenterococcal transfer of this large chromosomal element appears to be the primary mechanism for vanB operon spread in northeast Ohio. These results expand the known family of Tn916-related transposons, suggest a mechanism for vanB operon entry into and dissemination among enterococci, and provide an explanation for the nearly universal association of vancomycin and high-level ampicillin resistance in clinical E. faecium strains.     

Inhibition of Vancomycin-Resistant <Emphasis Type="Italic">Enterococcus</Emphasis> by Continuous-Flow Cultures of Human Stool Microflora With and Without Anaerobic Gas Supplementation

A continuous-flow competitive exclusion (CFCE) culture model of human stool microflora was used to examine whether supplemental anaerobic gas is necessary for maintenance of anaerobes and inhibition of vancomycin-resistant Enterococcus (VRE). CFCE cultures of human stool microflora were maintained with supplemental nitrogen, without supplemental nitrogen, or with percolated room air. Cultures with or without supplemental nitrogen maintained >9 log₁₀ CFU mL^–1 of obligate anaerobes and eliminated 10⁶ CFU mL^–1 of VRE. When room air was percolated into the culture, anaerobes were detected at 2 log₁₀ CFU mL^–1, and the same VRE inoculum was not eliminated (P < 0.001). These data demonstrate that human stool CFCE cultures maintain high levels of obligate anaerobes and inhibit VRE without the addition of supplemental anaerobic gas.     

Genome-wide linkage analysis replicates susceptibility locus for fasting plasma triglycerides: NHLBI Family Heart Study

Recent reports implicate chromosomal regions linked to inter-individual variation in plasma triglycerides. We conducted genome-wide scans to replicate these linkages and/or identify other loci influencing plasma triglycerides in the NHLBI Family Heart Study (FHS). Data were obtained for 501 three-generational families. Genotyping was done by the Utah Molecular Genetics Laboratory and NHLBI Mammalian Genotyping Service; markers from both were placed on one genetic map. Analysis was done using multipoint variance components linkage. Fasting plasma triglycerides were log-transformed and age-, sex-, and field center-adjusted; suggestive linkage evidence was found on chromosome 8 (LOD=2.80 at 89 cM, marker D8S1141). Further adjustment for waist girth, BMI, diabetes, hypertension, and lipid-lowering drugs suggested linkage regions on chromosomes 6 (LOD=2.29 at 79 cM, marker D6S295) and 15 (LOD=1.85 at 43 cM, marker D15S659). Since HDL is correlated with triglycerides and because it was linked to this region on chromosome 15 in FHS, we created a composite triglyceride–HDL phenotype. The combined phenotype LOD score was 3.0 at the same marker on chromosome 15. Chromosome 15 likely harbors a susceptibility locus with an influence on triglycerides and HDL. Regions on chromosomes 6 and 8 may also contain loci contributing to inter-individual variation in plasma triglycerides.     

The circular dichroism of tumor necrosis factor-α: Measurement into the vacuum UV and analysis for secondary structure

The circular dichroism (CD) spectrum of tumor necrosis factor-α has been measured into the vacuum UV to 168 nm. Analysis of the CD for secondary structure is in good agreement with X-ray diffraction results, but the analysis is somewhat unstable. Adding the CD of this protein together with its X-ray determined secondary structure to the basis set should improve subsequent analyses of CD spectra for other all-β proteins.     

A large, dominant pedigree of atrioventricular septal defect (AVSD): exclusion from the Down syndrome critical region on chromosome 21.

We describe a large pedigree of individuals with autosomal dominant atrioventricular septal defect (AVSD). The pedigree includes affected individuals and individuals who have transmitted the defect but are not clinically affected. AVSDs are a rare congenital heart malformation that occurs as only 2.8% of isolated cardiac lesions. They are the predominant heart defect in children with Down syndrome, making chromosome 21 a candidate for genes involved in atrioventricular septal development. We have carried out a linkage study in the pedigree by using 10 simple-sequence polymorphisms from chromosome 21. Multipoint linkage analysis gives lod scores of less than -2 for the region of trisomy 21 associated with heart defects, which excludes a locus within this region as the cause of the defect in this family.     

TRANSVERSE ELECTRIC IMPEDANCE OF NITELLA

Alternating current measurements have been taken on single Nitella cells over a frequency range from 30 to 2,500,000 cycles per second with the current flow perpendicular to the axis of the cell. The measuring cells were so constructed that electrolytes of any desired concentration could be circulated during the course of the measurements. The cellulose wall which surrounds the cell is found to play an important part in the interpretation of the results obtained. In a mature cell, this cellulose has a specific resistance of about 1000 ohm cm. which is independent of the medium in which the cell is suspended. The thickness of the wall is computed to be about 10 µ. The cell membrane is found to be virtually non-conducting, and to have a capacity of 0.94 µf./cm.² ± 10 per cent and a phase angle of 80° ± 4°. The specific resistances of the sap were difficult to compute from data on living cells and were unsatisfactory because they were very much dependent upon the medium, while measurements on extracted sap gave 58 ohm cm. ± 8 per cent which was independent of the medium. There are indications that the chloroplasts have impedance properties similar to those of living cells.     

Growth and senescence in plant communities exposed to elevated CO2 concentrations on an estuarine marsh

Summary Three high marsh communities on the Chesapeake Bay were exposed to a doubling in ambient CO₂ concentration for one growing season. Open-top chambers were used to raise CO₂ concentrations ca. 340 ppm above ambient over monospecific communities of Scirpus olneyi (C₃) and Spartina patens (C₄), and a mixed community of S. olneyi, S. patens, and Distichlis spicata (C₄). Plant growth and senescence were monitored by serial, nondestructive censuses. Elevated CO₂ resulted in increased shoot densities and delayed sensecence in the C₃ species. This resulted in an increase in primary productivity in S. olneyi growing in both the pure and mixed communities. There was no effect of CO₂ on growth in the C₄ species. These results demonstrate that elevated atmospheric CO₂ can cause increased aboveground production in a mature, unmanaged ecosystem.     

The reliability of the ankle-brachial index in the Atherosclerosis Risk in Communities (ARIC) study and the NHLBI Family Heart Study (FHS)

Background

Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro.

Methods

To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin^® and the polycation transduction enhancer Transfectam^®. The EGFP-positive transduced cells were then enriched by flow cytometry.

Results

More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes.

Conclusion

The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients' own skeletal myoblasts.