Direct detection of Borrelia burgdorferi in Ixodes scapularis (Acari: Ixodidae) nymphs by hybridization to ribosomal RNA.

A method for direct detection of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner has been developed. Cells are lysed to facilitate release of ribosomal RNA. Lysates are filtered onto nylon membranes that are hybridized with probes specific for sequences in B.

Induction of apoptosis and cell cycle-specific change in expression of p53 in normal lymphocytes and MOLT-4 leukemic cells by nitrogen mustard.

DNA damage in the cell activates expression of the p53 tumor suppressor gene, whose role is associated with cell arrest in G1 or apoptosis. The aim of this study was to examine the cell cycle position-related changes in expression of p53, as well as induction of apoptosis, in mitogen-stimulated normal human lymphocytes and in human leukemic MOLT-4 cells (which express mutated p53), following DNA damage by the alkylating agent nitrogen mustard. Measurement of p53 expression and DNA content by flow cytometry followed by bivariate analysis of the data made it possible to correlate the drug-induced changes in p53 expression in individual cells with their cell cycle position without the need for cell synchronization.

Functional analysis of a CArG-like promoter element in cardiac myosin light chain 2 gene.

We have examined the functional properties of a putative regulatory sequence, CCAAAAGTGG, (element A) in chicken cardiac myosin light chain 2 (MLC2) gene promoter by deletion/substitution mutagenesis and transcriptional analysis of RNA by S1 nuclease mapping. The results indicate that the element A sequence, which resembles the evolutionarily conserved A/T-rich CArG box, plays a role in defining the transcription initiation site in MLC2 gene. This is accomplished via repression of a potential transcription initiation at site -40 and promoting the initiation at +1.

Native low density lipoprotein. Endothelial cell recruitment of mononuclear cells.

The effect of native low density lipoprotein (LDL) on human umbilical vein endothelial cell (EC) recruitment of mononuclear cells (Monos) was investigated. ECs were exposed to LDL at atherogenic concentrations (240 mg cholesterol [Chol]/dl) for as long as 4 days (LDL-treated ECs). LDL-treated ECs bound substantially greater amounts of freshly isolated human monocytes and U937 cells than did control ECs.

Studies on the assembly and secretion of fibrinogen.

cDNAs of fibrinogen A alpha and gamma chains were individually subcloned into a eukaryotic expression vector by using the polymerase chain reaction. Triple cotransfection into COS cells of the two plasmids together with a B beta chain expression plasmid, constructed as described previously (Danishefsky, K.J.

Early proto-oncogene expression in rat aortic smooth muscle cells following endothelial removal.

To study the mechanism(s) of vascular smooth muscle cell proliferation in vivo, mRNA levels of c-fos, c-jun, and c-myc were determined by Northern blot analysis following vascular balloon de-endothelialization (BDE). Medial smooth muscle cells (SMC) were separated and studied by enzymatic digestion of the vessel wall. mRNA levels of c-fos and c-jun from aortic smooth muscle cells (SMC) were simultaneously induced within 30 minutes of BDE and declined to baseline by 1.

Intracellular fate of fibrinogen B beta chain expressed in COS cells.

Full-length fibrinogen B beta cDNA was subcloned into an expression vector, pBC12BI, and transfected into COS cells. B beta chain expression was measured by pulse-labelling cells with L-[35S]methionine, immunoprecipitating the B beta chain with antibody to fibrinogen and separating the nascent radioactive protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). B beta chain was expressed in transfected COS cells but was not secreted into the medium.

Characterization of 5'-flanking region of heart myosin light chain 2A gene. Structural and functional evidence for promoter activity.

Two recombinant clones, lambda LC5 and lambda LC13, encompassing the entire regulatory myosin light chain 2 (MLC2A) gene of chicken heart muscle were isolated. Of these, lambda LC5 which contains a large 5'-flanking sequence of about 7.0 kb, was characterized by a partial nucleotide sequence analysis.

Formation of a stable complex of thrombin and the secreted platelet protein glycoprotein G (thrombin-sensitive protein, thrombospondin) by thiol-disulfide exchange.

When 125I-labeled thrombin was incubated with washed human platelets or with the supernatant solution of activated platelets, it formed a NaDodSO4-stable complex of apparent mass greater than 450 000 daltons. Formation of the complex was temperature dependent; with 20 nM thrombin incubated with the supernatant solution of ionophore-activated platelets, the initial rate of formation of the stable complex was 1 nM thrombin/min at 37 degrees C, 50 times the rate at 22 degrees C. Thrombin with all free amino groups methylated was still reactive.

Photoaffinity labeling of platelet thrombin-binding proteins.

The interaction of thrombin and platelets was studied with a heterobifunctional photoactivable crosslinking agent. Radiolabeled thrombin that was modified with ethyl-N-5-azido-2-nitrobenzoylaminoacetimidate formed two types of complex with platelet proteins: platelet-associated complexes and supernatant complexes. The platelet-associated complexes formed within 20 s.