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. burgdorferi 23S rRNA. The technique is rapid and does not require any enzymatic amplification steps. With the use of a cocktail containing five different probes, approximately 1,000 organisms could be detected. The assay was successfully applied to direct detection of B. burgdorferi in Ixodes scapularis Say nymphs.

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. Expression of p53 was detected immunocytochemically using the AB-6 mAb, which reacts with the product of the wild-type p53 tumor suppressor gene and with most of its mutated forms. Exposure of normal lymphocytes to 5 microM nitrogen mustard caused their arrest in G1, an increase in p53 expression which was maximal in such cells, and significant apoptosis in cells located beyond the arrest point (S and G2 + M cells). In contrast, neither arrest in G1 nor significant apoptosis of MOLT-4 cells was seen after administration of either 0.5 or 5 microM nitrogen mustard for up to 24 h, although the drug reduced the rate of cell progression in the S-phase at both concentrations. Expression of p53 was highest for S and G2 + M MOLT-4 cells in response to the nitrogen mustard. Although a severalfold lower level of p53 was detected in lymphocytes compared to MOLT-4 cells prior to drug treatment, the relative increase in p53 expression in response to the drug was 2-fold higher in lymphocytes. These data suggest that DNA damage caused by nitrogen mustard provides a signal that results in stabilization of wild-type p53, preferentially in G1 cells, causes cell arrest in G1, and induces apoptosis of the cells that either were in the S-phase at the time of drug administration and/or escaped G1 arrest. The increase in expression of mutated p53, in response to DNA damage, is unrelated to the cell cycle position, and neither provides a signal for cell arrest in G1 nor a trigger for immediate apoptosis.

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. One of the two other dA-dT-rich sequences (element C), located proximal to initiation site (+1), serves as the basal promoter while the distal A/T rich element B participates in tissue specific transcription of the gene.

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. The enhanced Mono binding was time and LDL concentration dependent. LDL-induced binding was reduced to control levels when cycloheximide was added together with LDL, indicating that de novo protein synthesis was required. Furthermore, this LDL effect was not a general feature of apolipoproteins, as high density lipoprotein in physiologically relevant concentrations (45 mg Chol/dl, 4 days) had no effect on EC-Mono binding. Conditioned media from LDL-treated EC cultures did not increase EC binding of Monos. In contrast, minimally modified LDL increased EC-Mono binding more than eightfold. In conclusion, LDL in concentrations associated with the premature development of atherosclerosis increased EC affinity for Monos. Such LDL-induced alterations in EC physiology likely represent a proinflammatory response and an early step in atherogenesis.

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., Hartwig, R., Banerjee, D., and Redman, C. (1990) Biochim. Biophys. Acta 1048, 202-208), resulted in the secretion of complete fibrinogen into the media and the formation of four additional intracellular complexes which we also showed to be present in the hepatocyte cell line Hep 3B. The complexes, which have Mr = 232, 150, 135, and 128 (x 10(-3) conform with the Mr expected for A alpha B beta gamma 2, B beta gamma 2 and gamma 3, respectively. A A mechanism of assembly is proposed based on the assumption that all these complexes are precursors of complete fibrinogen. Each of the expressed fibrinogen chains in transfected COS cells interacts noncovalently with binding protein (BiP, GRP 78), but not to the same extent; gamma chain binds less BiP than the A alpha and B beta chains. Assembly of fibrinogen is not absolutely required for its secretion. In addition to complete fibrinogen, the conditioned media of hepatocytes and of transfected COS cells contained free A alpha, free gamma, and two of the above-mentioned complexes, A alpha gamma 2 and A alpha B beta gamma 2.

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 1.5 hours, c-myc mRNA did not begin to increase until 1 hour after vascular injury. Levels of c-myc peaked at 2 hours and were sustained for an additional 4 hours before gradually declining. Smooth muscle cells derived from enzyme-treated control aortae that did not undergo BDE expressed c-fos and c-jun, but showed no evidence of c-myc message. In contrast, nonenzymatically treated, non-BDE whole aortae (containing both media and adventitia) demonstrated a prominent c-myc signal, but failed to express c-fos and c-jun. Corresponding examination of adventitia derived from enzyme-treated aortae showed this tissue to be a source of all three proto-oncogenes. The results of this study demonstrate the earliest in vivo molecular markers of vascular injury reported to date and implicate SMC proto-oncogene expression in the initiation of SMC proliferation. Furthermore these findings suggest two avenues for proto-oncogene induction, that are due to (1) vessel wall manipulation and (2) humoral stimulation.

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. Treatment with endoglycosidase H showed that non-secreted B beta chain contains mannose-rich carbohydrates rather than the complex form of carbohydrate which occurs in plasma fibrinogen and indicates that B beta chain is not transported to the Golgi apparatus. In transfected COS cells, antibody to fibrinogen co-immunoprecipitated B beta chain and 78 kDa immunoglobulin-binding protein (BiP) and antibody to BiP immunoprecipitated BiP and nascent B beta chains. Non-secreted B beta chain was degraded intracellularly with a half-life of 5 h by enzymes which were not affected by incubating transfected cells with NH4Cl, which indicates a non-lysosomal pathway of degradation. These studies indicate that B beta chain by itself does not contain the signal for fibrinogen secretion and that non-secreted B beta chain is associated with BiP and degraded in the rough endoplasmic reticulum.

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. A TATA-like sequence (TATTTTTA) and a CAAT-box (CAAAAGT) are located at positions -32 and -59, respectively, which most likely constitute the functional promoter region in the gene. Based on primer extension reaction with a synthetic 20-mer corresponding to the 5'-leader sequence and total poly(A+) RNA, the probable transcription initiation site in the gene was located. The gene promoter activity was demonstrated following transient expression of recombinant genomes containing the chicken upstream sequence fused to the bacterial chloramphenicol acetyltransferase (CAT) or to the rat preproinsulin II genes. The extracts from a Quail fibroblast cell line (QT35) transfected with the construct (pLCo5.2iCat) containing the putative chicken promoter, and the CAT gene promoted the formation of 3'-acetate chloramphenicol. Another construct (pBC12LC5.2f) contains the rat preproinsulin II gene placed under the control of chicken promoter and a simian virus 40 origin of replication. Transfection of COS cell line with pBC12LC5.2f DNA resulted in an efficient expression of rat preproinsulin mRNA initiating from the chicken promoter. The transfection assay also allowed detection of chicken MLC2A gene transcripts by S1-nuclease protection of end-labeled DNA probes. A comparison of the MLC2A upstream gene sequence with those available for skeletal myosin light chains revealed no common sequence elements, suggesting that cardiac MLC2A gene promoter region has diverged considerably from its counterparts in skeletal muscle.

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. Active-site-blocked thrombin formed the complex only slowly. The complex that formed with active thrombin was not dissociated by hydroxylamine in urea. Reduction with 2-mercaptoethanol dissociated the complex, and its formation was blocked by the sulfhydryl-blocking agents iodoacetamide and 4,4'-dithiodipyridine. The complex was thus unlike those of thrombin and alpha 2-macroglobulin or antithrombin III, but it had characteristics of a disulfide-linked complex. Of the secreted proteins, albumin and glycoprotein G adhered to an activated thiol-Sepharose column, indicating that they contained free thiol groups. Purified glycoprotein G and thrombin formed a complex similar to the complex formed when thrombin was incubated with the supernatant solution of activated platelets. The purified glycoprotein bound 2.6 mol of radioactive N-ethylmaleimide/mol of protein, indicating three sulfhydryl groups per mole. After reacting with purified glycoprotein G, thrombin developed a new sulfhydryl group. It is concluded that glycoprotein G (thrombin-sensitive protein, thrombospondin) and thrombin form a dissociable complex that leads to a covalent complex by thiol-disulfide exchange of a thiol group on glycoprotein G and a disulfide on thrombin.

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. Autoradiography after electrophoresis with sodium dodecyl sulfate indicated that these complexes had apparent masses of 210, 185, 155 and 125 kDa. Formation of the complexes was blocked by hirudin; this is consistent with crosslinking that was a direct consequence of the binding of thrombin to a specific receptor, since hirudin blocks thrombin-induced platelet activation and the saturable binding of thrombin to platelets. The labeled supernatant complex had an apparent mass of about 490 kDa. It also formed in the supernatant solution of platelets after activation with a divalent cation ionophore, suggesting a complex of thrombin with a secreted protein. The supernatant complex did not involve fibrinogen or alpha 2-macroglobulin, but a similar complex was formed with partially purified secreted glycoprotein G (thrombin-sensitive protein, thrombospondin). Formation of the complex was blocked by hirudin. A similar complex was formed after prolonged (1 h) incubation without photoactivation. It is concluded that thrombin forms high-affinity, hirudin-sensitive complexes with secreted glycoprotein G, as well as with platelet surface proteins.