Benzo[b]fluoranthene: tumorigenicity in strain A/J mouse lungs, DNA adducts and mutations in the Ki-ras oncogene

The polycyclic aromatic hydrocarbon benzo[b]fluoranthene (B[b]F) is a pervasive constituent of environmental combustion products. We sought to examine the lung tumorigenic activity of B[b]F in strain A/J mice, to study the relationship between formation and decay of B[b]F-DNA adducts and to examine mutations in the Ki-ras proto-oncogene in DNA from B[b]F-induced tumors. Mice were given i.p. injections of 0, 10, 50, 100 or 200 mg/kg body wt and lung adenomas were scored after 8 months. B[b]F induced significant numbers of mouse lung adenomas in a dose-related fashion, with the highest dose (200 mg/kg) yielding 6.95 adenomas/ mouse, with 100% of the mice exhibiting an adenoma. In mice given tricaprylin, the vehicle control, there were 0.60 adenomas/mouse, with 55% of the mice exhibiting an adenoma. Based on dose, B[b]F was less active than benzo[a]pyrene. DNA adducts were analyzed qualitatively and quantitatively by 32P-post-labeling in lungs of strain A/J mice 1, 3, 5, 7, 14 and 21 days after i.p. injection. Maximal levels of adduction occurred 5 days after treatment with the 200 mg/kg dose group, producing 1230 amol B[b]F-DNA adducts/microgram DNA. The major B[b]F-DNA adduct was identified by co-chromatography as trans-9, 10-dihydroxy-anti-11, 12-epoxy-5-hydroxy-9, 10, 11, 12-tetra-hydro-B[b]F-deoxyguanosine. Approximately 86% of the tumors had a mutation in codon 12 of the Ki-ras oncogene, as determined by direct DNA sequencing of PCR-amplified exon 1 and single-stranded conformation polymorphism analysis. Analysis of the Ki-ras mutation spectrum in 25 of 29 B[b]F-induced tumors revealed the predominant mutation to be a G-->T transversion in the first or second base of codon 12, congruous with the DNA adduct data. Our data are consistent with previous reports in mouse skin implicating a phenolic diol epoxide as the proximate carcinogenic form of B[b]F that binds to guanine.     

Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods

Previous studies have indicated that the conventional tests used for the identification of mycobacteria may (i) frequently result in erroneous identification and (ii) underestimate the diversity within the genus Mycobacterium. To address this issue in a more systematic fashion, a study comparing phenotypic and molecular methods for the identification of mycobacteria was initiated. Focus was given to isolates which were difficult to identify to species level and which yielded inconclusive results by conventional tests performed under day-to-day routine laboratory conditions. Traditional methods included growth rate, colonial morphology, pigmentation, biochemical profiles, and gas-liquid chromatography of short-chain fatty acids. Molecular identification was done by PCR-mediated partial sequence analysis of the gene encoding the 16S rRNA. A total of 34 isolates was included in this study; 13 of the isolates corresponded to established species, and 21 isolates corresponded to previously uncharacterized taxa. For five isolates, phenotypic and molecular analyses gave identical results. For five isolates, minor discrepancies were present; four isolates remained unidentified after biochemical testing. For 20 isolates, major discrepancies between traditional and molecular typing methods were observed. Retrospective analysis of the data revealed that the discrepant results were without exception due to erroneous biochemical test results or interpretations. In particular, phenotypic identification schemes were compromised with regard to the recognition of previously undescribed taxa. We conclude that molecular typing by 16S rRNA sequence determination is not only more rapid (12 to 36 h versus 4 to 8 weeks) but also more accurate than traditional typing.     

Comparison of production of choriogonadotropin inhibitory protein, prolactin and insulin-like growth factor binding protein-1 by human decidua in vitro

BACKGROUND: Tissue plasminogen activator (tPA) is elevated in cancer patients and is thought to promote tumor angiogenesis by facilitating endothelial cell migration through plasmin-mediated degradation of extracellular matrix. Due to the presence of an epidermal growth factor (EGF)-finger domain in the tPA A-chain and the existence of an endothelial cell (EC) receptor that binds this domain, it was hypothesized that tPA has a direct receptor-mediated effect on EC proliferation, independent of plasmin. METHODS AND RESULTS: Using cultured canine ECs, tPA (7.25 microg/ml, approximately 107 nM) increased proliferation as much as 50 and 170% in the absence and presence of growth factors, respectively. tPA-induced increases in EC proliferation occurred independent of plasmin generation, as the plasmin inhibitor, aprotinin (10 microg/ml) did not inhibit tPA-induced proliferation. However, tPA-induced proliferation was inhibited dose-dependently to a maximum of 78% using a monoclonal antibody against the tPA EGF-finger domain. This antibody, known to inhibit tPA binding to its receptor, did not inhibit tPA-induced plasmin generation. To investigate the role of potential signal transduction pathways, ECs were exposed to lavendustin A, a tyrosine kinase inhibitor, at 33.5 microM (IC50 for basic fibroblast growth factor). Lavendustin A did not inhibit tPA-induced EC proliferation. However, Rp-cAMP, an inhibitor of cAMP-dependent kinases, specifically inhibited tPA-induced EC proliferation in a dose-dependent manner (IC50 = 50.5 microM). Pertussis toxin at maximal concentrations for this system (0.5 ng/ml) did not inhibit tPA-induced EC proliferation. CONCLUSION: These results lend support to the hypothesis that tPA may have a direct receptor-mediated effect on EC proliferation and that this effect occurs independent of plasmin and may be dependent upon protein kinase A activity.     

Effects of fibinolysis on neurite growth from dorsal root ganglia cultured in two- and three-dimensional fibrin gels

The mechanism of neurite penetration of three-dimensional fibrin matrices was investigated by culturing embryonic chick dorsal root ganglia (DRGs) within fibrin gels, upon fibrin gels, and upon laminin. The length of neurites within three-dimensional matrices of fibrin was decreased in a concentration-dependent manner by agents that inhibited plasmin, e.g. aprotinin, or that inhibited plasminogen activation, e.g., epsilon-aminocaproic acid (EACA), or plasminogen antiserum. In contrast, such agents increased the length of neurites growing out from DRGs cultured upon two-dimensional substrates of fibrin and had no effect on the length of neurites growing out from DRGs cultured upon laminin. Visualization of neurites within three-dimensional fibrin matrices demonstrated that the distance between fibrin strands was much smaller than the diameter of neurites. All these data were consistent with the hypothesis that fibrinolysis localized to the region of the neurite tip is an important mechanism for neurite penetration of a physical barrier of fibrin strands arranged in a three-dimensional matrix.     

The mitochondrial genome integrity gene, MGI1, of Kluyveromyces lactis encodes the beta-subunit of F1-ATPase

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the beta-subunit of the mitochondrial F1-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the gain-of-function phenotype found in mgi1 point mutants. The location of Arg435 in the beta-subunit, as deduced from the three-dimensional structure of the bovine F1-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the beta- and alpha- (MGI2) subunits with the gamma-subunit (MGI5) is likely to be affected by the mutations.     

Amplitude-dependent modulation of brush border enzymes and proliferation by cyclic strain in human intestinal Caco-2 monolayers

Little is known about the effects of repetitive deformation during peristaltic distension and contraction or repetitive villus shortening on the proliferation and differentiation of the intestinal epithelium. We sought to characterize the effects of repetitive deformation of a physiologically relevant magnitude and frequency on the proliferation and differentiation of human intestinal epithelial Caco-2 cells, a common cell culture model for intestinal epithelial biology. Human intestinal epithelial Caco-2 cells were cultured on collagen-coated membranes deformed by -20 kPa vacuum at 10 cycles/minute, producing an average 10% strain on the adherent cells. Proliferation was assessed by cell counting and 3H-thymidine incorporation. Alkaline phosphatase and dipeptidyl dipeptidase specific activity were measured in cell lysates. Since cells at the membrane periphery experience higher strain than cells in the center, the topography of brush border enzyme histochemical and immunohistochemical staining was analyzed for strain-dependence. Cyclic strain stimulated proliferation compared to static cells. Proliferation was highest in the membrane periphery where strain was maximal. Strain also modulated differentiation independently of its mitogenic effects, selectively stimulating dipeptidyl dipeptidase while inhibiting alkaline phosphatase. Strain-associated enzyme changes were also maximal in areas of greatest strain. The PKC inhibitors staurosporine and calphostin C ablated strain mitogenic effects while intracellular PKC activity was increased by strain. The strain-associated brush border enzyme changes were attenuated but not blocked by PKC inhibition. Thus, strain of a physiologically relevant frequency and magnitude promotes proliferation and modulates the differentiation of a well-differentiated human intestinal epithelial cell line in an amplitude-dependent fashion. PKC may be involved in coupling strain to increased proliferation.     

Inhibition of N-methyl-D-aspartate glutamate receptor subunit expression by antisense oligonucleotides reveals their role in striatal motor regulation

N-methyl-D-aspartate (NMDA) glutamate receptors have an established role in the regulation of motor behavior by the basal ganglia. Recent studies have revealed that NMDA receptors are heteromeric assemblies of structurally related subunits from two families: NMDAR1, which is required for channel activity, and NMDAR2A-D, which modulate the properties of the channels. In the rat, the NMDA receptor subunits exhibit anatomically restricted patterns of expression, so that each component of the basal ganglia has a distinct NMDA receptor subunit mRNA phenotype. We have used in vivo intrastriatal injection of synthetic antisense oligodeoxynucleotides (ODNs) to examine the roles of particular NMDA receptor subunits in the regulation of motor behavior in rats. Injection of 15 nmol of a 20-mer ODN targeted to the NMDAR1 subunit induced spontaneous ipsilateral rotation. Smaller doses of NMDAR1 antisense ODN did not lead to spontaneous rotation, but prominent ipsilateral rotation was observed after systemic administration of D-amphetamine. An antisense ODN to NMDAR2A was also effective in eliciting amphetamine-inducible rotation, although the magnitude of the effect was less than that seen with NMDAR1, whereas ODNs targeted to NMDAR2B, NMDAR2C and an NMDAR1 sense strand ODN had no effect on behavior. In situ hybridization demonstrated that injection of the NMDAR1, NMDAR2A or NMDAR2B antisense ODNs produced specific reductions in target mRNA signal intensity in the injected striatum. After NMDAR1 antisense ODN injection, striatal binding of 3H-glutamate target mRNA signal intensity in the injected striatum. After NMDAR1 antisense ODN injection, striatal binding of 3H-glutamate to NMDA sites was not altered, although strychnine-insensitive 3H-glycine binding sites exhibited a small but significant reduction. These observations suggest that NMDA receptor complexes containing NMDAR1 and, to a lesser extent, NMDAR2A subunits play particularly important roles in the regulation of motor behavior by neostriatal neurons.     

Lorazepam-valproate interaction: studies in normal subjects and isolated perfused rat liver

Valproate (VPA) has been shown to interact with all the major antiepileptic drugs (AEDs) through two mechanisms of action: displacement from albumin binding sites and inhibition of drug metabolism. More recently, evidence showed that VPA inhibits the elimination of drugs metabolized by glucuronide conjugation. Lorazepam (LZP), which is primarily eliminated by conjugation with glucuronic acid, is administered concurrently with VPA both in treatment of epilepsy and in patients treated with VPA for psychiatric disorders. Therefore, a significant drug interaction is likely. We investigated such interaction both in in vitro isolated perfused rat liver (IPRL) and in normal subjects. LZP [2 mg, intravenous (i.v.) bolus] was administered to 8 normal volunteers before and after chronic dosing with VPA. In 6 of 8 subjects, VPA significantly decreased LZP plasma clearance by an average of 40% (p < 0.05) and increased LZP concentrations by decreasing formation clearance of the LZP glucuronide. In the IPRL studies, VPA also significantly decreased formation of LZP glucuronide (from 0.72 +/- 0.14 to 0.22 +/- 0.15 ml/h/kg, p < 0.05), indicating that IPRL is a useful tool for evaluation of the effect of VPA on drugs eliminated by glucuronide conjugation.     

Prosthetic valve endocarditis: superiority of surgical valve replacement versus medical therapy only

The objective of our study was to assess the long-term outcome of patients with prosthetic valve endocarditis. We used a multicenter, prospective, observational study design. Six university teaching hospitals with high volume cardiothoracic surgery participated. Seventy-four patients with prosthetic valve endocarditis as defined by explicit, objective criteria were selected for participation. All patients were followed up prospectively for 1 year. Thirty-one percent and 69% had development of endocarditis within 60 days of valve insertion ("early") and after 60 days ("late"), respectively. The most common causes were Staphylococcus epidermidis (40%), Staphylococcus aureus (20%), streptococcal species (18%), and aerobic gram-negative bacilli (11%). Physical signs of endocarditis (new or changing murmur, stigmata, emboli) were seen in 58%. At 6 months and 12 months, mortality was 46% and 47%, respectively. Surgical replacement of the infected valve led to significantly lower mortality (23%) as compared with medical therapy alone (56%), as assessed by both univariate and multivariate analyses (p < 0.05). Improved outcome was seen for the surgical group even when controlling for severity of illness at time of diagnosis. From these findings we conclude that accurate assessment of outcome in prosthetic valve endocarditis requires long-term follow-up of at least 6 months following diagnosis. Surgical therapy warrants greater scrutiny; evaluation in controlled clinical trials is appropriate.     

Detailed active site configuration of a new crystal form of methanol dehydrogenase from Methylophilus W3A1 at 1.9 A resolution

The three-dimensional structure of a new crystal form of methanol dehydrogenase from Methylophilus W3A1 has been obtained in the presence of substrate using data recorded at a synchrotron. The structure of this approximately 140 kDa heterotetramer, refined at 1. 9 A resolution, reveals the detailed configuration of its redox cofactor, pyrroloquinoline quinone (PQQ). C4, one of the oxygen-bearing atoms of this orthoquinone is in a planar configuration while C5, which bears the other quinone oxygen, is tetrahedral, suggesting that the PQQ is in the semiquinone redox state. The substrate binding site has been identified close to PQQ and to the side chain of Asp297, the putative active site base. The proximity of the hydroxyl of methanol to C5 of PQQ compared to the greater separation of the substrate methyl group from C5 supports the addition-elimination reaction mechanism involving a hemiketal intermediate.