Chitinolytic activity in Chromobacterium violaceum: substrate analysis and regulation by quorum sensing

Quorum sensing control mediated by N-acyl homoserine lactone (AHL) signaling molecules has been established as a key feature of the regulation of exoenzyme production in many gram-negative bacteria. In Chromobacterium violaceum ATCC 31532 a number of phenotypic characteristics, including production of the purple pigment violacein, hydrogen cyanide, antibiotics, and exoproteases are known to be regulated by the endogenous AHL N-hexanoyl-L-homoserine lactone (HHL). In this study we show that C. violaceum produces a set of chitinolytic enzymes whose production is regulated by HHL. The chitinolytic activity was induced in strains grown in the presence of chitin as the sole carbon source and quantitated in the secreted proteins by using p-nitrophenol analogs of disaccharide, trisaccharide, and tetrasaccharide oligomers of N-acetylglucosamine. By using 4-methylumbelliferyl analogs of the same oligomers of N-acetylglucosamine as substrates for proteins separated and renatured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least six enzymes were detected: a chitobiase with high specificity to a dimeric substrate of 87 kDa, two N-acetylglucosaminidases with apparent molecular masses of 162 and 133 kDa, two endochitinases of 108 and 67 kDa, and a chitobiosidase of 56 kDa. In addition, two unidentified bands of >205 kDa were found where a tetrameric chitin derivative was used as a substrate. A pleiotropic mini-Tn5 mutant of C. violaceum (CV026) that is defective in HHL production and other quorum-sensing-regulated factors was also found to be completely deficient in chitinolytic activity. Growth of this mutant on minimal medium with chitin supplemented with culture supernatant from the C. violaceum wild-type strain or 10 microM synthetic HHL restored chitinase production to the level shown by the parental strain. These results constitute the most complete evidence so far for regulation of chitinolytic activity by AHL signaling in a gram-negative bacterium.     

Polycyclic aromatic hydrocarbon-degrading capabilities of Phanerochaete laevis HHB-1625 and its extracellular ligninolytic enzymes

The ability of Phanerochaete laevis HHB-1625 to transform polycyclic aromatic hydrocarbons (PAHs) in liquid culture was studied in relation to its complement of extracellular ligninolytic enzymes. In nitrogen-limited liquid medium, P. laevis produced high levels of manganese peroxidase (MnP). MnP activity was strongly regulated by the amount of Mn2+ in the culture medium, as has been previously shown for several other white rot species. Low levels of laccase were also detected. No lignin peroxidase (LiP) was found in the culture medium, either by spectrophotometric assay or by Western blotting (immunoblotting). Despite the apparent reliance of the strain primarily on MnP, liquid cultures of P. laevis were capable of extensive transformation of anthracene, phenanthrene, benz[a]anthracene, and benzo[a]pyrene. Crude extracellular peroxidases from P. laevis transformed all of the above PAHs, either in MnP-Mn2+ reactions or in MnP-based lipid peroxidation systems. In contrast to previously published studies with Phanerochaete chrysosporium, metabolism of each of the four PAHs yielded predominantly polar products, with no significant accumulation of quinones. Further studies with benz[a]anthracene and its 7,12-dione indicated that only small amounts of quinone products were ever present in P. laevis cultures and that quinone intermediates of PAH metabolism were degraded faster and more extensively by P. laevis than by P. chrysosporium.     

Syntenic assignment of dopamine tautomerase (DCT) to bovine chromosome 12

Heterologous primers were used to amplify an exon and intron-containing segment of the bovine homologue of the human dopachrome tautomerase gene. After confirmation of homology by sequence analysis (exon sequence similarity greater than 90%), bovine-specific primers were developed for synteny mapping purposes. The dopachrome tautomerase gene was assigned to bovine chromosome 12 (BTA12) with 97% concordance to the coagulation factor 10 locus. Together with previous synteny mapping of bovine chromosome 12 genes, fms-related tyrosine kinase, esterase D and 5-hydroxytryptamine receptor 2, this assignment further indicates conservation between human chromosome 13q and bovine chromosome 12.     

The modulation of cellular responses to poly(2-hydroxyethyl methacrylate) hydrogel surfaces: phosphorylation decreases macrophage collagenase production in vitro

We examined the regulation of collagenase production by the monocyte/macrophage THP-1 cell line when these cells were exposed to poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel surfaces with different chemistries and morphologies. Tissue culture modified polystyrene (TCP), used as a control surface, induced the maximum collagenase response. Copolymer hydrogels containing 2-ethoxyethyl methacrylate (EMA) or methyl methacrylate (MMA) also induced a high response, while PHEMA hydrogels induced a low level response and the phosphorylated hydrogel induced no response. This pattern was altered when the morphology of the hydrogels was changed to that of a sponge. The overall enzyme response to the sponge hydrogels was lower than that to the homogeneous hydrogels. Sponges containing EMA and MMA produced low level response relative to the TCP control. PHEMA and phosphorylated sponges produced little and no response respectively. The dramatically reduced enzyme response to phosphorylated surfaces was not a consequence of cell death, and may be a phenomenon related to changes in cell surface charge.     

Pathologic seminal vesicle invasion after radical prostatectomy for patients with prostate carcinoma: effect of early adjuvant radiation therapy on biochemical control

BACKGROUND: The authors evaluated the effect of postoperative radiation therapy on freedom from biochemical failure (bNED) in men with prostate carcinoma who had pathologic seminal vesicle invasion after radical prostatectomy and negative pelvic lymph node dissection (pT3cN0). METHODS: Between 1989 and 1995, 375 men underwent radical prostatectomy at Thomas Jefferson University Hospital. Fifty-three men (13%) had pT3cN0 prostate carcinoma and were the subject of this analysis. Men in whom prostate specific antigen (PSA) could not be detected were deemed free of biochemical failure. RESULTS: Of the 53 men with pT3cN0 prostate carcinoma, 18 had an elevated PSA immediately after surgery and received salvage radiation therapy (RT). The 3-year bNED rate for this group was only 38%. At 3 months, PSA could not be detected in the other 35 men. Fifteen of those 35 men underwent early adjuvant RT, and the other 20 were observed for biochemical failure. The 3-year bNED rate for the 15 patients treated with immediate adjuvant RT was 86%, compared with 48% for the 20 men who were observed (P = 0.01). CONCLUSIONS: These data suggest that early adjuvant RT for men with pT3cN0 prostate carcinoma and no detectable PSA postoperatively reduces the likelihood of future biochemical failure. Men with pT3cN0 prostate carcinoma and a persistently elevated postoperative PSA level are less likely to benefit from RT and should be considered for systemic therapy.     

Calcium regulation of Ndr protein kinase mediated by S100 calcium-binding proteins

Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases identified as being critical for the regulation of cell division and cell morphology. The regulatory mechanisms that control Ndr activity have not been characterized previously. In this paper, we present evidence that Ndr is regulated by EF-hand calcium-binding proteins of the S100 family, in response to changes in the intracellular calcium concentration. In vitro, S100B binds directly to and activates Ndr in a Ca2+-dependent manner. Moreover, Ndr is recovered from cell lysates in anti-S100B immunoprecipitates. The region of Ndr responsible for interaction with Ca2+/S100B is a basic/hydrophobic motif within the N-terminal regulatory domain of Ndr, and activation of Ndr by Ca2+/S100B is inhibited by a synthetic peptide derived from this region. In cultured cells, Ndr is rapidly activated following treatment with Ca2+ ionophore, and this activation is dependent upon the identified Ca2+/S100B-binding domain. Finally, Ndr activity is inhibited by W-7 in melanoma cells overexpressing S100B, but is unaffected by W-7 in melanoma cells that lack S100B. These results suggest that Ndr is regulated at least in part by changes in the intracellular calcium concentration, through binding of S100 proteins to its N-terminal regulatory domain.     

Sarcoidosis and thyrotoxicosis: A study of five patients

Regional blood flow of the gastrointestinal (GI) tract and cardiac output have been determined in male Sprague-Dawley rats weighing from 30-726 g. The cardiac output (ml/min per kg) was highest in rats weighing 80-100 g. In heavier rats the cardiac output decreased proportionally with the body weight. The gradient of blood flow to the different parts of the GI tract develops step by step. In the weaning period the blood flow (ml/min per g tissue) through the stomach was less than that through the distal parts of the GI tract. However, the blood flow through the small intestine, cecum and large intestine was uniform at this age. In rats weighing 80-100 g the blood flow through both the cecum and large intestine was less than that through the small intestine. The gradient in blood flow through the various segments of small intestine developed last.