Functional alterations induced by fusarium T-2 toxin in Madin-Darby bovine Kidney (MDBK) and primary fetal bovine Kidney (PFBK) cultures

We recently reported that primary fetal bovine Kidney (PFBK) cells were consistently more sensitive to the cytotoxic effects of fusarium T-2 toxin than Madin-Darby bovine kidney (MDBK) cells in culture. The present report examined the influence of T-2 on selected biochemical parameters of these two culture types. T-2 toxin inhibited incorporation of labeled thymidine, uridine, and leucine in both culture types; at lower concentrations of the toxin, PFBK cells were affected to a greater extent than MDBK cells. T-2 toxin inhibited both the transport of thymidine as well as thymidine incorporation into macromolecules in MDBK cells during initial periods, but did not affect uridine incorporation. The cellular enzymes, K⁺- dependent phosphatase and succinic dehydrogenase were inhibited in MDBK but not in PFBK cultures; acid phosphatase was not influenced in either culture types. In a cell-free system none of the above enzymes were affected by T-2 until the toxin concentration exceeded 10^?5M.     

Effect of ganglionic stimulating and blocking agents on the fast components of colonic myoelectrical activity in the rat

The presence of two types of fast myoelectrical activities, medium fast activity and fast activity, has been demonstrated previously in the electromyogram of colon in normal children and in the rat by the authors. An absence of medium fast activity in Hirschsprung's disease and in experimental aganglionosis of colon in the rat has also been described. In the present study the fast components of colonic myoelectrical activity were analysed during the procedures affecting ganglionic transmission. It was observed that ganglionic stimulants, such as balloon inflation, and intra-arterial injections of acetylcholine and small amounts of nicotine, increased the spike activity and the frequency of medium fast activity without affecting fast activity. The intra-arterial injections of ganglionic blocking agents, such as nicotine in large amounts and pentolinium tartrate, completely abolished the medium fast activity. These observations suggest that the ganglionic activity is responsible for the genesis of medium fast activity and that the absence of cholinergic ganglionic transmission is the most important single factor for the reported altered electromyogram pattern in aganglionosis.     

Isolation of heparin-binding growth factors from bovine, porcine and canine hearts

Fresh bovine, porcine and canine hearts were homogenized and mitogens for mesoderm-derived cells were purified in three different steps. Extraction by two different ammonium sulfate precipitations was followed by cation-exchange chromatography and by heparin-Sepharose affinity chromatography. A heparin-Sepharose fraction from heart (eluted at 1.1 M NaCl) increased mitotic activity in serum-deprived cultures of porcine aortic endothelial and smooth muscle cells, and in human fibroblasts. This mitogenic activity is potentiated by heparin and inhibited by gamma-interferon. The heart mitogenic fraction showed one double peak on HPLC at A215 and one polypeptide band on SDS/PAGE. These peaks and bands were identical to those obtained from bovine brain. The heart acidic fibroblast growth factor (aFGF) showed a positive signal in Western blots using antibodies raised against brain aFGF. Gas-phase amino acid sequencing established that the mitogens were identical to aFGF and the N-terminally truncated aFGF. Extraction in the presence of a protease inhibitor (pepstatin A) produced a higher-molecular mass form of aFGF with a blocked amino terminus. Another mitogen, eluted at 1.6 M NaCl from heparin-Sepharose, reacted with polyclonal antiserum against human recombinant basic fibroblast growth factor (bFGF) and showed a 66% (12 from 18 amino acids determined by gas-phase sequencing) similarity with bFGF. This polypeptide increased the mitotic activity of the same cell lines but was more potent than aFGF.     

Mercaptan and dicarboxylate inhibitors of hamster dihydroorotase

In mammals, dihydroorotase is part of a trifunctional protein, dihydroorotate synthetase, which catalyzes the first three reactions of de novo pyrimidine biosynthesis. Dihydroorotase catalyzes the formation of a peptide-like bond between the terminal ureido nitrogen and the beta-carboxyl group of N-carbamyl-L-aspartate to yield heterocyclic L-dihydroorotate. A variety of evidence suggests that dihydroorotase may have a catalytic mechanism similar to that of a zinc protease [Christopherson, R. I., & Jones, M. E. (1980) J. Biol. Chem. 255, 3358-3370]. Tight-binding inhibitors of the zinc proteases, carboxypeptidase A, thermolysin, and angiotensin-converting enzyme have been synthesized that combine structural features of the substrates with a thiol or carboxyl group in an appropriate position to coordinate a zinc atom bound at the catalytic site. We have synthesized (4R)-2-oxo-6-thioxohexahydropyrimidine-4-carboxylate (L-6-thiodihydroorotate) and have found that this analogue is a potent competitive inhibitor of dihydroorotase with a dissociation constant (Ki) in the presence of excess Zn2+ ion of 0.17 +/- 0.02 microM at pH 7.4. The potency of inhibition by L-6-thiodihydroorotate in the presence of divalent metal ions decreases in the order Zn2+ greater than Ca2+ greater than Co2+ greater than Mn2+ greater than Ni2+; L-6-thiodihydroorotate alone is less inhibitory and has a Ki of 0.85 +/- 0.14 microM. 6-Thioorotate has a Ki of 82 +/- 8 microM which decreases to 3.8 +/- 1.4 microM in the presence of Zn2+. Zn2+ alone is a moderate inhibitor of dihydroorotase and does not enhance the potency of other inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton nuclear magnetic resonance characterization of the aromatic residues in the variant-3 neurotoxin from Centruroides sculpturatus Ewing

The amino acid sequence for the variant-3 (CsE-v3) toxin from the venom of the scorpion Centruroides sculpturatus Ewing contains eight aromatic residues. By use of 2D NMR spectroscopic methods, the resonances from the individual protons (NH, C alpha H, C beta H',H", and the ring) for each of the individual aromatic residues have been completely assigned. The spatial arrangement of the aromatic ring systems with respect to each other has been qualitatively analyzed by 2D-NOESY techniques. The results show that Trp-47, Tyr-4, and Tyr-42 are in close spatial proximity to each other. The NOESY contacts and the ring current induced shifts in the resonances of the individual protons of Tyr-4 and Trp-47 suggest that the aromatic ring planes of these residues are in an orthogonal arrangement. In addition, the spatial proximity of the rings in the pairs Tyr-4, Tyr-58; Tyr-42, Tyr-40; and Tyr-40, Tyr-38 has also been established. A comparison with the published crystal structure suggests that there is a minor rearrangement of the aromatic rings in the solution phase. No 2D-NOESY contacts involving Phe-44 and Tyr-14 to any other aromatic ring protons have been observed. The pH dependence of the aromatic ring proton chemical shifts has also been studied. These results suggest that the Tyr-58 phenolic group is experiencing a hydrogen-bonding interaction with a positively charged group, while Tyr-4, -14, -38, and -40 are experiencing through-space interactions with proximal negatively charged groups. The Trp-47 indole NH is interacting with the carboxylate groups of two proximal acidic residues. These studies define the microenvironment of the aromatic residues in the variant-3 neurotoxin in aqueous solution.     

Clinical and experimental mycotic keratitis caused by Aspergillus terreus and the effect of subconjunctival oxiconazole treatment in the animal model

Aspergillus terreus was isolated from a case of Keratomycosis. The patient, a 50 year old, female presented with a large corneal ulcer with hypopyon. The direct microscopic examination of the scrapings revealed hyaline, thin, septate and branched hyphae. In vitro some antimycotics (amphotericin B,5-fluorocytosine, oxiconazole, amorolfine and ketoconazole) were tested against A. terreus by agar dilution method. Ketoconazole with MIC of 3 gmg/ml after 7 days of incubation was most effective followed by oxiconazole (10g/ml). Experimental corneal ulcer was produced by injecting intralamellary 0.1 ml of the spore suspension containing 10×10₆ cfu/ml into the eyes of previously immunocompressed albino rabbits. Histopathologic examination showed infiltration and large destruction of the corneal stroma. Subconjunctival oxiconazole therapy exhibited complete cure. Based on our findings, a clinical evaluation of oxiconazole in human keratomycosis seems to be justified.     

Genetic evidence for α2-adrenergic receptor subtypes in rat brain,heart and adrenal gland

Summary A complementary DNA (cDNA) clone - cA₂-47 - corresponding to a new ₂-adrenergic receptor subtype has been isolated from a rat brain cDNA library and used as a hybridization probe to scrutinize the ₂-receptor poly(A⁺) RNAs in rat brain, heart and adrenal gland. Hybridization of the 5 half of the coding region of this cDNA at 37°C to rat brain poly(A+) RNA revealed a single band at 5.8 kb as the size of its corresponding mRNA. Under identical hybridization conditions, a human platelet ₂-receptor genomic probe failed to hybridize to any rat brain mRNAs.Under lower stringency conditions, hybridization of the full-length cDNA, cA₂-47, to selected rat tissue poly(A⁺) RNA showed the presence of four different sized mRNAs in brain and three in both heart and adrenal gland. Messages of 1.3 kb and 2.1 kb were common in all three tissues (although the band at 2.1 kb was slightly higher in the heart and adrenal gland). A 5.8 kb mRNA was unique to the brain and a slightly higher band at 6.0 kb was consistently present in heart and adrenal gland but was absent in the brain. A fourth message at 3.4 kb was found predominantly in the brain and was either absent or present at very low levels in the other tissues examined. Under the same conditions, a human platelet ₂-receptor probe hybridized to similar sized messages of 2.1 and 5.8 kb in rat brain and 2.2 and 6.0 kb in rat heart and adrenal gland. This probe, however, failed to detect the abundant 1.3 kb mRNA common to all tissues or the 3.4 kb message in rat brain. The extent of homology of these messages with cA₂-47 is not confined to limited regions of the cDNA since similar hybridization patterns were observed using either 5-noncoding or 5-coding regions of the probe.These results provide the first direct evidence of a surprisingly large range of mRNA sizes for members of the ₂-receptor family in brain, heart, and adrenal gland. The unique nature of certain members of the family in each of the tissues examined raises the curious possibility that these members might contribute to some of the individualized functions of the brain, cardiovasculature and adrenal gland.     

Search for chromosomal variations among gas-exposed persons in Bhopal

Summary A chromosomal survey using standard lymphocyte cultures employing different media and G-banding techniques was initiated in 1984. This study became particularly important following the tragic gaseous exposure of the population in Bhopal at midnight on 2 December 1984. We have been able to formulate a chromosomal profile for each person whom we have studied; during 1986–1988, 154 persons were examined twice. Among seemingly normal individuals, as many as 20% might possess some chromosomal abnormality; of these, 50% may develop, at a later date, some kind of pathological complication (such as tumours, recurrent abortion or transmission of defects to their offspring). The people exposed to methyl isocyanate have repeatedly shown Robertsonian translocations, mostly in acrocentric chromosomes 13 and 21. Other types of translocations have been studied among all exposed (53) and normal (101) persons; the involvement of chromosomes 5, 9, 11, 14 and 16 is statistically significant (P= <0.001). One of the major clinical symptoms is dyspnoea; we have estimated that almost all seriously dyspnoeic patients have developed at least two categories of chromosomal aberrations, one of which is Robertsonian translocation, in at least 10% metaphases. Our chromosomal survey will be of significance because we are able to identify people with chromosomal aberrations that might be correlated with future pathological consequences of the accident. The chromosomal load that can be sustained with an apparently normal phenotype can also be measured.