Temperature-Light Intensity Effect on the Antagonism of Species of Cladosporium to Melampsora larici-populina on Cultivars of Populus X euramericana (Dode) Guinier

The antagonistic and colonistic effect of post-inoculation temperature and light intensity was investigated in a factorial experiment involving four Cladosporium spp., three poplar cultivars, two levels of temperature and light intensity, anda race of Melampsora larici-populina. The species of Cladosporium were more antagonistic to uredinial production on the inoculated leaf discs of the cultivars when these were incubated in high temperature – high light intensity than in any other regime. Whilst colonisation of the uredinia was usually minimal in discs incubated on the high temperature – high light intensity regime, the condition of incubation favouring maximum colonisation varied with the cultivar and the species of Cladosporium employed. All the major factors and most of their two-, three- and four-way interactions were significant determinants of variation in both antagonism to uredinial production and in colonisation of the uredinia produced. The validity ofextrapolation from such experiments to the field environment is discussed.     

Reaction of nitric oxide with heme proteins and model compounds of hemoglobin

Rates for the reaction of nitric oxide with several ferric heme proteins and model compounds have been measured. The NO combination rates are markedly affected by the presence or absence of distal histidine. Elephant myoglobin in which the E7 distal histidine has been replaced by glutamine reacts with NO 500-1000 times faster than do the native hemoglobins or myoglobins. By contrast, there is no difference in the CO combination rate constants of sperm whale and elephant myoglobins. Studies on ferric model compounds for the R and T states of hemoglobin indicate that their NO combination rate constants are similar to those observed for the combination of CO with the corresponding ferro derivatives. The last observation suggests that the presence of an axial water molecule at the ligand binding site of ferric hemoglobin A prevents it from exhibiting significant cooperativity in its reactions with NO.     

Age-dependent regulation of glucocorticoid receptors in the liver of male rats

Specific binding of [3H]dexamethasone to cytosol and the activation of bound hormone-receptor complexes were studied in the liver of immature (3 weeks old) and mature (26 weeks old) Long-Evans male rats. The concentration of specific binding sites was significantly higher (33%) in the liver of immature rats as compared to mature, while dissociation constants (Kd) remain unaltered at both ages. Heat activation (for 45 min at 25 degrees C) significantly enhances the binding of [3H]dexamethasone-receptor complexes to DNA-cellulose and purified nuclei at both the ages, with a greater magnitude in mature rats. Cross mixing experiments (i.e., binding of activated cytosol from mature rats to nuclei of immature and vice-versa) show receptor specificity. Ca2+ activation (20 mM Ca2+ for 45 min at 0 degree C) also enhances the nuclear and DNA-cellulose binding at both the ages, but to a similar extent. Differences in the number of specific binding sites and some of the physiochemical properties of glucocorticoid receptors presented here between immature and mature rats may underlie the functional changes in tissue response with age.     

Extracellular enzymes of mycobacteria

Abstract Extracellular enzymes were studied in different mycobacteria using a plate substrate assay. All the pathogenic mycobacteria included in the study showed the presence of protease, while lipase, ribonuclease, mucinase and β-lactamase could also be detected in some strains. In contrast, no protease was detected in the 3 saprophytic mycobacteria studied. DNase was not detected in any of the species studied. Thus, the demonstration of extracellular enzymes, in particular of protease, in mycobacteria may be relevant in understanding their role in pathogenicity.     

A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups

A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986) J. Biochem. Biophys. Methods 12, 349-354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5'-O-(4,4'-dimethoxytrityl)-thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4,4'-dimethoxytrityl cation into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption at 498 nm (epsilon 498 = 70,000), is then determined spectrophotometrically. A comparative study of DTDS, N-succinimidyl-3-(2-pyridyldithio)propionate and 4,4-dimethoxytrityl chloride is also included. The method was found to be very useful to determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionalization of solid supports for affinity chromatography and synthesis of biopolymers.     

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)