Enhancement of carotenoid synthesis by fungal metabolites

Carotenoids, mainly the all-trans--carotene, may be produced by the fungus Phycomyces blakesleeanus under normal fermentation conditions. A number of chemical compounds that stimulate or inhibit fungal carotenogenesis have been reported earlier. A procedure for the screening of organisms that enhance carotenogenesis on co-culturing with P. blakesleeanus is described. A water-soluble stimulator of carotenogenesis was obtained from the fermentation broth of an unidentified Aspergillus sp. isolated from soil. The crude preparation appears to be ten times more potent than dimethylphthalate.     

Probing pH and pressure effects on the apomyoglobin heme pocket with the 2''-(N,N-dimethylamino)-6-naphthoyl-4-trans-cyclohexanoic acid fluorophore.

The environmentally sensitive fluorophore 2'-(N,N-dimethylamino)-6-naphthoyl-4-trans-cyclohexanoic acid (DANCA) has been used to probe the apomyoglobin heme pocket. The unexpected polarity of this domain is generally interpreted as arising from dynamic dipolar relaxation of the peptide dipoles surrounding the heme pocket. In the present work we reexamine the photophysical properties of DANCA in a variety of solvents and complexed with apomyoglobin (apoMb) to further probe the heme pocket environment as a function of external solvent conditions. Absorption and excitation spectra in a number of solvents are consistent with the well-known pi*<--pi (LE) and pi*<--n (CT) electronic absorption transitions observed for naphthylamine derivatives. Dual emission is also a well-documented property of such derivatives. Based on the time scale of the heterogeneity in the decay of the DANCA fluorophore observed in a series of solvents, we propose that the emission properties of DANCA in apoMb are not uniquely attributable to dynamic relaxation events, but also reflect dual emission from both a long-lived, red CT state and the shorter-lived, blue LE state. The pH studies in the range of pH 5-9 of the emission properties of DANCA in apoMb support this hypothesis. They also suggest a specific interaction of DANCA with one or both of the pocket histidyl residues, which leads to a drastic static quenching and red shift of the bound DANCA fluorescence upon protonation. Similar effects are observed with increasing pressure, indicating that these two perturbations alter the DANCA-apoMb complex in a similar fashion. The pressure-induced form of the protein is distinct both energetically and structurally from the previously characterized acid intermediate, in that it is populated above pH 5 and retains a significant degree of integrity of the heme pocket.     

The effect of prostaglandin synthetase inhibitors on renal function: studies in normal rats during sodium or water diuresis and in rats with chronic renal insufficiency

The effect of acute infusion of the prostaglandin synthetase inhibitors - meclofenamate or indomethacin - was examined in awake rats. Studies were performed in normal rats undergoing either sodium or water diuresis and in salt-replete rats with chronic renal insufficiency. Prostaglandin synthetase inhibitors had no effect on renal plasma flow, glomerular filtration rate or fractional excretion of sodium in any of the groups. Absolute urinary excretion rates for sodium and potassium decreased only in the normal, salt-replete rats. In contrast, prostaglandin synthetase inhibitors consistently decreased urinary flow and osmolar clearance under all experimental conditions studied. In the normal, salt-replete rats the fall in urine flow was preceded by an increase in urinary excretion of cyclic AMP. These results show that inhibitors of prostaglandin synthesis enhance the ability of the kidney to reabsorb water. This effect may be secondary to increased cyclic AMP generation and to increased urea recirculation resulting in higher urea accumulation in the renal medulla.     

Fe-heme conformations in ferric myoglobin.

X-ray absorption near-edge structure (XANES) spectra of ferric myoglobin from horse heart have been acquired as a function of pH (between 5.3 and 11.3). At pH = 11.3 temperature-dependent spectra (between 20 and 293 K) have been collected as well. Experimental data solve three main conformations of the Fe-heme: the first, at low pH, is related to high-spin aquomet-myoglobin (Mb+OH2). The other two, at pH 11.3, are related to hydroxymet-myoglobin (Mb+OH-), and are in thermal equilibrium, corresponding to high- and low-spin Mb+OH-. The structure of the three Fe-heme conformations has been assigned according to spin-resolved multiple scattering simulations and fitting of the XANES data. The chemical transition between Mb+OH2 and high-spin Mb+OH-, and the spin transition of Mb+OH-, are accompanied by changes of the Fe coordination sphere due to its movement toward the heme plane, coupled to an increase of the axial asymmetry.     

Subunit interactions in hemoglobin probed by fluorescence and high-pressure techniques

The dissociation of the subunits of human adult oxyhemoglobin has been investigated by using steady-state fluorescence anisotropy, multifrequency phase fluorometry, and high hydrostatic pressure. Human hemoglobin obtained by using two purification procedures (bulk preparation by centrifugation or further fractionation using anion-exchange chromatography) was labeled with an extrinsic fluorescent probe, 5-(dimethylamino)naphthalene-1-sulfonyl chloride (DNS-Cl). The long fluorescence lifetime of this probe allows for the observation of the macromolecular tumbling, and thus provides a method for observing changes in the size of the complex upon subunit dissociation under differing solution conditions of proton and organic phosphate concentration. At pH 7, the dansylated preparations of bulk and fractionated hemoglobin showed a concentration-dependent decrease in the anisotropy which though not identical can only arise from the tetramer to dimer dissociation. We observed primarily the dimer at pH 9 and a small destabilization of the tetramer in the presence of saturating inositol hexaphosphate (IHP). High-pressure experiments allowed for the observation of the dissociation of the hemoglobin dimer into monomers. From these measurements, we estimate the dimer dissociation constant to be between 0.1 and 1 nM. We compare the present results on the subunit affinities in hemoglobin obtained from steady-state and time-resolved fluorescence data with those obtained previously by using gel filtration, sedimentation, and kinetic techniques. These comparisons are indicative of a certain degree of conformational heterogeneity in the hemoglobin preparations.     

Nature of Transient Inhibition of Deoxyribonucleic Acid Synthesis in HeLa Cells by Parainfluenza Virus 1 (Sendai)

Adsorption of ultraviolet-inactivated Sendai virus, at high or low multiplicity, to HeLa cells caused a transient increased incorporation of (3)H-thymidine into the cellular deoxyribonucleic acid (DNA). In HeLa cells synchronized by a double-thymidine block, this increased incorporation of thymidine during the S phase lasted from about 30 to 90 min after virus adsorption. The observations that the kinetics of accumulation of radioactive thymidine in the nucleotide pool did not differ in control and in the virus-treated cells and that the (32)P incorporation into the DNA of the virus-treated cells was inhibited at the same time indicate that the augmented incorporation of (3)H-thymidine into DNA results from a transient block in the endogenous pathway of thymidine synthesis. Chromatographic analysis of the nucleotide pool of the virus-treated cells labeled with (14)C-formate indicates that methylation of deoxyuridine monophosphate to thymidine monophosphate is inhibited. It is suggested that the inhibition is caused by a block of either the thymidilate synthetase or some step in the tetrahydrofolate cycle.     

Transfer of plasmids pBR322 and pBR325 in wastewater from laboratory strains of Escherichia coli to bacteria indigenous to the waste disposal system

Laboratory strains of Escherichia coli containing plasmid pBR325 (or pBR322) were coincubated with a mobilizer strain of E. coli (containing the conjugative plasmid R100-1) and a recipient strain of bacteria. Bacterial strains isolated from raw wastewater or a plasmid-free E. coli laboratory strain served as recipients. Transfer of the pBR plasmid into the recipient strain occurred during a 25-h coincubation in either L broth or sterilized wastewater; transfer frequencies were several orders of magnitude lower in wastewater. After the coincubation, recipients exhibited both plasmid-encoded phenotypic characteristics and an altered plasmid profile, as shown by agarose gel electrophoresis of purified plasmid DNA.