Isolation and preliminary characterization of a 2-chlorobenzoate degrading Pseudomonas

Pseudomonas sp. strain B-300, which is able to utilize 2-chlorobenzoic acid, was isolated from a soil sample by enrichment culture. This strain was shown to grow on 2-chlorobenzoic acid and to completely degrade the substrate with concomitant chlorine ion release. Concentrations of 2-chlorobenzoic acid higher than 0.5% (w/v) were toxic to the cells. Our study also suggested that in the presence of glucose, 2-chlorobenzoic acid is converted to catechol or chlorocatechol; these are in turn transformed to muconic and chloromuconic acid, respectively, suggesting a repression by glucose of some of the degradation pathway enzymes. A similar scheme was already described for 3-chlorobenzoate degradation by pAC25 plasmid.     

Effect of cholesterol in various liposomal compositions on the in vivo toxicity, therapeutic efficacy, and tissue distribution of amphotericin B

The effect of cholesterol in neutral, positively and negatively charged liposomes on the toxicity, therapeutic efficacy, and alteration in the tissue distribution pattern of amphotericin B (Amp-B) in normal and infected mice was studied. It was observed that inclusion of cholesterol (CHOL) into egg phosphatidylcholine (EPC) liposomes increased the LD50 of Amp-B from 5.3 to 8.5 mg/kg body weight. In the case of phosphatidylserine (PS) liposomes as well as stearylamine (SA) liposomes, cholesterol incorporation had no effect in altering the toxicity of the drug. The survival pattern of animals with all types of liposomal formulation of Amp-B was similar. The tissue distribution studies indicated that in the case of normal mice, cholesterol inclusion in all types of liposomes increased the organ concentration of the drug in various tissues. In infected animals, the concentration of Amp-B in all organs was increased when cholesterol was included in EPC and EPC/PS liposomes. The organ concentration of Amp-B in lung and liver after 1 h of injection was the same in the case of EPC/SA and EPC/SA/CHOL liposomes. Considering the observations on toxicity, therapeutic efficacy, and tissue distribution, it was suggested that cholesterol had a beneficial therapeutic effect on neutral EPC liposomes.     

Characterization of [3H]palmitate- and [3H]ethanolamine-labelled proteins in the multicellular parasitic trematode Schistosoma mansoni.

Biosynthetic labelling experiments with cercariae and schistosomula of the multicellular parasitic trematode Schistosoma mansoni were performed to determine whether [3H]palmitate or [3H]ethanolamine was incorporated into proteins. Parasites incorporated [3H]palmitate into numerous proteins, as judged by SDS/polyacrylamide-gel electrophoresis and fluorography. The radiolabel was resistant to extraction with chloroform, but sensitive to alkaline hydrolysis, indicating the presence of an ester bond. Further investigation of the major 22 kDa [3H]palmitate-labelled species showed that the label could be recovered in a Pronase fragment which bound detergent and had an apparent molecular mass of 1200 Da as determined by gel filtration on Sephadex LH-20. Schistosomula incubated with [3H]ethanolamine for up to 24 h incorporated this precursor into several proteins; labelled Pronase fragments recovered from the three most intensely labelled proteins were hydrophilic and had a molecular mass of approx. 200 Da. Furthermore, reductive methylation of such fragments showed that the [3H]ethanolamine bears a free amino group, indicating the lack of an amide linkage. We also evaluated the effect of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: [3H]palmitate-labelled proteins of schistosomula and surface-iodinated proteins were resistant to hydrolysis with this enzyme. In conclusion, [3H]palmitate and [3H]ethanolamine are incorporated into distinct proteins of cercariae and schistosomula which do not bear glycophospholipid anchors. The [3H]ethanolamine-labelled proteins represent a novel variety of protein modification.     

Effect of alkylated and intercalated DNA on the generation of superoxide anion by riboflavin

Superoxide anion (O ₂ ^.– ) was photogenerated upon illumination of riboflavin in fluorescent light. The rate of O ₂ ^.– formation was stimulated by double stranded DNA but not by denatured DNA or RNA. Depurinated DNA, which was predominantly depleted in guanine residues, did not exhibit the stimulatory effect, indicating an interaction of riboflavin, or active oxygen species derived from it, with guanine bases. Also, the stimulation of O ₂ ^.– photogeneration was not observed with ethidium bromide but was seen with proflavin-intercalated DNA. Since ethidium bromide intercalates preferentially between purines and pyrimidines, and proflavin prefers dA-dT rich sites, these results were interpreted to suggest that the interaction of riboflavin with DNA is mainly with GC or CG base pairs.     

New prospects for deducing the evolutionary history of metabolic pathways in prokaryotes: Aromatic biosynthesis as a case-in-point

Metabolic pathways of prokaryotes are more biochemically diverse than is generally recognized. Distinctive biochemical features are shared by phylogenetic clusters. The hierarchical levels of characterstate clustering depends upon evolutionary events which fortuitously became fixed in the genome of a common ancestor. Prokaryotes can now be ordered on a phylogenetic tree. This allows the evolutionary steps that underlie the construction and regulation of appropriately complex biochemical pathways to be traced in an evolutionary progression of prokaryote types that house these pathways. Essentially the approach is to deduce ancestral character states at ever deeper phylogenetic levels, utilizing logical principles of maximum parsimony. The current perspective on the evolution of the biochemical pathway for biosynthesis of aromatic amino acids is developed as a case-in-point model for analyses that should be feasible with many major metabolic systems. Phenylalanine biosynthesis probably arose prior to the addition of branches leading to tyrosine and tryptophan. An evolutionary scenario is developed that begins with non-enzymatic reactions which may have operated in primitive systems, followed by the evolution of an enzymatic system that pre-dated the divergence of major lineages of modern eubacteria (Gram-positive bacteria, Gram-negative purple bacteria, and cyanobacteria).Florida Agricultural Experiment Station, Journal Series No. 8251.     

Synthesis and biological activity of novel C-terminal-extended and biotinylated growth hormone-releasing factor (GRF) analogs.

A series of novel hGRF(1-29)-NH2 analogs were synthesized and biotinylated. The immunological and biological activities of these analogs were then characterized. To distance the biotin moiety from the putative bioactive core, a C-terminal spacer arm consisting of -Gly-Gly-Cys-NH2 (-GGC) was added to hGRF(1-29)-NH2 (hGRF29) and analogs, with subsequent biotinylation performed at the cysteine residue. Neither addition of the C-terminal spacer arm nor biotinylation affected affinity of these analogs for GRF antibody. Relative to hGRF(1-44)-NH2 (hGRF44: potency = 1.0), the biotinylated analogs were equipotent in vitro to their nonbiotinylated, parent compounds: [desNH2Tyr1,D-Ala2,Ala15]hGRF29-GGC-(tpBiocyt in)-NH2 (4.7) = [Ala15]hGRF29-GGC-(tpBiocytin)-NH2 (3.9) greater than hGRF29-GGC-(tpBiocytin)-NH2 (0.8). Based upon cumulative GH release data in vivo (0-60 min postinjection), [desNH2Tyr1,D-Ala2,Ala15]hGRF29-GGC-(tpBiocyt in)-NH2, [Ala15]hGRF29-GGC-(tpBiocytin)-NH2, and hGRF29-GGC-(tpBiocytin)-NH2 displayed 8.6, 5.5, and 0.8 times, respectively, the potency of hGRF44. These in vivo potency values were not significantly different from the corresponding parent compounds (i.e., with or without the C-terminal spacer arm). In summary, biotinylated hGRF analogs have been developed that retain full immunoreactivity and potent bioactivity (in vitro and in vivo), thus permitting their use in GRF receptor isolation, ELISA, and histochemical procedures.     

Rat liver lowMr phosphotyrosine protein phosphatase isoenzymes: Purification and amino acid sequences

Two lowM _r phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as lowM _r acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH₂-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40–73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.     

Change of speciation of Cu(II) in growth medium due to uptake of ammonia byPseudomonas testosteroni during growth

Growth ofPseudomonas testosteroni in a medium containing 1mm Cu(II) causes a color change from blue to green. The spectrum of the supernatant solution from the blue culture shows an absorption at 660 nm, identical to that of 1mm [Cu(II)] in the medium. The green supernatant solution shows a UV absorption, which tails into the visible and so is responsible for the green color, and ad-d absorption at 720 nm. The absorption at 660 nm for the blue supernatant solution is probably due to [Cu(NH₃)₃(H₂O)₃]²⁺. Growth of the organism causes loss of ammonia and a speciation change to [Cu(NH₃)₂(H₂O)₄]²⁺, with a shift in absorption maximum from 660 to 720 nm. These conclusions are based upon the spectra of known aquaammine complexes of Cu(II) and calculations of the speciation of Cu(II) before and after growth. Change in metal speciation owing to nutrient uptake by an organism does not appear to have been recognized previously.     

Leucasin, a triterpene saponin from Leucas nutans.

A new saponin, leucasin, has been isolated from Leucas nutans and characterized on the basis of chemical investigation and spectroscopic studies as 3-O-[β- -glucopyranosyl(1→2)β- -glucopyranosyl]2,3β-dihydroxylup-20(29)-ene. Lupeol palmitate, sitosterol and stigmasterol were also isolated.