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.     

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.     

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), cyperquat (MPP+) and paraquat on isolated mitochondria from rat striatum, cortex and liver

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), its metabolite 1-methyl-4-phenyl pyridinium ion (MPP+, cyperquat) and a structurally-related compound paraquat on mitochondrial functions were investigated in isolated organelles from rat striatum, cortex and liver. MPTP (0.1-1.0 mM) had no significant effect on various parameters of mitochondrial oxidative phosphorylation. In contrast, MPP+ (0.5 mM) inhibited the oxidation of the nicotinamide adenine dinucleotide (NAD+)-linked substrates pyruvate and malate but not that of the flavin adenine dinucleotide (FAD+)-linked substrate succinate. Paraquat (5.0 mM) significantly stimulated basal oxygen consumption (state 4) without influencing the oxygen utilization (state 3) associated with adenosine diphosphate (ADP) phosphorylation. Thus, these structurally-related compounds have different effects on mitochondrial oxidative phosphorylation, but the organelles from striatum, cortex and liver were affected in a similar manner by these compounds.     

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.     

Mini-exon derived RNA gene ofLeishmania donovani: structure,organization and expression

Mini-exon derived RNA is a small nuclear RNA of trypanosomatid protozoa such asLeishmania which donates its 5′-terminal 39 nucleotides to the 5’-ends of cellular messenger RNAs by trans-splicing. We have cloned a mini-exon derived RNA gene fromLeishmania donovani and studied its organization and expression. About 200 copies of the gene per haploid genome are organized as a tandem repeat on a single chromosome. The gene is transcribed as a 95-nucleotide RNA. The first 39 nucleotides of mini-exon derived RNA is also found at the 5′-terminus of a cellular mRNA (Β-tubulin), thus confirming its identity. Sequence analysis of the gene and its flanking regions showed that while classical RNA polymerase II promoter elements such as TATA and CAAT are absent from the 5′-upstream region, intragenic sequence motifs resembling RNA polymerase III promoter elements are present. The implications of this finding for mini-exon derived RNA expression are discussed.     

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.     

Characterization oftrans-acting regulatory elements affecting the expression of Mn-superoxide dismutase (sodA) inEscherichia coli

Escherichia coli strains harboringtrans-acting mutations affecting the expression of Mn-superoxide dismutase (SOD) gene (sodA) were used to studysodA regulation. Complementation studies revealed that eitherarc (aerobic respiratory control) orfur (ferric uptake regulation) loci independently complemented anaerobic expression of asodA::lacZ protein fusion in one mutant strain (UV16). This mutant exhibited phenotypes (i.e., elevated outer membrane proteins, enzyme activity, and dye sensitivity) typical offur andarc mutants. When these mutations were introduced into an otherwise wild-type background, anaerobicsodA expression occurred only when botharc andfur mutations were present simultaneously, suggesting cooperative roles of Fur and Arc insodA repression. The reconstructedfur arcA andfur arcB double mutants were still inducible by iron chelators, suggesting the possible involvement of another iron-containing repressor protein. A second independent mutant strain harboring atrans-acting regulatory mutation (UV14) was only partially complemented by multicopy plasmids carryingfur ⁺ orarc ⁺ genes, implicating other genetic elements insodA regulation.     

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.