Mass-spectrometric study of the azopigments obtained from bile pigments with diazotized ethyl anthranilate

The structures of some azopigments obtained by diazotization of conjugated and unconjugated bile pigments with diazotized ethyl anthranilate were studied by mass spectrometry. The alpha(0)-azopigments derived from rat bile and human bile were shown to be identical (t.l.c. and mass spectra) with azobilirubin derived from unconjugated bilirubin. The presence of two methyl vinyl isomers (Ia) and (Ib) in equal amounts was shown by t.l.c. and mass spectrometry. The structure of the delta-azopigment derived from rat bile was studied by two methods: (a) ammonolysis gave rise to an amide having a CH(2).CH(2).CO.NH(2) side chain as shown by its mass spectrum; (b) the mass spectrum of a trimethylsilyl derivative of the delta-azopigment methyl ester confirmed the ester to be a beta-d-monoglucuronide ester of azobilirubin I.     

A well-characterized plasma membrane fraction obtained from mouse peritoneal macrophages

A new method was developed to isolate a plasma membrane fraction from lipopolysaccharide-stimulated mouse peritoneal macrophages. Colchicine treatment was followed by sucrose density-gradient centrifugation. Total yield of Na,K-ATPase, a marker of plasma membrane, was 60 +/- 1% with the specific activity of 37 +/- 3 mumol of Pi/mg of protein/h. The preparation contained 1 +/- 1% pinosomes, 2 +/- 1% lysosomes, 17 +/- 2% endoplasmic reticulum, 6 +/-1% mitochondria, and a negligible number of nuclei, as judged by distribution of markers.     

Leishmanicidal activity of a supercritical fluid fraction obtained from Tabernaemontana catharinensis

The branches and leaves of Tabernaemontana catharinensis were extracted with supercritical fluid using a mixture of CO(2) plus ethanol (SFE), and the indole alkaloid enriched fraction (AF3) was selected for anti-Leishmania activity studies. We found that AF3 exhibits a potent effect against intracellular amastigotes of Leishmania amazonensis, a causative agent of New World cutaneous leishmaniasis. AF3 inhibits Leishmania survival in a dose-dependent manner, and reached 88% inhibition of amastigote growth at 100 microg/mL. The anti-parasite effect was independent of nitric oxide (NO), since AF3 was able to inhibit NO production induced by IFN-gamma plus LPS. In addition, AF3 inhibited TGF-beta production, which could have facilitated AF3-mediated parasite killing. The AF3 fraction obtained from SFE was nontoxic for host macrophages, as assessed by plasma membrane integrity and mitochondrial activity. We conclude that SFE is an efficient method for obtaining bioactive indole alkaloids from plant extracts. Importantly, this method preserved the alkaloid properties associated with inhibition of Leishmania growth in macrophages without toxicity to host cells.     

Chemical and biological properties of a peptido-glucan fraction from Armillaria mellea (Basidiomycetes).

A peptide-glucan fraction from Armillaria mellea (Basidiomycetes) was isolated and some aspects of its chemical structure were determined. The glucan is linked to the peptide portion which represents 30% w/w of the complex. Treatment with alkali destroys most of the threonine and leads to the separation of the peptide and carbohydrate moieties indicating the involvement of the hydroxyl group of threonine in the peptide-glucan linkage. The results of partial hydrolysis, methylation studies and Smith degradation involving periodate oxidation, borohydride reduction and acid hydrolysis indicate that the polysaccharide moiety consists of beta(1 leads to 3) and beta(1 leads to 6)-linked D-glucose residues. This peptide-rich glucan fraction showed a significant antitumor activity.     

Dicarboxylate transport in the inner membrane matrix fraction obtained from rat liver mitochondria

Dicarboxylate transport was studied in the inner membrane matrix fraction (mitoplasts) and compared to that in intact rat-liver mitochondria from which the former was obtained.It is concluded that, kinetics of dicarboxylate exchange measured in mitoplasts, are very similar to those observed with mitochondria. These results would indicate that the preparation technique preserves the integrity of the inner membrane and that neither the outer membrane nor the components of the peripheral space affect these results.     

Adaptogenic activity of glyco-peptido-lipid fraction from the alcoholic extract of Trichopus zeylanicus Gaerten (part II).

Anti-stress activity was carried out on glyco-peptido-lipid (AF) fraction from the alcoholic extract of Trichopus zeylanicus Gaerten and demonstrated against a battery of tests in rats and mice. AF exhibited significant anti-stress activity in dose-related manners in all the parameters studied against different models used to induce non-specific stress viz physical and chemically. The major parameters studied were immobilization induced gastric ulceration, adjuvant-induced trauma (Stress); humoral antibody synthesis in normal and immuno-suppressed mice and delayed type of hypersensitivity (DTH) reaction, chemically stress-induced alteration in hepatic function and anti-oxidant activity. The extract of Withania somnifera root (a commercial preparation available locally, Dabur India ltd.) was used to compare the results. In the safety evaluation study the maximum tolerance dose (MTD) and oral LD50 were found to be more than 3000 mg/kg, with no signs of abnormalities or any mortality observed for 15 days period under observation after single dose of drug administration. Feeding behaviour and fecal output were normal.     

Activation of mammalian oocytes by a factor obtained from rabbit sperm

In this study a fraction was prepared from rabbit sperm that activated rabbit and mouse oocytes following injection into the cytoplasm. The sperm factor activated oocytes exhibited cortical granule exocytosis, pronuclear formation, and cleavage. The sperm factor was soluble in aqueous solution and was not active extracellularly. Unlike most artificial activation methods that are only effective with aged oocytes, the sperm factor activated recently ovulated oocytes. The factor appears to be a protein or associated with a protein but not an acrosomal protein. Fractions from both mouse and bull sperm did not activate rabbit or mouse oocytes. Their inactivity may be owing to the techniques used to recover the fractions or differences between species in sperm morphology and fertilization processes. These observations support the hypothesis that oocyte activation is induced by a factor within sperm that is released into the cytoplasm of the oocyte at the time of sperm-oocyte fusion.     

Transfer of phospholipids by a protein fraction obtained from canine pulmonary lavage

Surfactant phospholipid exists in multicompartment pools within the subphase of the lung. Movement among these pools and back into type II alveolar cells may be catalyzed by a phospholipid transfer protein resident in the subphase. We demonstrate here that a protein fraction obtained from canine lung lavage catalyzes the intermembrane transfer of all the major surfactant phospholipids. The protein is probably not derived from serum and is unrelated to surfactant proteins that have already been described.     

Interaction of bilirubin with the synaptosomal plasma membrane

The interaction of the neurotoxic pigment bilirubin with synaptosomal plasma membrane vesicles (SPMV) isolated from rat brain was investigated. The interaction seems to involve three steps: (a) a rapid formation of an electrostatic complex between bilirubin and polar lipid head groups; (b) a slow inclusion of the pigment into the hydrophobic core of the membrane; and (c) a SPMV-induced bilirubin aggregation, observed when membrane capacity for bilirubin is exceeded. The association constant of the initial complex increased markedly when pH was lowered below 7.4, particularly in SPMV isolated from newborn rats. A preferential binding of bilirubin to pure gangliosides and sphingomyelin was observed, thus suggesting a role for these lipids as first targets of the pigment in the synaptic membrane. The inclusion of bilirubin into the membranes was gradually enhanced when decreasing the pH or the age of the rats from which SPMV were isolated. In addition, membranes from 2-day-old rats have a higher capacity for bilirubin incorporation compared to those from adult rats. Experiments with reconstituted liposomes of varying protein and cholesterol contents suggest that the effect of age may be related to changes in synaptosomal membrane fluidity during development. Our results support the hypothesis that the interaction of bilirubin with the synaptic membrane plays an important role in the molecular mechanisms of bilirubin neurotoxicity.     

Properties of triacylglycerol lipase in a mitochondrial fraction from baker's yeast (Saccharomyces cerevisiae).

A triacylglycerol lipase in a mitochondrial fraction isolated from yeast (Saccharomyces cerevisiae) has been characterized and the hydrolysis studied kinetically using an insoluble artificial triacylglycerol suspension. 1. The triacylglycerol was hydrolyzed almost completely to fatty acids and glycerol. The lipase activity was inhibited by potassium fluoride and the sodium salts of -chloride, -glycocholate and -pyrophosphate as well as by protamine sulfate but at concentrations much too high to indicate that the lipase is a non specific esterase or a lipoprotein lipase. Also parachloromercuribenzoate inhibited the lipase activity. Inhibitory effect of fatty acid was observed at concentrations above 1mM. This inhibition may provide a regulatory mechanism of the lipase in vivo. 2. On the day of isolation the lipase activity of intact mitochondria at pH 7.5 and 30 degrees C was 400 nmol free fatty acid -h-1 - mg-1 at a triacylglycerol concentration of 9.0 mM. Sonication of the mitochondria increased the activity 2-3 fold. Freezing of the mitochondria also activated the lipase and this activation was dependent upon the freezing method, the concentration of mitochondrial protein and the presence of bovine serum albumin. 3. The particulate nature of the assay system was illustrated by the observation that the apparent Km value of the lipase increased with the concentration of mitochondrial protein. For each protein concentration the lipase had two apparent Km values when the activity was assayed with intact mitochondria, but only one when assayed with submitochondrial particles. At the same protein concentration the Km value for the latter was identical with the "low affinity" Km for the lipase in intact mitochondria.     

F390 synthetase and F390 hydrolase from Methanobacterium thermoautotrophicum (strain delta H).

Factor F390 is the 8-OH adenylated form of the deazaflavin coenzyme F420, which is a central electron carrier in methanogenic bacteria. The enzymes catalysing the formation of F390 from ATP and F420 (F390 synthetase) and its hydrolysis into AMP and F420 (F390 hydrolase) were isolated and partially purified from Methanobacterium thermoautotrophicum. Both enzymes were oxygen-stable. The F390 synthetase tended to coelute with coenzyme F420 reducing hydrogenase during all purification steps. The 30-fold purified enzyme was still contaminated with the hydrogenase. The F390 hydrolase was purified 135-fold to a specific activity of 8.6 mumol/min/mg protein. The colourless enzyme consisted of one polypeptide of approximately 27,000 kd.