Assessment of bacterial diversity in agricultural by-product compost by sequencing of cultivated isolates and amplified rDNA restriction analysis

An investigation of bacterial diversity in compost was performed using molecular chronometer in order to reveal its phylogeny. Thirty-three bacterial isolates isolated from compost were analyzed by 16S rRNA gene sequencing which revealed phylogenetic lineage of class Bacilli, γ, β-Proteobacteria, and Actinobacteria. Among these lineages, isolates belonging to class Bacilli consisted of species from genera Staphylococcus, Bacillus, Terribacillus, and Lysinibacillus. From phylum Actinobacteria: Microbacterium barkeri and Kocuria sp. were identified. Other bacterial groups had phylogenetic linkage with genera Comamonas and Acidovorax (class β-Proteobacteria); Serratia, Klebsiella, and Enterobacter (class γ-Proteobacteria). Similar isolates were analyzed through ARDRA. Amplified product of 16S rRNA gene from each isolates was subjected to cleavage by enzymes HpaII, HinfI, and MspI in separate reaction tubes. HpaII generated 2–6 bands ranging from 90–688 bp, HinfI generated 2–5 bands of 71–1,038 bp, and MspI 2–7 bands of 69–793 bp. The restriction patterns from HpaII, HinfI, and MspI were normalized separately and combined by means of pattern recognition software “Diversity Database.” HpaII had highest discrimination index (0.72) than HinfI (0.68) and MspI (0.65), and the combination of all three showed discrimination index (0.69). Numerical analysis of ARDRA patterns demonstrated sufficient phylogenetic information for characterizing bacterial diversity. Phylogenetic relationship obtained among isolates through ARDRA was compared with 16S rRNA gene sequence and ARDRA results showed sufficiently similar 16S rRNA gene sequence analysis, but not an overlapping. It has been observed that ARDRA technique facilitates the identification of bacteria in less than 36 h as compared to traditional 16S rRNA gene sequencing.     

Measurement of serum glycosylated proteins and glycosylated low density lipoprotein fraction in diabetes mellitus

A simple method was developed for estimating serum glycosylated protein levels using gel filtration with Bio-Gel P6 by determining the protein and sugar content in the void volume fraction. The glycosylated protein levels (GSP) correlated well with fasting blood sugar levels and glycosylated albumin level (G-ALB) determined by affinity chromatography with Blue Sepharose CL6B. The glycosylation level of heparin-citrate precipitable fraction of serum which predominantly contained low density lipoprotein (G-LDL) also correlated well with GSP and LDL-cholesterol levels. Significantly different values were obtained for GSP, G-ALB, and G-LDL between normals and diabetics.     

Cholesterol binding reserve of hyperlipemic rat serum lipoproteins in chronic ethanol administration

Chronic administration of ethanol in rats caused the reduction of serum cholesterol binding reserve. The very low density and high density lipoproteins, main serum cholesterol binding reserves, were slightly increased with corresponding increases in their lipid and protein components during initial stage of alcohol consumption. However, these capacities get deminished during reversal of hyperlipemia induced by prolonged action of ethanol. This situation may be an early indicator for the initiation of hepatic damage and a variety of secondary effects of ethanol.     

Synthesis of phosphatidylcholine by rat lung during choline deficiency

The effect of choline deficiency on the de novo pathway for phosphatidylcholine (PC) synthesis in the lung was investigated in rats fed a washed soy protein (lipotrophic) diet deficient in choline and methionine for 2-3 wk. Lungs from lipotrophic rats showed a decreased content of choline and choline-phosphate (P less than 0.05) compared with control but no change in content of cytidine 5'-diphosphocholine or PC. Isolated perfused lungs from lipotrophic rats were evaluated for choline and fatty acid utilization for PC synthesis. Lipotrophic lungs perfused with 5 microM [14C-methyl]-choline chloride showed increased incorporation into PC while there was no significant effect at saturating levels of choline (100 microM). There was increased incorporation of [1-14C]-palmitic acid into PC and diglyceride and increased incorporation of D-[U-14C]glucose into fatty acids of PC. Increased choline and glucose incorporation was not due to alteration of intracellular specific activity of these substrates. This study indicates the utilization of choline and fatty acid for PC synthesis is stimulated as a result of choline deficiency while lung CDP-choline concentration is maintained, possibly through regulation of choline phosphate cytidyl transferase activity. These mechanisms compensate for decreased choline availability to maintain the PC content of lungs.     

Uptake hydrogenase in fast-growing strains of Rhizobium sp. (Sesbania) in relation to nitrogen fixation

Eight of 104 fast-growing strains of Rhizobium sp. ( Sesbania ) tested possessed a hydrogen recycling system. Depression of uptake hydrogenase occurred in poor as well as in rich carbon media. A statistically significant increase (> 22%) in total plant nitrogen content and dry matter yield was observed by the inoculation of hup + strains over hup ^- strains.     

Isolation, characterization and metal-ion-binding properties of the alpha-subunit from S-100a protein.

The present experiments were undertaken to investigate the role of the phosphoinositides phosphatidylinositol 4-phosphate (PtdIns-4-P) and phosphatidylinositol 4,5-biphosphate (PtdIns-4,5-P2) in the alpha 1-adrenergic stimulation of respiration in isolated hamster brown adipocytes. Exposure of isolated brown adipocytes to the alpha-adrenergic-receptor agonist phenylephrine provoked a breakdown of 30-50% of the PtdIns-4-P and PtdIns-4,5-P2 after prelabelling of the cells with [32P]Pi. Coincident with the breakdown of phosphoinositides was an accumulation of labelled phosphatidic acid, which continued for the duration of the cell incubation. The time course of phosphoinositide breakdown was defined more precisely by pulse-chase experiments. Under these conditions, phenylephrine caused radioactivity in phosphatidylinositol, PtdIns-4-P and PtdIns-4,5-P2 to fall by more than 50% within 30 s and to remain at the depressed value for the duration of the incubation (10 min). This phospholipid response to alpha-adrenergic stimulation was blocked by exposure of the cells to phorbol 12-myristate 13-acetate (PMA); likewise phenylephrine stimulation of respiration was prevented by PMA. beta-Adrenergic stimulation of respiration and inhibition of respiration by 2-chloroadenosine and insulin were, however, unaffected by treatment with PMA. On the assumption that PMA is acting in these cells as an activator of protein kinase C, these results suggest the selective interruption of alpha-adrenergic actions in brown adipocytes by activated protein kinase C. These findings suggest that breakdown of phosphoinositides is an early event in alpha-adrenergic stimulation of brown adipocytes which may be important for the subsequent stimulation of respiration. The results from the pulse-chase studies also suggest, however, that phenylephrine-stimulated breakdown of inositol phospholipids is a short-lived event which does not appear to persist for the entire period of exposure to the alpha 1-adrenergic ligand.     

Interaction of heavy metal toxicants with brain constitutive nitric oxide synthase

This study was designed to evaluate thein vitro effects of transition heavy metal cations on activity of constitutive isoform of nitric oxide synthase (cNOS) in rat brain. NOS activity was determined in the cytosolic fractions of rat cerebral hemispheres by conversion of³H-L-arginine to³H-L-citrulline. Different concentrations of mercury (Hg²⁺), nickel (Ni²⁺), manganese (Mn²⁺), zinc (Zn²⁺), cadmium (Cd²⁺), lead (Pb²⁺) and calcium (Ca²⁺) were tested on NOS activity. While all the cations caused inhibition, there were differences in the apparent inhibition constants (Ki) among the cations. With the exception of calcium ion no other cation required preincubation with the enzyme preparation. These results indicate that while calcium ion modulate cNOS activity at regulatory site(s), inhibitory influence of toxic heavy metal cations may be exerted on the catalytic site(s) either by direct binding to it or by interfering with the electron transfer during catalysis.