Glycine Betaine as a Direct Substrate for Methanogens (Methanococcoides spp.)

Nine marine methanogenic Methanococcoides strains, including the type strains of Methanococcoides methylutens, M. burtonii, and M. alaskense, were tested for the utilization of N-methylated glycines. Three strains (NM1, PM2, and MKM1) used glycine betaine (N,N,N-trimethylglycine) as a substrate for methanogenesis, partially demethylating it to N,N-dimethylglycine, whereas none of the strains used N,N-dimethylglycine or sarcosine (N-methylglycine). Growth rates and growth yields per mole of substrate with glycine betaine (3.96 g [dry weight] per mol) were similar to those with trimethylamine (4.11 g [dry weight] per mol). However, as glycine betaine is only partially demethylated, the yield per methyl group was significantly higher than with trimethylamine. If glycine betaine and trimethylamine are provided together, trimethylamine is demethylated to dimethyl- and methylamine with limited glycine betaine utilization. After trimethylamine is depleted, dimethylamine and glycine betaine are consumed rapidly, before methylamine. Glycine betaine extends the range of substrates that can be directly utilized by some methanogens, allowing them to gain energy from the substrate without the need for syntrophic partners.     

Insulin, not glucose, controls hepatocellular glycogen deposition. A re-evaluation of the role of both agents in cultured liver cells

The direct effects of insulin and glucose on glycogen accumulation were compared using monolayers of chicken embryo hepatocytes which, when cultured in chemically defined medium without hormones, retain viability for several days but become depleted of glycogen. The data strongly suggest that insulin is the major direct signal for hepatic glycogen synthesis, while glucose supports glycogen accumulation primarily in its role as a substrate. Insulin alone, when added to the cells in physiological concentrations, either shortly after isolation or throughout culture, restored glycogen to the maximal levels found in the liver of the fed chicken. Addition of increasing amounts of glucose in the absence of insulin, in contrast, yielded proportional but limited increases in glycogen deposition attaining not more than 30% of the maximal storage capacity of the cells. This hormone-independent glycogenesis was characterized by a 30-min burst of glycogen deposition immediately following a stepped increase of glucose, with no detectable change in glycogen synthase activity. Insulin-dependent glycogenesis evidenced a much slower rate of glycogen deposition and was accompanied by a near tripling of glycogen synthase activity. Insulin-induced glycogen stores were broken down following removal of the hormone, even when glucose was present in great excess, indicating that the cells require insulin to maintain as well as build up maximal levels of glycogen. In the presence of glucagon, insulin-induced glycogen stores were rapidly degraded, but glucose-induced glycogenesis was not inhibited. The actions of insulin and glucose in this system are both qualitatively and quantitatively similar to those that have been observed in the diabetic animal.     

Utility of the white gene in estimating phylogenetic relationships among mosquitoes (Diptera: Culicidae)

The utility of a nuclear protein-coding gene for reconstructing phylogenetic relationships within the family Culicidae was explored. Relationships among 13 species representing three subfamilies and nine genera of Culicidae were analyzed using a 762-bp fragment of coding sequence from the eye color gene, white. Outgroups for the study were two species from the sister group Chaoboridae. Sequences were determined from clone PCR products amplified from genomic DNA, and aligned following conceptual intron splicing and amino acid translation. Third codon positions were characterized by high levels of divergence and biased nucleotide composition, the intensity and direction of which varied among taxa. Equal weighting of all characters resulted in parsimony and neighboring-joining trees at odds with the generally accepted phylogenetic hypothesis based on morphology and rDNA sequences. The application of differential weighting schemes recovered the traditional hypothesis, in which the subfamily Anophelinae formed the basal clade. The subfamily Toxorhynchitinae occupied an intermediate position, and was a sister group to the subfamily Culicinae. Within Culicinae, the genera Sabethes and Tripteroides formed an ancestral clade, while the Culex-Deinocerites and Aedes- Haemagogus clades occupied increasingly derived positions in the molecular phylogeny. An intron present in the Culicinae- Toxorhynchitinae lineage and one outgroup taxon was absent in the basal Anophelinae lineage and the second outgroup taxon, suggesting that intron insertions or deletions may not always be reliable systematic characters.      

Ascorbic acid accumulation by cultured rat adrenocortical cells

Summary The influence of adrenocorticotrophin (ACTH) on radiolabeled ascorbic acid (AA) accumulation by adrenocortical cells was examined in primary cultures of collagenase dissociated glands from adult male rats. The cells were ACTH responsive by morphological and steroidogenic criteria. After 5 d in AA-free medium, cells pretreated with 100 mU/ml ACTH for 3 d took up two to three times more AA over a 2 h period than did untreated controls (4.0 to 10.0 nmol versus 1.7 to 3.4 nmol AA/μg DNA). In contrast, ACTH administered on Day 6 concurrently with AA inhibited AA accumulation compared to cultures exposed to AA alone. This acute inhibitory effect of ACTH was in the order of 30% in cultures pretreated with ACTH for 3 d but was not significant (7%) without ACTH pretreatment. The results show that ACTH has distinct long term stimulatory and acute inhibitory effects on AA accumulation by adrenocortical cells and suggest that both maximal AA accumulation and the responsiveness to acute inhibition of AA accumulation by ACTH may depend on the maintenance of the differentiated state of the adrenal cortex. This work was supported by a grant and research associateship to N. A. from the National Cancer Institute of Canada.     

Isolation and fractionation of porcine peripheral lymphocytes

A method is described for the separation and purification of porcine peripheral blood lymphocytes and their subsequent sub-cellular fractionation. The isolated cell population consisted of 99% lymphocytes with a viability of 100% and an overall recovery of 30%. The lymphocyte : thrombocyte ratio was 56 : 1 and the population contained no detectable erythrocytes. The lymphocytes proved resistant to mechanical breakage but a method of homogenisation in a hypotonic medium was developed which resulted in complete cell rupture with minimal damage to sub-cellular components.     

The reaction of iodine and thiol-blocking reagents with human complement components C2 and factor B. Purification and N-terminal amino acid sequence of a peptide from C2a containing a free thiol group.

Human complement components C2 and Factor B each contain one free thiol group/molecule. Reaction with p-chloromercuribenzoate destroyed the haemolytic activity of C2 but had no effect on Factor B. Reaction of C2 with I2 gave a 16-fold enhancement of its haemolytic activity. The pH optimum for the reaction was 7.0. The I2 reacted at the thiol group in C2 with a stoicheiometry of 1 mol of I2/mol of C2. The product of the reaction was unaffected by millimolar concentrations of dithiothreitol; however, azide and cyanide were inhibitory. Reaction with azide did not result in re-expression of the thiol group. Mild oxidation of the thiol group with m-chloroperbenzoic acid did not enhance the haemolytic activity. The results suggest that reaction with I2 causes intramolecular covalent, but not disulphide, bond formation. I2 reacted with Factor B at the free thiol group without affecting the haemolytic activity. A CNBr-cleavage peptide from C2a (obtained by cleavage of C2 by subcomponent C1s) containing the free thiol group was isolated. Automated Edman degradation of the peptide showed that it was the N-terminal peptide of C2a. The free thiol group was identified at position 18.