Adipose-tissue-specific increase in glyceraldehyde-3-phosphate dehydrogenase activity and mRNA amounts in suckling pre-obese Zucker rats. Effect of weaning.

The regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was studied during the onset of obesity in the genetically obese (fa/fa) rat by determination of GAPDH activity and hybridizable mRNA amounts in adipose tissue and liver from suckling and weanling rats. GADPH activity remained low throughout the suckling period, and a burst of activity occurred after weaning in both lean and obese pups. As early as 7 days of age, adipose tissue from pre-obese rats displayed a significant increase in enzyme activity, whereas no difference could be detected in the liver. In both suckling (16 days of age) and weanling (30 days of age) obese rats a proportionate increase in GAPDH activity and mRNA amounts was observed in adipose tissue, but not in liver. It is concluded that the obese genotype influences GAPDH gene expression at a pretranslational level and in a tissue-specific manner. This phenomenon could partly contribute to the hyperactive fat accretion in the obese rat, since glycolysis is the major metabolic pathway for lipogenic substrates in adipose tissue.     

Transposition of the Streptococcus lactis ssp. lactis Z270 lactose plasmid to pVA797: demonstration of an insertion sequence and its relationship to an inverted repeat sequence isolated by self-annealing

The lactose plasmid pUCL22 of the single plasmid strain Streptococcus lactis ssp. lactis Z270 was demonstrated to fuse with the heterologous conjugative plasmid pVA797. The fusion of pUCL22 with pVA797 occurred by recombination between a specific sequence of pUCL22 and different sites of pVA797. The cointegrates of pUCL22::pVA797 were unstable: in the absence of lactose selection, they segregated plasmids that corresponded to pVA797 enlarged by one sequence of 1.2 kb, common to all derivative plasmids. This resolution sequence (RS) was shown to originate in the 9.7 kb BstEII restriction fragment of pUCL22 and to duplicate during replicon fusion. In addition, after nuclease S1 treatment of pUCL22 DNA, a self-annealing sequence was isolated; the two copies of this inverted repeat (IR) sequence were located on the 18 kb BamHI segment of the plasmid. This latter sequence was distinct from the RS with which it hybridized weakly. The RS was responsible for the transposition of the entire lactose plasmid; the role of the IR remains to be elucidated.     

Species-specific TNF induction of thymocyte proliferation

Summary Recombinant murine (rMu) tumor necrosis factor (TNF), in a standard comitogenic assay with phytohemagglutinin, induced murine thymocyte proliferation, while up to 10,000-fold higher concentrations of recombinant human TNF did not. The induction of thymocyte proliferation was dependent upon TNF concentration in a biphasic manner. Thus, 100 to 1000 units/ml TNF were near optimal while concentrations 1,000 units/ml caused apparent down regulation. The effect was abrogated by neutralizing antibody to rMu-TNF but not by neutralizing antibody to rMu-interleukin 1 or . The rMu-TNF did not induce proliferation of the mature murine T-helper cell line, D10.G4.1, in the presence of mitogen. Taken together the results indicate that TNF, in a strictly species-specific manner, can regulate thymocyte proliferation independently of interleukin 1.Supported in part by Asahi Chemical Industry Co., Inc. and by USPHS Grants CA-24538, CA-15142 and CA-09072 awarded by the National Cancer Institute, Department of Health and Human Services     

Interactions of piperidine derivatives with the nicotinic cholinergic receptor complex from Torpedo electric organ

The interactions of eight piperidine derivatives with nicotinic receptor complexes fromTorpedo californica electric organ were studied using [¹²⁵I]alpha-bungarotoxin ([¹²⁵I]BGT) as a probe for the acetylcholine binding site and [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) as a probe for a site associated with the receptor-gated ion channel.Cis- andtrans-2-methyl-6-n-undecanyl piperidines (MUP), major constituents of fire ant venom, had a high-affinity for [³H]H₁₂-HTX binding sites (K_i=0.08–0.24 M), but had no affect on receptor binding. MUP affinity for [³H]H₁₂-HTX binding sites was approximately doubled in the presence of 1 M carbamylcholine. Introduction of a 2-hydroxyl group to the undecanyl side channel had little effect on activity of the alkaloid. The analog 2,6- (but not 3,5-) dimethylpiperidine was a moderately active inhibitor of [³H]H₁₂-HTX binding (K _i-8.8 M). 2-Methylpiperidine was considerably less active (K _i=600 M), although it was more potent than either 3- or 4-methylpiperidine. The affinities of 2,6-dimethylpiperidine and 2-methylpiperidine for [³H]H₁₂-HTX binding sites were decreased in the presence of 1 M carbamylcholine. Carbamylcholine affinity for the receptor was increased by up to 7 fold in the presence of 10 and 32 M MUP, but was decreased in the presence of 2,6-dimethylpiperidine and 2-methylpiperidine. Thecis- andtrans-isomers of MUP were equipotent in producing each of its effects. In these actions, MUP resembles a variety of other compounds derived from 2,6-disubstituted piperidines, including histrionicotoxins, gephyrotoxins and pumiliotoxins. These studies establish the importance of alkyl substitutions in theortho position of the piperidine ring in conferring ion channel specificity, and the importance of substantial alkyl side chains in conferring the ability of channel blockers to stabilize the nicotinic receptor complex in high affinity, desensitized conformations.     

Carnivora: the primary structure of the common otter (Lutra lutra, Mustelidae) hemoglobin

The hemoglobin of the Common Otter (Lutra lutra, Carnivora) contains only one component. The complete primary structures of the alpha- and beta-chains are presented. They were separated by high-performance liquid chromatography and the sequences determined by automatic liquid and gas-phase Edman degradation of the chains and their tryptic peptides. The alpha-chains show 18 and the beta-chains 13 substitutions compared to human alpha- and beta-chains, respectively. In the alpha-chains one heme- and two alpha 1/beta 1-contacts are exchanged. In the beta-chains the replacements involve one heme-, one alpha 1/beta 1-, and one alpha 1/beta 2-contact. The alpha- and beta-chains of the Common Otter are compared to those of other Carnivora hemoglobins. The unexpected low number of substitutions between Common Otter hemoglobin and that of Lesser Panda as well as of Harbor Seal is discussed.     

Microtubule and microfilament distribution and tubulin content in the cell cycle of Indian muntjac cells

Using DAPI, rabbit antitubulin antibody, FITC-labeled goat anti-rabbit IgG, and TRITC-phalloidin to stain individual cells, the microspectrophotometric analysis showed that three markers that represent the nucleus, microtubules (MT), and microfilaments (MF), respectively, could be recognized in individual cells without interference. The phase of the cell cycle was determined by DNA content. We found that in Indian muntjac (IM) cells, the amount of tubulin in G2 and M phases was about twice as much as that in G1 phase. In G2 cells, the cytoplasmic microtubule complex (CMTC) became denser than in G1 cells. The cytoplasmic MT extent in basically the same orientation as MF bundles in interphase. The regions where the MT is denser also have a denser MF distribution.     

Effects of ATP on Phosphatidylinositol-Phospholipase C and Inositol 1-Phosphate Accumulation in Rat Brain Synaptosomes

Incubation of rat brain synaptosomes prelabeled with [2-3H]inositol resulted in a time-dependent release of labeled inositol 1-phosphate. This process was Ca2+ dependent, and ATP (1 mM) enhanced the inositol 1-phosphate formation three- to fivefold. Using [1-14C]arachidonoyl-phosphatidylinositol which was introduced into saponin-permeabilized synaptosomes, ATP (1 mM) and free Ca2+ (approximately 20 microM) enhanced the phospholipase C hydrolysis of this substrate to form labeled diacylglycerol. When the same permeabilized synaptosomal preparation was incubated with [2-3H]inositol-phosphatidylinositol, ATP not only enhanced the formation of labeled inositol 1-phosphate, but also inhibited the conversion of inositol 1-phosphate to inositol. Furthermore, ATP appeared to reduce the Ca2+ requirement of the phosphatidylinositol-phospholipase C. Inhibition of the conversion of inositol 1-phosphate to inositol could not be overcome by increasing the Mg2+ concentration in the incubation medium. Although the ATP effect is not viewed as a receptor-mediated event, it is possible that such an event may occur in synaptosomes under conditions in which intrasynaptic Ca2+ concentration becomes elevated.