Modification of liver mitochondrial lipids and of adenine nucleotide translocase and oxidative phosphorylation by cold adaptation

The decrease in respiration rate following thyroidectomy is preceded by changes in the lipid composition of the mitochondrial membrane (Hoch, F.L., Subramanian, C., Dhopeshwarkar, G.A. and Mead, J.F. (1981) Lipids 16, 328–334) and in concert, changes in the kinetic parameters of the adenine nucleotide translocase (Mak, I.T., Shrago, E. and Elson, C.E. (1981) Fed. Proc. 40, 398). To demonstrate that physiological adaptation also involves this sequence of events, rats were housed at 8°C for 3–4 weeks. Cold adaptation resulted in a modest (5%) increase in the unsaturation index for the mitochondrial fatty acids comprised of a significant increase in arachidonic acid and a reciprocal decrease in linoleic acid. Phospholipid analysis indicated that cold adaptation increased the mitochondrial phosphatidylethanolamine and reciprocally decreased the phosphatidylcholine content. Concomitantly, cold adaptation resulted in 25–30% increases in rat liver mitochondrial respiratory activities without changing the respiratory control or ADP/O ratios. The kinetic parameters of the adenine nucleotide translocase were determined by the back-exchange method (Pfaff, E. and Klingenberg, M. (1968) Eur. J. Biochem. 6, 66–79). At 0–4 and 10°C, the V_max and K_m of the cold-adapted rat liver adenine nucleotide translocase were not distinguishable from the control values. The K_i values determined by Dixon plot studies for atractylate and palmitoyl-CoA were also comparable between the two groups. However, at 25 and 37°C, cold-adapted rat liver adenine nucleotide translocase exhibited a 20% increase in V_max and a 20% decrease in K_m for external ADP. The results suggest that one adaption to a cold environment involves hormone-mediated changes in the lipid composition in the mitochondrial membranes which in turn modulate the adenine nucleotide translocase and subsequent respiratory activities.     

Expansion of myelopoietic precursors and inhibition of B-cell precursors in mice that express a T-cell receptor gamma (V gamma 1.1J gamma 4C gamma 4) transgene.

Knowledge of the genetic determinants that can affect renewal of multipotential stem cells and their commitment to specific cell lineages is essential to our understanding of multicellular development. However, despite the vast amount of accumulated knowledge in this area, genetic determinants that affect renewal and commitment of precursor cells are unknown. In this study, we demonstrate that three independently derived founder mouse strains, transgenic for the TcR V gamma 1.1J gamma 4C gamma 4 (TcR gamma 4) chain gene, differed significantly from normal mice in their development of T and B cells as well as myelopoietic precursor cells. Ontogenic programs consistent with an acceleration of T-cell development and a delayed appearance and suppressed levels of pre-B- and B-cell precursors were evident in these transgenic mice. In addition, TcR gamma 4 transgenic mice possessed a significantly elevated level of myelopoietic pluripotential precursors. 3H-thymidine cell suicide studies suggest that higher percentages of pluripotent precursors from the bone marrow of the TcR gamma 4 transgenic mice were in the S phase of the cell cycle. These modulations of the lymphoid and myelopoietic compartments, however, were not found in other T-cell receptor transgenic mice (e.g., TcR V gamma 1.2J gamma 2C gamma 2, TcR gamma 2; or V beta 8.1D beta J beta 2.4C beta 2, TcR beta) constructed with the same or similar cDNA expression vector. The results suggest that the expression of a specific T-cell receptor gamma chain gene, and/or an elevated level of particular subset of TcR gamma delta cells, may affect the proliferation and relative proportions of haemopoietic and lymphoid precursors.     

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621

The role of pyruvate metabolism in the triggering of aerobic, alcoholic fermentation in Saccharomyces cerevisiae has been studied. Since Candida utilis does not exhibit a Crabtree effect. this yeast was used as a reference organism. The localization, activity and kinetic properties of pyruvate carboxylase (EC 6.4.1.1), the pyruvate dehydrogenase complex and pyruvate decarboxylase (EC 4.1.1.1) in cells of glucose-limited chemostat cultures of the two yeasts were compared. In contrast to the general situation in fungi, plants and animals, pyruvate carboxylase was found to be a cytosolic enzyme in both yeasts. This implies that for anabolic processes, transport of C4-dicarboxylic acids into the mitochondria is required. Isolated mitochondria from both yeasts exhibited the same kinetics with respect to oxidation of malate. Also, the affinity of isolated mitochondria for pyruvate oxidation and the in situ activity of the pyruvate dehydrogenase complex was similar in both types of mitochondria. The activity of the cytosolic enzyme pyruvate decarboxylase in S. cerevisiae from glucose-limited chemostat cultures was 8-fold that in C. utilis. The enzyme was purified from both organisms, and its kinetic properties were determined. Pyruvate decarboxylase of both yeasts was competitively inhibited by inorganic phosphate. The enzyme of S. cerevisiae was more sensitive to this inhibitor than the enzyme of C. utilis. The in vivo role of phosphate inhibition of pyruvate decarboxylase upon transition of cells from glucose limitation to glucose excess and the associated triggering of alcoholic fermentation was investigated with 31P-NMR. In both yeasts this transition resulted in a rapid drop of the cytosolic inorganic phosphate concentration. It is concluded that the relief from phosphate inhibition does stimulate alcoholic fermentation, but it is not a prerequisite for pyruvate decarboxylase to become active in vivo. Rather, a high glycolytic flux and a high level of this enzyme are decisive for the occurrence of alcoholic fermentation after transfer of cells from glucose limitation to glucose excess.     

Mapping of an adenovirus function involved in the inhibition of DNA degradation.

A function involved in the inhibition of DNA degradation has been assigned through complementation tests to a product of region E1b of the adenovirus genome (between 4.5 and 10.5 map units). DNA degradation induced by the adenovirus type 12 (Ad12) cyt mutant H12cyt70 and the Ad5 early deletion mutant dl313 (with the deletion between 3.5 and 10.7 map units) was inhibited by coinfection with Ad5 region E1a (between 0 and 4.5 map units) mutants dl312 and hr1 and region E1b mutant hr6. The defect of inhibition of DNA degradation in Ad5 dl313 was also complemented in 293 cells. This DNase-inhibitory function does not appear to involve polypeptide IX or the 58,000-dalton polypeptide. Wild-type Ad12 induced DNA degradation in hamster embryo cells, suggesting that the DNase-inhibitory function is not expressed in these nonpermissive cells. Additional evidence suggests the involvement of a second viral product which positively influences the DNase activity and which appears to be an early function.     

Larger precursors of mitochondrial translation products in Neurospora crassa. Indications for a precursor of subunit 1 of cytochrome c oxidase

Specific labeling in vivo of the formylated N termini of mitochondrial translation products revealed that some mitochondrially synthesized proteins were not labeled this way. As a consequence, it was worthwhile considering that larger precursor proteins of mitochondrial translation products exist. Although we used a rapid isolation procedure, only after 2-h of labeling in the presence of cycloheximide, could three additional mitochondrial translation products (molecular mass 45, 36, and 25 kilodaltons) be detected. Preincubation with cycloheximide indicated that these proteins might be larger precursors which were no longer processed due to the prolonged presence of cycloheximide. To prevent processing of the precursors during isolation, cells of the slime mutant were directly lysed in boiling sodium dodecyl sulphate solution. In this way, the same three additional mitochondrial translation products were detected after a pulse-labeling of 1 min. These proteins behave in a precursor-like fashion. Labeling at 9 degrees C resulted in a partial accumulation of the three additional proteins. Finally protein blots treated with antibodies and 125I-labeled protein A, support the idea that the 45-kDa protein is a precursor of subunit 1 of cytochrome c oxidase; 50-80% of this precursor could be detected in the post-mitochondrial supernatant, indicating that this polypeptide is not tightly bound to the membrane.     

Genetics of glucose phosphate isomerase and phosphoglucomutase in Aedes albopictus (diptera: culicidae)

Summary Glucose phosphate isomerase (E.C. 5.3.1.9) and phosphoglucomutase (E.C. 2.7.5.1) were found to be polymorphic in a laboratory colony of Aedes albopictus. The glucose phosphate isomerase locus is represented by two alleles resulting in three genotypes, while the phosphoglucomutase locus is represented by at least five alleles giving rise to a total of 15 genotypes. The inheritance of these two enzymes is of the Mendelian type with codominant alleles. Present data indicate that these genes are not linked.Of 105 mosquitoes analysed for these two gene-enzyme systems, the frequencies for glucose phosphate isomerase alleles are Gpi ^S=0.68 and Gpi ^F=0.32, while the frequencies for phosphoglucomutase alleles are Pgm ^A=0.16, Pgm ^B=0.11, Pgm ^C=0.19, Pgm ^D=0.30 and Pgm ^F= 0.24. The frequencies of the three glucose phosphate isomerase genotypes are in accord with Hardy-Weinberg expectations (X ₁ ² =2.74). Similarly, the frequencies of the 15 phosphoglucomutase genotypes probably do not differ significantly from Hardy-Weinberg expectations (X ₁₀ ² = 18.45).     

Genetic influences on female infidelity and number of sexual partners in humans: a linkage and association study of the role of the vasopressin receptor gene (AVPR1A).

In humans, in contrast to animals, the genetic influences on infidelity are unclear. We report here a large study of over 1600 unselected United Kingdom female twin pairs who confidentially reported previous episodes of infidelity and total lifetime number of sexual partners, as well as attitudes towards infidelity. Our findings demonstrate that infidelity and number of sexual partners are both under moderate genetic influence (41% and 38% heritable, respectively) and the genetic correlation between these two traits is strong (47%). Conversely, attitudes towards infidelity are driven by shared and unique environmental, but not genetic, influences. A genome-wide linkage scan identified three suggestive but nonsignificant linkage areas associated with infidelity and number of sexual partners on chromosomes 3, 7 and 20 with a maximum LOD score of 2.46. We were unsuccessful in associating infidelity or number of sexual partners with a locus implicated in other mammals' sexual behavior, the vasopressin receptor gene. Nonetheless, our findings on the heritability of sexual infidelity and number of sexual partners provide support for certain evolutionary theories of human sexual behavior, as well as justifying further genetic and molecular research in this domain.     

Cystic Fibrosis Transmembrane Conductance Regulator–associated ATP Release Is Controlled by a Chloride Sensor

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl⁻ that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl⁻ conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl⁻ conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl >> Br; R347P, Cl >> Br; R347E, Br >> Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl⁻ binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl⁻. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.