Microbial survivors: thermophiles, halophiles, and other prodigies.

When biologists encounter microbes flourishing in boiling water and other extreme habitats, they often consider such creatures as merely odd, and only search for possible protective mechanisms. But it may also be that extreme habitats resemble those where life first occurred, and that such organisms provide links with earlier evolution.

Thyroid hormone treatment alters phospholipid composition and membrane fluidity of rat brain mitochondria.

We examined effects of graded doses of thyroid hormones 3,3', 5-tri-iodo-L-thyronine (T3) and L-thyroxine (T4) on the lipid composition of rat brain mitochondria. Neither hormone significantly affected the mitochondrial cholesterol or total phospholipid content, but did increase phosphatidylethanolamine (PE) at the expense of phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylcholine (PC). The phosphatidic acid (PA) content was also elevated, suggesting enhanced phospholipid turnover. Changes in sphingomyelin (SPM) and diphosphatidylglycerol (DPG) were minimal. Mitochondrial membrane fluidity also increased after thyroid-hormone treatment, and the increase was closely correlated with PC/PE and SPM/PE molar ratios.

Is respiratory activity in the brain mitochondria responsive to thyroid hormone action?: a critical re-evaluation.

The effects of in vivo treatment with graded doses (0.5-1.5 micrograms/g body weight) of thyroid hormones, tri-iodothyronine (T3) and thyroxine (T4), for 4 consecutive days to euthyroid rats on the respiratory activity of isolated brain mitochondria were examined. T4 stimulated coupled State-3 respiration with glutamate, pyruvate + malate, ascorbate + tetramethyl-p-phenylenediamine and succinate, in a dose-dependent manner; T3 was effective only at the highest (1.5 micrograms) dose employed. T4 was more effective than T3 in stimulating respiratory activity. State-4 respiratory rates were in general not influenced except in the case of the ascorbate + tetramethyl-p-phenylenediamine system. Primary dehydrogenase activities, i.e. glutamate dehydrogenase, malate dehydrogenase and succinate dehydrogenase, were stimulated about 2-fold; interestingly mitochondrial but not cytosolic malate dehydrogenase activity was influenced under these conditions. The hormone treatments did not greatly influence the mitochondrial cytochrome content. The results therefore suggest that thyroid hormone treatment not only stimulates primary dehydrogenase activities but may also directly influence the process of mitochondrial electron transfer.

Comparison of Brucella abortus antigen preparations for in vitro stimulation of immune bovine T-lymphocyte cell lines.

Three Brucella abortus antigen preparations were tested for stimulatory activity with immune bovine T-lymphocyte cell lines in vitro. A total of 32 polyclonal T-lymphocyte cell lines were derived from two steers each from four immunization groups: (1) B. abortus Strain 19 (S19) alone, (2) heat-killed B. abortus whole bacterial cells (HKC) alone, (3) S19 with recombinant human interleukin 2 (rHuIL-2), (4) HKC with rHuIL-2. Peripheral blood mononuclear cells were isolated at 2 and 9 weeks post immunization and cultured in vitro with either HKC antigen or B. abortus soluble antigen (BASA) with recombinant bovine interleukin 2 (rBoIL-2) to initiate four cell lines per steer. Sixteen of the resulting T-lymphocyte cell lines (from the S19 and S19+IL-2 groups) were tested through indirect immunofluorescence for expression of cell surface markers CD2, CD4, CD6, CD8, major histocompatibility complex (MHC) Class II molecules and a marker expressed on a subset of helper T-lymphocytes (Th) as well as sIgM, CD1 and a MHC Class II+ monocyte/macrophage marker. The T-lymphocyte cell lines were used to evaluate antigen-induced lymphoproliferative (LP) responses in a titration assay with HKC, BASA and gamma-irradiated B. abortus (gamma BA) antigens. The results indicate that most of the cells in many of the cell lines were typical activated T-lymphocytes as determined by surface marker expression and included cells positive for all T-lymphocyte markers tested. The cell lines contained no B-lymphocytes or mononuclear phagocytes. However, two cell lines contained significant populations (> 80%) of CD2-, CD4-, CD6-, CD8- cells that were both responsive to exogenous rBoIL-2 and were capable of exhibiting antigen-induced LP responses. In 22 of the 32 cell lines tested, gamma BA was superior to HKC at nearly every concentration tested in stimulating LP responses. This observation was independent of the immunization used to prime the T-lymphocytes in vivo. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins with relative molecular masses common to all three antigen preparations as well as significant (P < 0.05) quantitative and qualitative differences in individual proteins between HKC and gamma BA. Together, the results suggest gamma BA may provide an in vitro antigenic stimulus which is deficient in HKC.

Uptake of inorganic pyrophosphate by Bacillus megaterium.

Cells of Bacillus megaterium take up inorganic pyrophosphate, employing a saturable carrier which is sensitive to sulfhydryl reagents, orthophosphate, and arsenate. Uptake is stimulated by proton ionophores, including CCCP and nigericin, indicating that proton cotransport can lead to an opposing gradient. Inhibitor sensitivity, as well a relatively high Km for inorganic pyrophosphate render it likely that uptake is mediated by an orthophosphate transport system.

Evidence for the ordered release of rubidium ions occluded within the Na,K-ATPase of mammalian kidney.

When Na,K-ATPase containing occluded rubidium ions is exposed to orthophosphate, in the presence of magnesium ions, there is a rapid release of half or all of the occluded ions. This behaviour is observed irrespective of whether the occluded-rubidium form of the enzyme is generated by putting the unphosphorylated enzyme in a sodium-free medium containing rubidium ions, or by allowing rubidium ions to catalyse the hydrolysis of phosphoenzyme made by adding ATP to enzyme suspended in a medium containing sodium and magnesium ions. The release of occluded rubidium ions by orthophosphate requires the presence of magnesium, presumably because phosphorylation is necessary. Whether the addition of orthophosphate causes the rapid release of all or of half of the occluded rubidium depends on whether free rubidium (or potassium, thallium or (probably) caesium ions) are present in the medium at the time the orthophosphate is added. In the absence of free ions of these species, all of the occluded rubidium is released. In their presence (in adequate concentration), only half of the occluded rubidium is released. The relative effectiveness of the different potassium congeners in preventing the rapid release of 50% of the occluded rubidium when orthophosphate is added is: thallium greater than rubidium greater than potassium greater than caesium. Lithium and sodium are ineffective even at high concentrations, and sodium ions strongly antagonize the effect of free rubidium ions. In a sodium-free, Tris medium, the concentration of free rubidium ions necessary for a half-maximal effect is about 30 microM. In a medium containing 250 microM-free rubidium, the concentration of sodium necessary to reduce the effect of free rubidium by 50% is about 500 microM. These figures are compatible with the hypothesis that the free rubidium or other ions act at the potassium-loading sites at the extracellular face of the pump. By starting with enzyme occluding unlabelled rubidium, and using 86Rb-labelled free rubidium, it is possible to show that the free ions that prevent the rapid release of half of the occluded ions themselves become occluded. These experiments are significant in two ways. First, they provide direct evidence for the existence of a second route for the release of occluded rubidium (and therefore presumably of occluded potassium) ions. Secondly, they seem to require that the release of occluded ions by this route occurs in an ordered fashion.

Influence of anion flux upon mitochondrial oxidative phosphorylation.

We have measured mitochondrial ATP synthesis during passive anion influx and find that influx of phosphate leads to diminished efficiency (as reflected in the ATP:0 ratio) whereas influx of acetate produced enhanced efficiency. The anions, sulfate, propionate, and thiocyanate, are without influence on the ATP:0 ratio. It is likely that the opposite effects of phosphate and acetate on the ATP:0 ratio reflect phosphate-acetate exchange, and that acetate influx produces its positive effect on ATP synthesis by promoting phosphate efflux. Thus, phosphate efflux may be associated with increased, and phosphate influx, with decreased energy conservation.

Inhibition of erythrocyte plasma membrane NADH dehydrogenase by nucleotides and uncouplers.

Erythrocyte ghost NADH dehydrogenase is inhibited in a competitive fashion by ATP and ADP whereas other nucleoside di- and triphosphates, cyclic nucleosides, as well as non-phosphorylating ATP analogs are relatively ineffective. In addition, this enzyme, measured with ferricyanide as electron acceptor, is inhibited by uncouplers of oxidative phosphorylation (proton-conducting reagents), the inhibition being competitive in character (i.e., the uncouplers were without influence upon maximum velocity). The effectiveness of the uncouplers was in the order of their hydrophobic character with the presence of the alkyl side chain rendering nonyl-dinitrophenol much more active than 2,6-dinitrophenol itself. Hydrophobic compounds that are not protonophores (e.g., eosin, proflavin or valinomycin) were not inhibitory. Whereas adenine nucleotides probably inhibit NADH oxidation competitively through structural similarity with the substrate, it appears unlikely that uncouplers compete at the NADH site directly. Rather, the apparently-competitive inhibition in the latter case may reflect competition for proton transfer to an acceptor residing in a hydrophobic region of the enzyme complex.

Sphingolipid metabolism during infection of human fibroblasts by herpes simplex virus type 1.

Results from several experiments support the view that sphingomyelin turnover and glycosphingolipid synthesis play a role in herpes simplex virus infection of cells in culture. Inhibition of sphingomyelinase by phosphatidylcholine or by a synthetic ceramide, N-palmitoyl-DL-dihydrosphingosine, interferes substantially with virus reproduction as does a genetic defect in the enzyme (Niemann-Pick disease). In Niemann-Pick cells, yields of infectious virus are about 3% of the normal level. An inhibitor of ceramide: UDP glucose glucosyltransferase was also used to test the effect of altered glycosphingolipid synthesis on infection. This compound, DL-2-decanoylamino-3-morpholinopropiophenone, decreased virus yields to a level about 0.1% of normal at the highest concentration tested.

Incorporation of 32P-phosphate into membrane phospholipids during infection of cultured human fibroblasts by herpes simplex virus type 1.

Infection of cultured human skin fibroblasts by herpes simplex virus leads to increased incorporation of labeled inorganic phosphate into host membrane sphingomyelin. Incorporation into the other major membrane phospholipids, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine, was unaffected. Since sphingomyelin has been shown to serve as precursor to ceramides (via sphingomyelinase) in monkey kidney cells and since enhanced synthesis of ceramide-based glycolipids has been shown to occur after herpes simplex virus infection, we suggest that the observed increase in labeling of sphingomyelin may reflect mobilization for glycolipid synthesis. The distribution of phosphate label among the phospholipids of the viral envelope was identical to that among the phospholipids of the cellular cytoplasmic membrane fraction and differed from that of the nuclear fraction.

Energy coupling in liver mitochondria from dystrophic mice: differential sensitivity of oxidative phosphorylation and Ca2+ uptake to K+.

Studies were carried out to examine oxidative phosphorylation, cation uptake, and electrokinetic properties of liver mitochondria from genetically dystrophic mice in comparison with those from livers of littermate controls. While no differences were seen with respect to the rates of substrate oxidation, ADP/oxygen ratio, and RCl and cytochrome content, the mitochondria from the dystropic group were characterized by an elevated basal ATPase activity in the presence of NaCl. Additionally, these mitochondria were highly sensitive to high concentrations of exogenously added K+ that, besides stimulating state 4 respiration, caused uncoupling in the mitochondria. These mitochondria accumulated Ca2+ at a higher rate, and unlike the controls, Ca2+ uptake was not sensitive to exogenously added K+. It was also observed that the net negative charge on mitochondria decreased significantly in the dystrophic state. It is thus apparent that muscular dystrophy manifests itself also in terms of alteration in the membrane properties of liver mitochondria.

Erythrocyte lipids in heterozygous carriers of duchenne muscular dystrophy.

Erythrocyte membranes from heterozygous carriers of Duchenne muscular dystrophy exhibit a diminished amount of palmitoleic acid when compared to membranes from normal subjects. A similar, but more variable, diminution is observed in the case of patients with this disorder. The change in fatty acid composition appears related to a low membrane triglyceride content and may provide both a possible technique for carrier detection and a clue regarding pathogenesis.

Erythrocyte surface membrane alterations.

Electrophoretic mobility measurements were made of red blood cells obtained from patients with Duchenne and myotonic muscular dystrophy, from dystrophic mice and chickens, and from corresponding controls. Alterations in the erythrocyte surface electrokinetic properties were found in dystrophic mice and chickens and in many, but not all, patients with muscular dystrophy. The results are consistent with the concept of muscular dystrophy as a systemic membrane disease not limited to muscle.

Elevated erythrocyte phospholipase A associated with Duchenne and myotonic muscular dystrophy.

Erythrocytes from patients with Duchenne or myotonic muscular dystrophy exhibit substantially increased phospholipase A activity when compared to those from normal subjects. Incubation of dystrophic erythrocyte membranes with diphenylhydantoin lowers the phospholipase A activity to within normal limits while the drug does not diminish activity in membranes from normal cells. Elevated phospholipase A activity may be expected to lead to increased concentrations of lysophospholipids which may contribute to the membrane dysfunction shown to be associated with these diseases.