Quantitation of the förster energy transfer for two-dimensional systems: II. Protein distribution and aggregation state in biological membranes

Analytical solutions are presented of the average rate of the Förster energy transfer for several processes affecting intrinsic membrane proteins within a phospholipid bilayer. The physical phenomena considered here are lateral phase separation of the protein, i.e., formation of eutectic mixtures, changes in the aggregation state of the protein and non-random distribution of protein molecules. It is shown that the average rate of energy transfer among protein and phospholipid molecules labelled with donor and acceptor molecules, respectively, allows differentiation between them and also that the average rate of energy transfer can be used to quantitate these phenomena.     

Quantitation of the förster energy transfer for two-dimensional systems: I. Lateral phase separation in unilamellar vesicles formed by binary phospholipid mixtures

An analytical solution is presented for the rate of energy transfer in unilamellar vesicles formed by binary mixtures of phospholipids showing lateral phase separation. The analytical approach developed here is mainly based on geometrical considerations and therefore, is formally different for lateral phase separation phenomena taking place in the gel and in the liquid crystalline states of the lipid system. The rate of energy transfer among donor and acceptor molecules attached to chemically different phospholipids is mathematically correlated to the average cluster size of the less-rich component of the binary mixture, thus allowing its calculation from experimental measurements. Moreover, the equations derived here permit the calculation of the average cluster size as a function of the concentration of each lipid component within certain ranges, and this can be used to improve our knowledge of the thermodynamics of these processes.     

Incorporation of adenosine into nucleotides of chromaffin cells maintained in primary cultures

Extracellular adenosine was incorporated into nucleotides of bovine chromaffin cells maintained in primary culture. In intact chromaffin tissue, a very low incorporation was found (0.8 pmol/10⁶ cells/h at an adenosine concentration of 11.45 μM), which increased 282 times in freshly isolated chromaffin cells. When maintained in primary culture, this value decreased to a value similar to that of chromaffin tissue, but later on, and in the presence of nerve growth factor (NGF), a time dependent increase of adenosine incorporation was observed which, in 84-h old cells reached up to 54 times more than that found in intact tissue. This incorporation might reflect changes in the adenosine transport at the cell membrane level, furthered by NGF effect. Incorporation, which was time-dependent, was weakly modified by stimulation of cells with 10^?4 M acetylcholine. However, acetylcholine-induced release of labelled nucleotides from chromaffin granules was observed, probably in relation to granule maturation.     

Phenylethanolamine-N-methyltransferase:alcohol and the mechanism of action

Mixed-solvent systems of methanol and other alcohols and water were used to study the properties of bovine phenylethanolamine-N-methyltransferase. The presence of methanol decreased the binding affinity of the enzyme for its amine substrate but did not alter the maximum velocity. The change in binding was accompanied by an alkaline shift in the pK of an ionizable group in the active site. The well-known property of enzyme inhibition by substrate was also alleviated. Increasing the pH of the medium, in the presence or absence of methanol, increased the maximum velocity but did not alter substrate inhibition. It is proposed that substrate inhibition is due in part to the ionic state of a single unidentified ionizable group in the active site of the enzyme and to a slow release of product. Evidence that an essential, pH-dependent sulfhydryl modulates product release is presented. The properties of phenylethanolamine-N-methyl-transferase are quite responsive to changes in pH, ionic strength, and water content so that the enzyme may well be regulated at the microenvironmental level.     

Exact versus heuristic models of kin selection

This paper examines the conditions under which the classical inclusive fitness formulation of Hamilton (1964) provides an adequate approximation to the dynamics of gene frequency change and to conditions for genetic equilibrium, in the “additive” model of altruism between sibs of Uyenoyama and Feldman (1981). It is concluded that the classical formulation is adequate, provided that either the effect of the gene on the probability of behaving altruistically is low or the costs and benefits of altruism are small, unless the benefit/cost ratio k is very close to 2, the value that must be exceeded for altruism to be favoured. In addition, the gene for altruism must be underdominant, recessive or partially recessive in its effect on the probability of behaving altruistically, for the inclusive fitness predictions to break down significantly.     

Reversible inactivation of Saccharomyces cerevisiae glutathione reductase under reducing conditions

Glutathione reductase from Saccharomyces cerevisiae was rapidly inactivated following aerobic incubation with NADPH, NADH, and several other reductants, in a time- and temperature-dependent process. The inactivation had already reached 50% when the NADPH concentration reached that of the glutathione reductase subunit. The inactivation was very marked at pH values below 5.5 and over 7, while only a slight activity decrease was noticed at pH values between these two values. After elimination of excess NADPH the enzyme remained inactive for at least 4 h. The enzyme was protected against redox inactivation by low concentrations of GSSG, ferricyanide, GSH, or dithiothreitol, and high concentrations of NAD(P)+; oxidized glutathione effectively protected the enzyme at concentrations even lower than GSH. The inactive enzyme was efficiently reactivated after incubation with GSSG, ferricyanide, GSH, or dithiothreitol, whether NADPH was present or not. The reactivation with GSH was rapid even at 0 degree C, whereas the optimum temperature for reactivation with GSSG was 30 degrees C. A tentative model for the redox interconversion, involving an erroneous intramolecular disulfide bridge, is put forward.     

Septate junctions between digestive vacuoles in human malacoplakia

Typical septate junctions between digestive vacuoles in phagocytic cells of human malacoplakia are described in this paper. Evidence for a honeycomb pattern of hexagonal subunits is presented for their cleft material. Junctions were not observed between other organelles or in cells other than phagocytes. It is assumed that the septate junctions described here may reflect a pathological change in the organization of the membrane components of digestive organelles.     

The effect of diabetes, nutritional factors, and sex on rat liver and kidney mevalonate kinase, mevalonate-5-phosphate kinase, and mevalonate-5-pyrophosphate decarboxylase

Isopycnic sucrose gradient separation of rat liver organelles revealed the presence of two distinct branched-chain α-keto acid decarboxylase activities; a mitochondrial activity, which decarboxylates the three branched-chain α-keto acids and requires CoA and NAD⁺ and a cytosolic activity, which decarboxylates α-ketoisocaproate, but not α-ketoisovalerate, or α-keto-β-methylvalerate. The latter enzyme does not require added CoA or NAD⁺. Assay conditions for the cytosolic α-ketoisocaproate decarboxylase activity were optimized and this activity was partially characterized. In rat liver cytosol preparations this activity has a pH optimum of 6.5 and is activated by 1.5 m ammonium sulfate. The decarboxylase activity has an apparent K_m of 0.03 mm for α-ketoisocaproate when optimized assay conditions are employed. Phenylpyruvate is a very potent inhibitor. α-Ketoisovalerate, α-keto-β-methylvalerate, α-ketobutyrate, and α-ketononanoate also inhibit the α-ketoisocaproate decarboxylase activity. The data indicate that the soluble α-ketoisocaproate decarboxylase is an oxidase. Rat liver cytosol preparations consumed oxygen when either α-ketoisocaproate or α-keto-γ-methiolbutyrate were added. None of the other α-keto acids tested stimulated oxygen consumption. 1-¹⁴C-Labeled α-keto-γ-methiolbutyrate is also decarboxylated by cytosol preparations. The α-ketoisocaproate oxidase was purified 20-fold from a 70,000g supernatant fraction of a rat liver homogenate. In these preparations the activity was increased 4-fold by the addition of dithiothreitol, ferrous iron, and ascorbate. The major product of this enzyme activity is β-hydroxyisovalerate. Isovalerate is not a free intermediate in the reaction. The data indicate an alternative pathway for metabolism of α-ketoisocaproate which produces β-hydroxyisovalerate.     

Studies on the induction of histocompatibility gene mutations in germ cells of mice by chemical mutagens and/or virus-inducing compounds

This work continues earlier studies concerning the use of histocompatibility mutations in mammalian germ cells as a mutagenicity test system (H test). The rate of spontaneous H mutations was re-examined using a new basis for the classification of H mutants. This procedure led to very high frequencies of suspected spontaneous H mutants: among C57Bl/6 mice, 6% and among C3H mice, 9%. F₂ hybrids of a cross between these strains revealed 1% suspected H mutants. Using the same procedure, the sensitivity of the H test was examined with the mutagens ethylnitrosourea, benzo[a]pyrene, 2-acetylaminofluorene (2-AAF), with the solvent dimethyl sulfoxide (DMSO) and with the antibacterial nitrofurantoin. It was possible to demonstrate the mutagenic potential of all mutagens tested as well as their specific action on the different stages of male germ cell development. We succeeded in demonstrating the mutagenicity of 2-AAF for the first time in germ cells of a mammal. In contrast to the negative result with benzopyrene (BP) in the specific locus test, BP induced H mutants even at the very low dose of 2 mg/kg. DMSO was found to induce H mutations in spermatogonia. This extraordinary result is possibly due to the virus-inducing properties of this compound. Nitrofurantoin which is often used in treating bacterial infections of the urinary tract in humans showed a very stage-specific action on maturing spermatids. The value of the H test for mutagenicity testing is discussed with respect to its sensitivity and economy. The very high spontaneous frequency of suspected H mutants and the ease of inducing incraased mutant frequencies by mutagens and by DMSO suggest the possibility that the majority of the histoincompatibilities found in the H test are due to induced antigenic gene products of endogenous viruses. This, however, does not interfere with the applicability of the H test for mutagenicity testing, but rather seems to augment its sensitivity to alkylating mutagens as well as mutagens which probably cause frameshift mutations.Other tests for mutations and/or inherited tumor proneness using mouse germ cells can easily be combined with the H test, because the test animal does not have to be killed, thus reducing the cost of the test.     

Mitochondrial respirasome works as a single unit and the cross-talk between complexes I,III2 and IV stimulates NADH dehydrogenase activity

Ustilago maydis is an aerobic basidiomycete that depends on oxidative phosphorylation for its ATP supply, pointing to the mitochondrion as a key player in its energy metabolism. Mitochondrial respiratory complexes I, III₂, and IV occur in supramolecular structures named respirasome. In this work, we characterized the subunit composition and the kinetics of NADH:Q oxidoreductase activity of the digitonine-solubilized respirasome (1600 kDa) and the free-complex I (990 kDa). In the presence of 2,6-dimethoxy-1,4-benzoquinone (DBQ) and cytochrome c, both the respirasome NADH:O₂ and the NADH:DBQ oxidoreductase activities were inhibited by rotenone, antimycin A or cyanide. A value of 2.4 for the NADH oxidized/oxygen reduced ratio was determined for the respirasome activity, while ROS production was less than 0.001% of the oxygen consumption rate. Analysis of the NADH:DBQ oxidoreductase activity showed that respirasome was 3-times more active and showed higher affinity than free-complex I. The results suggest that the contacts between complexes I, III₂ and IV in the respirasome increase the catalytic efficiency of complex I and regulate its activity to prevent ROS production.     

Effect of cysteine,glutamate and glycine supplementation to in vitro fertilization medium during bovine early embryo development

Glutathione (GSH) is an antioxidant synthesized from three constitutive amino acids (CAA): cysteine (Cys), glycine (Gly) and glutamate (Glu). Glutathione plays an important role in oocyte maturation, fertilization and early embryo development. This study aimed to investigate the effect of Cys (0.6 mM), Gly (0.6 mM) and Glu (0.9 mM) supplementation during in vitro fertilization (IVF) of cattle oocytes. In a Pilot Experiment, de novo synthesis of GSH in bovine zygote was evaluated using a modified TALP medium prepared without MEM-essential and MEM-non-essential amino acids (mTALP): mTALP + CAA (constitutive amino acids); mTALP + CAA+5 mMBSO (buthionine sulfoximide); mTALP + Cys + Gly; mTALP + Cys + Glu and mTALP + Gly + Glu. This evidence led us to investigate the impact of CAA supplementation to TALP medium (with essential and non-essential amino acids) on zygote viability, lipid peroxidation, total intracellular GSH content (include reduced and oxidized form; GSH-GSSG), pronuclear formation in zygotes and subsequent embryo development. IVF media contained a) TALP; b) TALP + Cys + Gly + Glu (TALP + CAA); c) TALP + Cys + Gly; d) TALP + Cys + Glu; e) TALP + Gly + Glu, were used. Total GSH-GSSG concentration was increased in TALP, TALP + CAA, and TALP + Cys + Gly. The viability of zygote was similar among treatments. Lipid peroxidation was increased in zygote fertilized with TALP + Cys + Gly; TALP + Cys + Glu; TALP + Gly + Glu and TALP + CAA. The percentage of penetrated oocytes decreased in TALP + CAA and TALP + Cys + Gly. The cleavage rate was lower in TALP + CAA and TALP + Gly + Glu. The percentage of embryos developing to the blastocyst stage was lower in TALP + Cys + Glu and TALP + CAA. In conclusion, we have demonstrated the synthesis of GSH during IVF. However, Cys, Gly and Glu supplementation to TALP medium had negative effects on embryonic development.     

Stimulatory effects of 5 beta-dihydrotestosterone on the sexual behavior in the domestic chick

During in vitro incubations, brain tissues of chicks transform testosterone mainly into 5β-reduced androgens including 5β-dihydrotestosterone (β-DHT). Although β-DHT is generally believed to have no androgenic effects, we showed recently that it stimulates sexual behavior in young chicks during hand thrust tests. This result was confirmed during three experiments presented here and the possible interactions of β-DHT with the endogenous sex steroids have been analyzed. There is no synergism between β-DHT and estradiol in the induction of sexual behavior in the chicks. β-DHT is still partly active in chicks injected with the antiandrogen, cyproterone acetate in doses sufficient to block all action of the endogenous testosterone. β-DHT is also active in castrated chicks. The effects of β-DHT on the sexual behavior thus do not seem to depend on an interaction with other sex steroids and the reasons why it is active in chicks during hand thrust tests and not in other test situations in other birds are briefly discussed.     

Random recirculation of small T lymphocytes from thoracic duct lymph in the mouse

The migration of ⁵¹Cr-labeled nylon-wool separated mouse thoracic duct T cells has been followed in order to determine whether there is a circulation of small (nondividing) T cells through the small intestine. Approximately 6% of the injected dose of T-TDL localized in the small intestine (minus Peyer's patches). Experiments revealed that this gut-localizing cell population consisted almost entirely, if not exclusively, of lymphoblasts present in mouse T-TDL. When lymphoblasts and small lymphocytes from mouse T-TDL were separated by velocity sedimentation, and the migration of separated fractions was studied, we found large cells (66% blasts) migrated well to the gut but poorly to the lymph nodes, whereas small cells (2% blasts) showed minimal migration to the gut but localized randomly in lymph nodes and spleen. The in vivo distribution of small cells from T-TDL was similar to that of T-PLN. Furthermore, the recirculatory patterns of both ⁵¹Cr-labeled T-TDL and T-PLN were found to be identical as accessed by their rate of recovery in the thoracic duct lymph of recipient mice. These results support the notion that the vast majority of T-TDL and T-PLN are part of a common pool of recirculating T cells which recirculate randomly through lymph nodes and spleen and not the small intestine.     

The effects of different thymidine concentrations on DNA replication in pea-root cells synchronized by a protracted 5-fluorodeoxyuridine treatment

Single-cell and DNA fiber autoradiography, cytophotometry and velocity sedimentation in alkaline sucrose gradients were used to analyse DNA replication and nascent replicon maturation in 5-fluorodeoxyuridine (FUdR)-synchronized cells of Pisum sativum. The replicon size was not significantly changed by the protracted FUdR treatment. When the synchronized cells were released from the inhibitor, labeled with [³H]TdR for 30 min, and chased in medium containing 1 × 10⁻⁶ M or lower concentrations of cold thymidine, DNA replication stopped after approx. 25% of the genome had replicated, and the nascent strands failed to grow above 9–12 × 10⁶ D single-stranded (ss) DNA. When the cells were chased in medium with 1 × 10⁻⁵ M cold thymidine, the DNA content of the labeled cells steadily increased with time and the size of the nascent molecules grew continuously until replicon size was achieved; then they were accumulated at replicon size until the cells arrived in late S or G2. When the FUdR-synchronized cells were chased in medium containing 1 × 10⁻⁴ M cold thymidine, the size of the nascent strands increased continuously with time, indicating that some neighbouring nascent replicons were joined as soon as they completed their replication. These observations led us to postulate that in FUdR-synchronized cells the rates of chain elongation, cell progression through the S phase and nascent replicon maturation are controlled by thymidine availability.     

A DNA-binding fraction of mouse kidney 3-ketosteroid reductase: Comparison with androgen receptors

Since approximately 1% of 3-ketosteroid reductase (which metabolizes dihydrotestosterone [17β-hydroxy-5α-androstan-3-one] to 5α-androstane-3α,17β-diol or 5α-androstane-3α,17β-diol) from mouse kidney cytosol adheres to DNA under conditions that allow virtually complete androgen receptor binding, these two DNA-binding activities were compared in cytosol extracts of mouse kidney and hypothalamus-preoptic area. This DNA-binding fraction of 3-ketosteroid reductase was distinguished from androgen receptor in several ways: (1) its pattern of elution from DNA-cellulose with steps of increasing NaC1 concentration differed from that for receptors from wild-type kidney; (2) it was influenced differently by the mutation Tfm, both in level and in DNA-cellulose elution pattern; (3) in mouse kidney cytosol it was relatively stable at moderate (25°C) temperatures which rapidly inactivated ligand-free androgen receptors in the same cytosols; (4) the DNA-binding was not proportional to androgen receptor levels between two wild-type tissues, the hypothalamus-preoptic area and kidney. By these criteria, a simple relationship of androgen receptors and a DNA-binding fraction of 3-ketosteroid reductase activity is unlikely.     

The influence of pH on Staphylococcus saprophyticus iron metabolism and the production of siderophores

Staphylococcus saprophyticus is a gram-positive coagulase negative bacteria which shows clinical importance due to its capability of causing urinary tract infections (UTI), as well as its ability to persist in this environment. Little is known about how S. saprophyticus adapts to the pH shift that occurs during infection. Thus, in this study we aim to use a proteomic approach to analyze the metabolic adaptations which occur as a response by S. saprophyticus when exposed to acid (5.5) and alkaline (9.0) pH environments. Proteins related to iron storage are overexpressed in acid pH, whilst iron acquisition proteins are overexpressed in alkaline pH. It likely occurs because iron is soluble at acid pH and insoluble at alkaline pH. To evaluate if S. saprophyticus synthesizes siderophores, CAS assays were performed, and the results confirmed their production. The chemical characterization of siderophores demonstrates that S. saprophyticus produces carboxylates derived from citrate. Of special note is the fact that citrate synthase (CS) is down-regulated during incubation at acid pH, corroborating this result. This data was also confirmed by enzymatic assay. Our results demonstrate that iron metabolism regulation is influenced by different pH levels, and show, for the first time, the production of siderophores by S. saprophyticus. Enzymatic assays suggest that citrate from the tricarboxylic acid cycle (TCA) is used as substrate for siderophore production.