The binding of carbon monoxide to cytochrome c oxidase.

The effect of CO on the optical absorbance spectrum of partially reduced cytochrome c oxidase has been studied. The changes at 432 and 590 nm suggest that the cytochrome alpha2/3+ - CO compound is formed preferentially and that concomitantly a second electron is taken up by the enzyme. From the CO-induced changes at 830 nm it is concluded that in the partially reduced enzyme addition of CO causes reoxidation of the copper component of cytochrome c oxidase. Addition of CO to partially reduced enzyme (2 electrons per 4 metal ions) also brings about a decrease in the intensities of electron paramagnetic resonance signals of high-spin heme iron near g = 6 and of the low-spin heme at g = 2.6. Concomitantly both the low-spin heme a signal at g = 3 and the copper signal at g = 2 increase in intensity. These results demonstrate that formation of the reduced diamagnetic cytochrome a3 - CO compound is accompanied by reoxidation of both the copper component detectable by electron paramagnetic resonance and possibly also by cytochrome a.     

Body composition and energetic efficiency in two lines of mice selected for rapid growth rate and their F1 crosses

Summary Correlated responses to selection for increased growth rate were compared in two mouse populations (M16 and H₆) of distinct genetic origin. Traits studied were body composition, feed intake, constituent gains and energetic efficiency. When compared with their respective controls (ICR and C₂) at 6 and 9 weeks of age, body weight increased more in M16 (57%and 69 % of the control mean) than in H₆ (40 % and 34%). The M16 showed correlated responses in fat percent of 2.6% (P <.05), 8.4% (P <.01) and 11.2% (P <.01) at 3, 6 and 9 weeks, respectively, whereas corresponding values in H₆ were –2.4% (P <.05), 3.3% (P <.05) and 2.09 % (P >.05). The correlated responses in fat percent were 2.7 and 4.7 times higher in M16 than H₆ at 6 and 9 weeks. The regression of ln fat weight on ln empty body weight was larger in M16 (P <.05) compared to ICR and larger (P <.01) in H₆ compared to C₂. Both M16 and H₈ exhibited positive correlated responses from 3 to 6 weeks of age in feed intake and gain and efficiency in fat, protein, calories and ash; fat and caloric gain and efficiency exhibited higher correlated responses in M16 than H₆. During the 6- to 9-week interval, the M16 population continued to evince positive correlated responses in gains and efficiencies of fat, protein and calories, whereas H₆ did not. Several possible explanations are presented to account for the differences in correlated responses between the selected populations. Partitioning of correlated response differences between M16 and H₆ into average direct and average maternal genetic effects indicated that average direct genetic effects, favoring M16, were responsible for the major difference between the selected populations. Direct heterosis in F₁ crosses of the selected populations were generally not significant, although there was a tendency for fat percent and fat weight to show heterosis.Paper No. 4929 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina 27607, Animal Research Institute Contribution No. 624 and Agricultural University at Wageningen Contribution No. 654-490-10. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Experiment Station of the Products named, nor criticism of similar ones not mentioned.On leave from Department of Animal Husbandry, Agricultural University, Wageningen, The Netherlands.On leave from Animal Research Institute, Agriculture Canada, Ottawa, Ontario K1A OC6.     

Direct and maternal genetic effects on body weight maturing patterns in mice

Summary Direct and maternal genetic effects were evaluated for maturing patterns of body weight in mice using a crossfostering design. Crossfostering was performed in one group using dams from populations selected for rapid growth rate (M16 and H₆) and their reciprocal F₁. crosses. A second crossfostering group consisted of dams from the respective control populations (ICR and C₂) and their reciprocal F₁. 's. Population differences were partitioned into direct and maternal effects due to genetic origin, correlated selection responses, heterosis and cytoplasmic or sex-linked effects. Degree of maturity was calculated at birth, 12, 21, 31 and 42 days of age by dividing body weight at each age by 63-day weight. Absolute and relative maturing rates were calculated in adjacent age intervals between birth and 63 days. Genetic origin effects (ICR vs. C₂; M16 vs. H₆) were significant for many maturity traits, with average direct being more important than average maternal genetic effects. In general, correlated responses to selection for maturity traits were larger in the M16 population (M16 vs. ICR) than in the H₆ population (H₆ vs. C₂) and correlated responses in average direct effects were larger than average maternal effects. Positive correlated responses in average direct effects were found for relative maturing rates at all ages and for absolute maturing rates from 31 to 63 days. Apparent correlated responses in degree of maturity were negative for M16 and H₆. However, further analysis suggested that the correlated response for degree of maturity in H₆ may be positive at later ages and negative at earlier ages. Direct and maternal heterosis for degree of maturity was positive in the selected and control crosses. Absolute and relative maturing rates showed positive heterosis initially, followed by negative heterosis. Reciprocal differences due to the cytoplasm or sex-linkage were not important for patterns of maturity.Paper No. 5244 the Journal Series of the North Carolina Agricultural Experiment Station, Ealeigh, Animal Research Institute Contribution No. 683 and Agricultural University at Wageningen Contribution No. 654–490–12On leave from the Animal Research Institute, Agriculture Canada at Ottawa, OntarioOn leave from the Department of Animal Husbandry, Agricultural University at Wagenitgen, the Netherlands     

Lymphokines in sensitized rats. III. Influence of prednisolone treatment of lymph node and spleen architecture in sensitized rats and upon MIF release in vitro.

The influence of prednisolone (corticosteroid, C.S.) treatment upon established cell-mediated immunity (CMI) induced by complete Freund's adjuvant (CFA) has been studied in rats by using in vitro migration inhibitory factor (MIF) assays, type IV skin reactions, and regional lymph node and spleen histology. Additionally, changes in the mononuclear-polymorphonuclear ratio of peripheral blood and T-cell accumulation in bone marrow in response to C.S. treatment have been determined. These results have been evaluated by comparison with equivalent experiments upon animals treated with anti-rat lymphocyte serum (ALS), oxisuran, or 2-[(methylsulfinyl)-acetyl]pyridine, which selectively suppresses CMI. The results suggest the existence of a population of “educated” T-cells in the thymic cortex of sensitized rats, and they suggest that prolonged C.S. administration does not suppress T-effector cells involved in established CMI but, rather, affects lymphocyte and monocyte migration patterns, including the migration of educated T-cells from the thymic cortex into other tissue compartments.     

Comparative plankton investigations in a brackish lake (L. Veere) and a brackish tidal estuary (Westerschelde) in the SW Netherlands

Summary As the data, presented in this lecture, have been published elsewhere (see references), only a summary is given here.A comparison was made between the plankton assemblages of the mesoand polyhaline ranges of Lake Veere and the Westerschelde estuary. Species composition, densities and seasonal distributions of the predominant planktonic organisms of the investigated brackish water ecosystems were quite different, depending on several other environmental factors than salinity.     

[3H]MK-801 Binding to N-Methyl-d-Aspartate Receptors Solubilized from Rat Brain: Effects of Glycine Site Ligands, Polyamines, Ifenprodil, and Desipramine

The N-methyl-D-aspartate (NMDA) receptor is thought to contain several distinct binding sites that can regulate channel opening. In the present experiments, the effects of ligands for these sites have been examined on [3H]MK-801 binding to a soluble receptor preparation, which had been passed down a gel filtration column to reduce the levels of endogenous small-molecular-weight substances. Glycine site agonists, partial agonists, and antagonists gave effects similar to those observed in membranes [EC50 values (in microM): glycine, 0.31; D-serine, 0.20; D-cycloserine, 1.46; (+)-HA-966, 4.06; and 7-chlorokynurenic acid, 1.81]. Spermine and spermidine enhanced [3H]MK-801 binding to the soluble receptor preparation (EC50, 4.3 and 20.1 microM, respectively), whereas putrescine and cadaverine gave small degrees of inhibitions. When spermine and spermidine were tested under conditions where [3H]MK-801 binding approached equilibrium, their ability to enhance [3H]MK-801 binding was much reduced, a result suggesting that the polyamines increase the rate to equilibrium. Putrescine antagonised the effects of spermine. Ifenprodil reduced [3H]MK-801 binding under both equilibrium and nonequilibrium conditions, although the high-affinity component of inhibition described in membranes was not observed. Ifenprodil antagonised spermine effects in an apparently noncompetitive manner. Desipramine was able to give total inhibition of specific [3H]MK-801 binding under nonequilibrium conditions with an IC50 of 4 microM, and this value was unaltered when [3H]MK-801 binding was allowed to reach equilibrium. These results suggest that the sites mediating the effects of glycine and its analogues, polyamines and desipramine are integral components of the NMDA receptor protein.     

Escherichia coli cells lacking methylation-blocking factor (leucine-responsive regulatory protein) have precise timing of initiation of DNA replication in the cell cycle.

A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown to be identical to the leucine-responsive regulatory protein (Lrp), is not responsible for the delayed methylation at oriC implicated in an eclipse period following initiation of DNA replication. Cells containing a transposon mutation within the mbf (lrp) gene initiate DNA replication at the correct time during the cell cycle, whereas cells with increased amounts of the Dam methyltransferase initiate DNA replication randomly throughout the cell cycle.     

Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.

Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitors of mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex III (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNF-mediated injury. In contrast, when the electron flow to ubiquinone was blocked, either at complex I (NADH-ubiquinone oxidoreductase) with amytal or at complex II (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitors significantly interfered with TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an early degeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is further supported by the protective effect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristic alterations of the mitochondrial ultrastructure.