Zur Regulation der NAD-abhängigen Hydrogenase-Aktivität

Zusammenfassung Ruhende Zellen von Hydrogenomonas H 16 enthalten je Gramm Trockengewicht 0,7 mg ATP und 0,9 mg NAD. Bei der Fixierung von Kohlendioxyd und der Synthese des Speicherstoffs Poly--hydroxybuttersäure sinkt die intracelluläre ATP-Konzentration um 30% und das Redoxverhältnis NADH₂/NAD von 1,4 auf 0,46. Die NAD-abhängige Hydrogenase enthält NAD als Coenzym relativ fest gebunden. In Gegenwart von Wasserstoff wird dieses zu NADH₂ reduziert und das Enzym in eine aktive, reaktionsfähige Form umgewandelt. Die Geschwindigkeit der NAD-Reduktion ist infolge einer allosterischen Hemmung der NAD-abhängigen Hydrogenase durch ihr Reaktionsprodukt NADH₂ von dem Redoxverhältnis NADH₂/NAD abhängig. Hierdurch erhält das Enzym eine regulatorische Funktion für den von Folgereaktionen abhängigen Wasserstofftransport.

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Summary Resting cells of Hydrogenomonas strain H 16 contain 0.7 mg ATP and 0.9 mg NAD/g dry weight. During the fixation of carbon dioxide and the synthesis of the storage product poly--hydroxybutyric acid, the intracellular concentration of ATP decreases by 30% and the redox-ratio (NADH₂/NAD) decreases from 1.4 to 0.46.In the NAD-dependent hydrogenase the coenzyme NAD is bound to the enzyme. In the presence of hydrogen NAD is reduced to NADH₂ and the enzyme is converted to a reactive state. The velocity of NAD-reduction is related to the concentration of NADH₂ and the redox-ratio NADH₂/NAD. This allosteric inhibition of the NAD-dependent hydrogenase by its reaction product NADH₂ is responsible for the control of hydrogen transport exerted by consecutive hydrogen requiring reactions.

     

Efficacy and stability of wettable powder amitraz in field and laboratory studies against Boophilus annulatus (Acari: Ixodidae) in south Texas

A study was done at the USDA-ARS, Cattle Fever Tick Research Laboratory, Mission, Tex., to determine the efficacy of a 50% wettable powder (WP) amitraz formulation applied as a whole-body spray in a standard dip vat, and in a laboratory bioassay against Boophilus annulatus (Say) on cattle. A study also was done at the King Ranch in Kleberg County, Tex., to determine the stability of 50% WP amitraz in a dip vat under South Texas conditions Cattle were infested with all parasitic life stages of B. annulatus and were sprayed or dipped with a concentration of 0.025% amitraz. As determined by calculations of the index of reproduction, the whole-body spray treatment provided 86% control of the ticks and the dip treatment provided 99.8% control. Laboratory bioassay results compared favorably with those obtained with the dip vat treatment. Amitraz WP settled very rapidly in the freshly charged ranch vat. However, as more cattle were dipped and the vat became polluted with dirt and excrement, settling occurred much more slowly. Overall, amitraz remained stable in the vat during the test period.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

A TnphoA insertion within the Bradyrhizobium japonicum sipS gene, homologous to prokaryotic signal peptidases, results in extensive changes in the expression of PBM-specific nodulins of infected soybean (Glycine max) cells

Bradyrhizobium japonicum mutant 132 was obtained by random TnphoA mutagenesis of strain 110spc4. A 6.5 kb BamHI kanamycin-resistance-coding DNA fragment of mutant 132 was used as a hybridization probe to clone the corresponding wild-type fragment. DNA sequence analysis of a 3213 bp BamHI—ClaI fragment revealed that three open reading frames (ORFs) were encoded in the same orientation. Based on sequence similarities to other proteins in the database, the second ORF was called sipS (signal peptidase). The TnphoA insertion in mutant 132 was found to be in frame near the 3′ end of sipS. The resulting SipS—PhoA hybrid polypeptide was shown to be expressed in free-living B. japonicum and in Escherichia coli cultures. An immunoblot analysis with a polyclonal antibody directed against the alkaline phosphatase revealed the appearance of a weak signal of a 70 kDa polypeptide both in B. Japonicum and in E. coli, in agreement with the calculated size of the hybrid polypeptide. A much stronger 52 kDa band was also detected. Mutant 132 was specifically disturbed in the interaction with soybean (Glycine max) when the bacteria were released from the infection threads. The bacteroids were not stably maintained within the symbiosome. Numerous vesicles were found in the plant cytosol, which finally resulted in the formation of large vacuoles within the infected nodule cells. Immunohistochemical analyses with antibodies directed against nodulins of the peribacteroid membrane indicated a lower expression of these proteins. The mutant phenotype was genetically complemented by 4.4 kb BamHI fragment including sipS. Subfragments thereof also complemented a temperature-sensitive E. coli lepB mutant, demonstrating that the B. japonicum fragment was functionally replacing Lep^ts in E. coli. Genetic studies suggested that the three genes are organized in one common operon which is expressed from a promoter upstream of the sequenced region. Inactivation of the gene downstream of sipS did not result in a detectable phenotype.     

Stage-related capacity for limb chondrogenesis in cell culture.

Cells from wing buds of varying-stage chick embryos were dissociated and grown in culture to test their capacity for cartilage differentiation. Micro-mass cultures were initiated with a cell layer greater than confluency, which occupied a restricted area of the culture dish surface (10–13 mm²). Cells from stage 24 chick embryo wing buds (prior to the appearance of cartilage in vivo) undergo cartilage differentiation in such cultures. Typically, during the first 1–2 days of culture, cells form aggregates (clusters of cells with a density 1.5 times greater than that of the surrounding nonaggregate area). By Day 3, virtually all aggregates differentiate into cartilage nodules which are easily recognized by their Alcian blue staining (pH 1.0) extracellular matrix. Subsequently, nodules increase in size, and adjacent nodules begin to coalesce. Micro-mass cultures were used to test the chondrogenic capacity of wing bud cells from chick embryos representing the different stages of limb development up to the appearance of cartilage in vivo (stages 17–25). Cells from embryo stages 21–24 form aggregates which differentiate into cartilage nodules in vitro with equal capacity (scored as number of nodules per culture). In contrast, cells from embryo stages 17–19 form aggregates in similar numbers, but these aggregates never differentiate into nodules under routine conditions. However, aggregates which form in cultures of stage 19 wing bud cells do differentiate into cartilage nodules if exposed to dibutyryl cyclic AMP and theophylline. Cells from stage 20 embryos manifest a varying capacity to form cartilage nodules; apparently, this is a transition stage. Cells from stage 25 embryos produce cartilage in vitro without forming either aggregates or nodules. Based on the results presented in this paper, the authors propose a model for cartilage differentiation from embryonic mesoderm cells involving: (1) aggregation, (2) acquisition of the ability to respond to the environment in the aggregate, (3) elevated intracellular cyclic AMP levels, and (4) stabilization and expression of cartilage phenotype.     

Evaluation of an isotope ratio method for measurement of cholesterol absorption in man

Recently an isotope ratio method (IRM) was developed for measuring cholesterol absorption in rats by analysis of radioactivity in peripheral blood (Zilversmit, D. B. 1972. Proc. Soc. Exp. Biol. Med. 140: 862-865). To validate it in man we have compared cholesterol absorption by a fecal radioactivity method (FRM) with that simultaneously measured by IRM in 14 patients (15 experiments) hospitalized on a metabolic ward. Cholesterol absorption by FRM was assayed as fecal recovery of orally administered [(14)C]cholesterol, after correction with markers for fecal flow (chromic oxide) and cholesterol degradation (beta-sitosterol). Simultaneously, [(3)H]cholesterol was administered intravenously, and the dose-normalized ratio of [(14)C]- to [(3)H]cholesterol was repeatedly assayed in plasma. After 72 hours the ratio became constant in each patient and remained so for as long as 63 weeks (five additional outpatient studies). In three patients the fecal data were unsatisfactory because of poor recoveries of chromic oxide and radioactive cholesterol. In the remaining 11 patients (12 experiments) the mean cholesterol absorption by IRM was 42.1% (range 15.7-62.9%) and by FRM 36.6% (range 13.8-58.8%). There was good to excellent agreement between the two methods in the same patient, except in one experiment. Statistical analysis of these 12 comparisons by estimating confidence intervals showed that we can be 95% confident that the two absorption methods will produce results within 5 percentage points, and 99% confident that the differences are less than 7 percentage points. Although we conclude that IRM affords results that are concordant with those obtainable by earlier validated methods, we urge that its suitability for outpatient studies be further examined in more extensive trials.     

Action Spectra for Conversions of Phycochrome c from Nostoc muscorum

The reversibly photochromic pigment, phycochrome c, was extracted from the blue-green alga Nostoc muscorum strain A. Action spectra were determined for in vitro conversions of the pigment from the short wavelength to the long wavelength form and vice versa. The action peak for the absorbance decrease at 650 nm is at 630 nm. During this decrease there is only a slight increase of the absorbance in the green region. Green and yellow light (maximum efficiency at 580 nm) completely restores absorbance at 650 nm. The observations are explained by the existence of three spectrally different forms of phycochrome c: P^c₆₃₀ and P^c₆₅₀ which equilibrate in darkness and P^c₅₈₀ which is reversibly photoconvertible to P^c₆₃₀. We have also measured the absorbance changes brought about by saturating irradiations with light of various wavelengths (“photostationary state spectrum”). Extreme photostationary states were obtained with about 650 nm and 500 nm light.     

Effects of Frost Hardening and Dehardening on Photosynthetic Electron Transport and Fluorescence Properties in Isolated Chloroplasts of Pinus silvestris

Photosynthetic electron transport and low-temperature fluorescence emission properties have been analyzed in isolated chloroplasts during the course of frost hardening and dehardening of Pinus silvestris L. Both the partial electron-transport reactions (H₂O DPIP and Asc./DPIP NADP) and the overall electron transport (H₂O — NAPD) showed decreasing capacities during the course of hardening. Upon exposing the plants to ?5°C and high irradiance a block in the electron-transport chain between the two photosystems developed, whereas the partial reactions still showed activities. The decrease in activity of PSl was accompanied by a decrease in P700 content, as determined by light oxidation of P700, which indicates a correlation between the two changes. Hardening also induced changes in the in vivo chlorophyll organization. During the course of hardening the fluorescence emission bands F692 and F726 decreased relative to F680. These changes were more pronounced if the plants were treated in high than in low irradiance. This suggests a greater destruction of the chlorophyll antennae in close association with the two photoreactions than in the so-called light-harvesting chlorophyll a/b antenna. During dehardening basically the reverse of the changes observed during hardening occurred. The recovery of secondary needles was complete, whereas primary needles only partly recovered.     

Adrenocortical cyclic GMP-dependent protein kinase: purification, characterization, and modification of its activity by calmodulin, and its relationship with steroidogenesis

Cyclic GMP-dependent protein kinase has been purified to apparent homogeneity from bovine adrenal cortex and its presence in the rat adrenal cortex has been demonstrated. Sucrose density sedimentation studies indicated that the M_r of the enzyme was 145,000. This protein was composed to two identical subunits each with M_r of 75,000. The enzyme molecule was asymmetric with a frictional coefficient of 1.54, Stokes radius of 53.5 Å and a sedimentation coefficient of 6.5. The enzyme self-phosphorylated and the stoichiometry of cyclic GMP binding was two molecules per holoenzyme. Calmodulin or troponin C markedly stimulated the apparent maximal velocity of cyclic GMP-dependent protein kinase without affecting its basal activity. This effect of protein modulators was independent of calcium. Sucrose density gradient studies indicated that the stimulatory effect of calmodulin was due to its interaction with histones. An interaction of calmodulin with the enzyme was not observed. The steroidogenic potential of cyclic GMP and its analogs correlated closely with their ability to stimulate cyclic GMP-dependent protein kinase; the order of potency for both activities was 8-bromocylic GMP > cyclic GMP > N²-monobutyryl cyclic GMP > N², O²-dibutyryl cyclic GMP. In each case, calmodulin enhanced the cyclic GMP-dependent protein kinase activity for histone phosphorylation. These results indicate that although cyclic GMP is the primary regulator of cyclic GMP-dependent protein kinase, other modulator proteins such as calmodulin could act as additional regulators of the phosphorylation of substrate proteins. In addition, the demonstration of cyclic GMP-dependent protein kinase in rat adrenal glands, and the results with cyclic GMP and its analogs relating to their activation of protein kinase and steroidogenesis are consistant with the concept that cyclic GMP is one of the mediators of adrenal steroidogenesis.