Respiration-driven proton translocation in Thiobacillus versutus and the role of the periplasmic thiosulphate-oxidizing enzyme system

The periplasmic location of enzymes A and B of the thiosulphate-oxidizing multienzyme system of Thiobacillus versutus has been further confirmed by differential radiolabelling of periplasmic and cytoplasmic proteins. The stoichiometries of respiration-driven proton translocation in T. versutus were determined using the oxygen pulse and the initial rate methods. A value for the H⁺/O quotient (number of protons translocated per oxygen atom reduced) of about 2.8 was found for the oxidation of thiosulphate, and of about 2.5 for sulphite. The H⁺/O quotient for endogenous respiration was about 5.7. The data are shown to be in good agreement with the scheme proposed previously for thiosulphate oxidation by this organism. Proton generation during the oxidation of thiosulphate or sulphite is indicated to occur in the periplasm rather than by pumping across the cytoplasmic membrane. The results also suggest that a H⁺/O quotient of six occurs during NADH oxidation (from endogenous metabolism measurements) and that the terminal cytochrome oxidase, aa₃, does not function as a proton pump.Abbreviations DCCD dicyclohexyl carbodiimide - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - HQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - IEF isoelectric focusing - HIC hydrophobic interaction chromatography - EAI ethyl acetimidate hydrochloride - IAI isethionyl acetimidate     

Behaviour of Nif− mutants of Rhodobacter capsulatus in photosynthetic, ammonia-limited chemostat cultures

Abstract Nif⁻ mutants of Rhodobacter capsulatus carrying mutations either in the nifR4 regulatory gene or in the nifH structural gene both outgrew the wild-type strain B10 in mixed chemostat cultures under conditions favouring nitrogenase-mediated H₂ production by the wild-type (ammonia as limiting nutrient, inert argon atmosphere, light as energy source), whereas under aerobic conditions in the dark, or in batch culture, the growth of Nif⁻ mutants was not favoured. Nitrogenase-mediated H₂ production therefore appears to be detrimental to the growth of R. capsulatus in nitrogen-limited continuous culture, as may also be the case for other nitrogen fixers.     

Decreased fertility in related females heterozygous for the 1/29 chromosome translocation

Eighteen heifers and 120 cows which were descendants of a presumed 1/29 carrier Simmental bull were karotyped. Nine heifers (50%) and 48 cows (40%) were found to be heterozygous for the 1/29 translocation (59, XX, t(1q;29q)). The other animals were chromosomally normal (i.e., 60, XX) or not karotyped. The 48 1/29 cows were compared with 72 chromosomally normal cows with regards to days to first conception, calving interval, percentage of calves conceived, percentage of calves weaned and production efficiency (% calved conceived × % calved weaned). Nine carrier heifers were compared to the nine noncarrier heifers as to pregnancy status. Carrier, noncarrier and nonkarotyped relatives were compared to each other and to contemporary females with regard to pregnancy status at their initial exposure to males. The percentage of calves conceived (calving efficiency) in the 72 noncarrier and the 48 females heterozygous for the 1/29 translocation were 81.5 and 74.8%, respectively (P<0.07). Although days to first conception was longer and percentage of calves weaned and production efficiency were lower in the female heterozygous for the 1/29 translocation, the differences were not statistically different (P>0.10) from the noncarriers. Pregnancy rate was 44.4 and 66.7% (P>0.10) for nine carrier and nine noncarrier heifers, respectively. The pregnancy rate of carrier (65.4%), noncarrier (73.2%) and nonkarotyped (77.8%) relatives of this sire at their mating as yearlings, did not differ (P>0.10). The pregnancy rate as yearlings of carrier females (65.4%) and contemporary heifers (79.8%) did differ (P<0.05). Comparing the pregnancy rate as yearlings of all descendants (72.0%) of the Simmental sire to contemporary heifers (79.8%), a significant decrease (P<0.05) was found indicating that fertility of this sire may have been lower than other sires or that other factors beside the translocation affected fertility.     

Detecting estrus in synchronized heifers-using tailpaint and an aerosol raddle

A synchronization treatment was initiated when each of 1227 heifers (four trials) was tailpainted. The tailpaint was sprayed with an aerosol raddle at the end of the treatment period. The heifers were in herds of 20 to 279 animals. Each herd was observed for estrus at selected post treatment intervals. A heifer was considered to be (or to have been) in estrus when the raddle was rubbed off. In three of the trials, animals which had the raddle removed were inseminated at 48h following the end of the synchronization treatment. The tailpaint of an inseminated animal was scored from 0 (less than 10% of the paint remained) to 5 (more than 90% of the paint remained) and was then reraddled with a second color. The detection-insemination sequence was always repeated at 72 and 96h, and sometimes at 120h. Animals which had been previously inseminated, but then had paint scores reduced by at least 2 units were reinseminated 24h later. Over the four trials, 94.5% of the heifers were detected in estrus through the use of the tailpaint and raddle system. The remaining 67 animals included only 10 (0.8%) which had ovulated without being detected in estrus. The reinsemination rate on consecutive days was 11.3% and was highest among animals that had a tailpaint score of 4 or 5 at 48h. The proportion of animals detected in estrus at selected posttreatment intervals varied with the different synchronization treatments used within one herd, or with the same treatment used in different herds. The combination of tailpaint, raddling, tailpaint scoring and reraddling is a simple sequence which can be effectively used to detect estrus among heifers synchronized in research or commercial herds.     

The influence of CO2 enrichment, phosphorus deficiency and water stress on the growth, conductance and water use of Pinus radiata D. Don

Abstract. Seedlings of Pinus radiata D. Don were grown in growth chambers for 22 weeks with two levels of phosphorus, under either well-watered or water-stressed conditions at CO₂ concentrations of either 330 or 660mm³ dm^?3. Plant growth, water use efficiency and conductance were measured and the relationship between these and needle photosynthetic capacity, water use efficiency and conductance was determined by gas exchange at week 22. Phosphorus deficiency decreased growth and foliar surface area at both CO₂concentrations; however, it only reduced the maximum photosynthetic rates of the needles at 660 mm³ CO₂ dm^?3 (plants grown and measured at the same CO₂ concentration). Water stress reduced growth and foliar surface area at both CO₂ concentrations. Increases in needle photosynthetic rates appeared to be partly responsible for the increased growth at high CO₂ where phosphorus was adequate. This effect was amplified by accompanying increases in needle production. Phosphorus deficiency inhibited these responses because it severely impaired needle photosynthetic function. The relative increase in growth in response to high CO₂ was higher in the periodically water-stressed plants. This was not due to the maintenance of cell volume during drought. Plant water use efficiency was increased by CO₂ enrichment due to an increase in dry weight rather than a decrease in shoot conductance and, therefore, transpirational water loss. Changes in needle conductance and water use efficiency in response to high CO₂ were generally in the same direction as those at the whole plant level. If the atmospheric CO₂ level reaches the predicted concentration of 660 mm³ dm^?3 by the end of next Century, then the growth of P. radiata will only be increased in areas where phosphorus nutrition is adequate. Growth will be increased in drought-affected regions but total water use is unlikely to be reduced.     

Carbon balance during CAM: an assessment of respiratory CO2 recycling in the epiphytic bromeliads Aechmea nudicaulis and Aechmea fendleri

Abstract The regulation of crassulacean acid metabolism (CAM) under controlled environmental conditions has been investigated for two tropical epiphytes, relating plant water and carbon balance to growth form and habitat preference under natural conditions. Aechmea fendleri is restricted to wet, upper montane regions of Trinidad, while A. nudicaulis has a wider distribution extending into more arid regions of the island. Morphological characteristics of these plants are related to habitat preference in terms of leaf succulence (0.44 and 0.94 kg m^?2 for the two species respectively) and a distinct layer of water storage parenchyma in A. nudicaulis In contrast, the thinner leaves of A. fendleri contain little water-storage parenchyma and less chlorenchyma per unit area, but the plants have a more open leaf rosette. The two species differ in expression of CAM, since the proportion of respiratory CO₂ recycled as part of CAM had been found to be much lower in A. fendleri This study compared the efficiency of water use and role of respiratory CO₂ recycling under two PAR regimes (300 and 120 μnol m^?2 s^?1) and three night temperatures (12, 18 and 25 °C). Dark CO₂ uptake rates for both species were comparable to plants in the field (maximum of 2.3 ± 0.2 μmol m^?2s^?1± SD, n= 3). Total net CO₂ uptake at night increased on leaf area basis with temperature for both species under higher PAR, although under the low PAR regime CO₂ uptake was maximal at 18 °C. Water-use efficiency (WUE) increased at 18 °C and 25 °C during dark CO₂ uptake (Phase I) and also during late afternoon photosynthesis (Phase IV) in both species. For A. fendleri, dawn to dusk changes in titrable acidity (ΔH ⁺) were similar under high and low PAR, although ΔH⁺ was correlated to night temperature and PAR in A. nudicaulis. The proportion of ΔH⁺ derived from respiratory CO₂ also varied with experimental conditions. Thus percentage recycling was lower in A. fendleri under high PAR (0–10%), but was only reduced at 18 °C under low PAR. Recycling by A. nudicaulis ranged from 32–42% under high PAR, but was also reduced to 6% under low PAR at 18 °C; at 12 °C and 25 °C, recycling was 37% and 52% respectively. Previous studies have suggested a relationship between the proportion of recycling and degree of water stress. This study indicated that CAM as a CO₂ concentrating mechanism regulates both water-use efficiency and plant carbon balance in these epiphytes, in response to PAR and night temperature. However, the precise relationship between respiratory processes and the balance between external and internal sources of CO₂ is as yet unresolved.     

Homologous and heterologous β-adrenergic desensitization in hepatocytes. Additivity and effect of pertussis toxin

In hepatocytes obtained from hypothyroid rats, phorbol myristate acetate (PMA) and vasopressin diminished the accumulation of cyclic AMP and the stimulation of ureagenesis induced by isoprenaline or glucagon without altering significantly the accumulation of cyclic AMP induced by forskolin. Pretreatment with PMA markedly reduced the stimulation of ureagenesis and the accumulation of cyclic AMP induced by isoprenaline or glucagon. In membranes from cells pretreated with PMA, the stimulation of adenylate cyclase induced by isoprenaline + GTP, glucagon + GTP or by Gpp[NH]p were clearly diminished as compared to the control, whereas forskolin-stimulated activity was not affected. The data indicate heterologous desensitization of adenylate cyclase. It was also observed that the homologous (García-Sáinz J.A. and Michel, B. (1987) Biochem. J. 246, 331–336) and this heterologous β-adrenergic desensitizations were additive. Pertussis toxin treatment markedly reduced the heterologous desensitization of adenylate cyclase but not the homologous β-adrenergic desensitization. It is concluded that the homologous and heterologous desensitizations involve different mechanisms. The homologous desensitization seems to occur at the receptor level, whereas the heterologous probably involves the guanine nucleotide-binding regulatory protein, Ns.     

A model for gramicidin A′-phospholipid interactions in bilayers

A model is proposed for the effect of gramicidin A on the order and structure of phospholipid dispersions. According to this model, the addition of gramicidin A influences the surrounding lipids via two independent mechanisms. The first arises from a drop in surface pressure for those lipids substantially bounded by gramicidin A. The second mechanism arises from the increase in the phospholipid headgroup spacing due to the small polar region of the polypeptide. The model provides an explanation for the currently available NMR, X-ray diffraction and Langmuir monolayer results. The model also suggests mechanisms for the ability of gramicidin A to trigger a transition of the lipid from the lamellar to hexagonal II phase, the dependence of this transition on the lipid chain length and the formation of a lamellar phase with lysophosphatidylcholine.Abbreviations NMR nuclear magnetic resonance - DMPC dimyristoylphosphatidylcholine - S molecular order parameter - CSA chemical shift anisotropy - DPPC dipalmitoylphosphati-dylcholine - LPC lysophosphatidylcholine     

Expression from symbiotic promoters ofRhizobium meliloti inAzotobacter vinelandii andAzospirillum brasilense

InRhizobium meliloti, the promoter P1 of thenif HDK operon, and also the promoter P2, have earlier been shown to be active in the bacteria present in alfalfa root nodules, but not in the bacteria grown aerobically in culture. Here we have looked at the expression from P1 and P2 in two non-symbiotic nitrogen-fixing bacteria,Azotobacter vinelandii andAzospirillum brasilense, using constructions in which the promoters are fused upstream of theβ-galactosidase gene. The promoter P1, but not P2, is active inA. vinelandii, while neither P1 nor P2 is active inAzospirillum brasilense.     

Utilization of the Synthetic Phosphagen Cyclocreatine Phosphate by a Simple Brain Model During Stimulation by Neuroexcitatory Amino Acids

The ability of 1-carboxymethyl-2-imino-3-phosphonoimidazolidine (cyclocreatine-P), accumulated by a simple brain model, to function as a supplemental synthetic phosphagen and respond to the decreases in cytosolic ATP/free ADP ratios that occur during prolonged stimulation by various excitatory amino acids was investigated. Suspensions of chopped whole brain from 11- to 14-day-old chick embryos were incubated with 30 mM cyclocreatine for 90 min, resulting in accumulation of 100 mumol/g dry weight of cyclocreatine-P, and then incubated for up to 1 h with a series of excitatory amino acids of widely differing potencies. Under these conditions net utilization of cyclocreatine-P was detected in response to stimulation by the following neuroexcitatory compounds at the indicated threshold concentrations: kainate (20 microM), N-methyl-DL-aspartate (20 microM), L-homocysteate (20 microM), L-glutamate (200 microM), D-glutamate (200 microM), L-aspartate (2 mM), DL-2-amino-3-phosphonopropionate (2 mM), and DL-2-amino-4-phosphonobutyrate (2 mM). Significant increases in water content of chick embryo brain minces accompanied stimulation by excitatory amino acids. It is suggested that changes in water content or cyclocreatine-P levels in this sensitive brain model might be utilized in automatable screening procedures for detecting novel antagonists and/or new agonists of excitatory amino acids.