Semen Characteristics of Purebred and Crossbred Male Rabbits

The aim of this investigation was to evaluate the semen quality traits of purebred male rabbits and their crosses under subtropical Egyptian conditions. A full 3 x 3 diallel crossing was performed for producing the first generation progeny of New Zealand White (N), Flander (F) and Rex (R) breeds. The highest ejaculate volume (p< 0.05) and percentage of live sperms (p<0.01) with the lowest percentage of sperm cell morphological abnormalities (p<0.05) had been recorded in the NF bucks. Moreover, they possessed positive estimates of direct heterosis for ejaculate volume, mass motility (Mm), individual motility (Im) and sperm cell concentration (SCC). On the contrary, pH had negative estimates of direct heterosis in all crosses and their reciprocal. Semen pH was negatively correlated with SCC (r = -0.18), Mm (r = -0.13) and Im (r = -0.23). In conclusion, the superiority of crossbreeding was particularly obvious in the New Zealand White x Flander males, which cumulated heterosis and favorable maternal effects of the Flander dams.     

The synthesis of fluorescent chlorotriazinylaminofluorescein-concanavalin A and its use as a glycoprotein stain on sodium dodecyl sulphate/polyacrylamide gels.

Concanavalin A, a specific glycoprotein probe, was optimally labelled to a maximum stoichiometry of 0.4 mol of chlorotriazinylaminofluorescein (CTAF)/mol of concanavalin A monomer under mild reaction conditions (pH 8.0, 6 h), and under these conditions the CTAF concanavalin A preparation retains its carbohydrate-binding ability and is able to penetrate SDS/7.5-15%-polyacrylamide gradient gels. CTAF-concanavalin A gives fluorescent bands for the glycoproteins transferrin, fetuin and deoxyribonuclease and shows no fluorescent response for the non-glycoproteins bovine serum albumin and soya-bean trypsin inhibitor. The detection limit of sensitivity for CTAF-concanavalin A, which is similar to that of fluorescein isothiocyanate-concanavalin A, is in the range 5-25 micrograms of glycoprotein. CTAF-concanavalin A is a suitable probe for the detection of glycoproteins in higher-percentage (greater than or equal to 10%) SDS/polyacrylamide gels, and will probably have other applications in, for example, fluorescent energy transfer and other structure-function studies.     

Purification by affinity chromatography of the dicarboxylate carrier from bovine heart mitochondria

Submitochondrial particles were prepared from bovine heart mitochondria, solubilized with Triton X-114 in the presence of lipids and submitted to hydroxylapatite chromatography. The eluate obtained, containing a mixture of mitochondrial carriers, was processed further by affinity chromatography using as ligand p-aminophenylsuccinate coupled via a diazo bond to aminohexyl-Sepharose 4B. The activity of the dicarboxylate exchanger was measured after reconstitution into asolectin vesicles at each step of the purification procedure. All samples studied were found to display substrate and inhibitor specificity similar to those described for the dicarboxylate carrier in mitochondria. The specific activity of the final material eluted from the affinity column was found to be about 1000-times higher than that of the Triton X-114 extract of submitochondrial particles. SDS-polyacrylamide gel electrophoresis analysis of the affinity chromatography eluate showed the presence of only two polypeptides.     

Fluorescent and photoactive probes for the study of protein kinase C

A fluorescent diacylglycerol, 2-(12-N-dansylaminododecanoyl)-1-myristoyl-sn-glycerol (dansyl-DAG) and a photoactive diacylglycerol, 2-(12-[N-(4-azido-2-nitrophenyl)] aminododecanoyl)-1-myristoyl-sn-glycerol (azido-DAG) have been synthesized. Both have been shown to bind to protein kinase C by inhibition of phorbol dibutyrate binding. Dansyl-DAG was able to activate protein kinase C at low calcium concentrations. Stimulation of neutrophils with dansyl-DAG resulted in a large release of superoxide radicals from the cells. The physicochemical properties of dansyl-DAG and azido-DAG may allow one to label and follow specifically changes in the location of protein kinase C and to understand some aspects of its function and regulation.     

Cyclin B targets p34cdc2 for tyrosine phosphorylation.

A universal intracellular factor, the 'M phase-promoting factor' (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase (H1K) originates from the concomitant tyrosine dephosphorylation of the p34cdc2 subunit and the phosphorylation of the cylin Bcdc13 subunit. We have investigated the role of cyclin in the formation of this complex and the tyrosine phosphorylation of p34cdc2, using highly synchronous mitotic sea urchin eggs as a model. As cells leave the S phase and enter the G2 phase, a massive tyrosine phosphorylation of p34cdc2 occurs. This large p34cdc2 tyrosine phosphorylation burst does not arise from a massive increase in p34cdc2 concentration. It even appears to affect only a fraction (non-immunoprecipitable by anti-PSTAIR antibodies) of the total p34cdc2 present in the cell. Several observations point to an extremely close association between accumulation of unphosphorylated cyclin and p34cdc2 tyrosine phosphorylation: (i) both events coincide perfectly during the G2 phase; (ii) both tyrosine-phosphorylated p34cdc2 and cyclin are not immunoprecipitated by anti-PSTAIR antibodies; (iii) accumulation of unphosphorylated cyclin by aphidicolin treatment of the cells, triggers a dramatic accumulation of tyrosine-phosphorylated p34cdc2; and (iv) inhibition of cyclin synthesis by emetine inhibits p34cdc2 tyrosine phosphorylation without affecting the p34cdc2 concentration. These results show that, as it is synthesized, cyclin B binds and recruits p34cdc2 for tyrosine phosphorylation; this inactive complex then requires the completion of DNA replication before it can be turned into fully active MPF. These results fully confirm recent data obtained in vitro with exogenous cyclin added to cycloheximide-treated Xenopus egg extracts.     

Cytochromec oxidase fromParacoccus denitrificans in Triton X-100: Aggregation state and kinetics

Cytochromec oxidase fromParacoccus denitrificans was homogenously dispersed in Triton X-100. Using gel exclusion chromatography and sucrose gradient centrifugation analysis a molecular weight of the detergent-protein complex of 155,000 was determined. After subtraction of the bound detergent (111 mol/mol hemeaa ₃) a molecular weight of 85,000 resulted, which agreed well with the model of a monomer containing two subunits. This monomer showed high cytochromec oxidase activity when measured spectrophotometrically in the presence of Triton X-100 (V _max=85 s^–1). The molecular activity, plotted according to Eadie-Hofstee, was monophasic as a function of the cytochromec concentration. AK _m of 3.6×10^–6 M was evaluated, similar to theK _m observed in the presence of dodecyl maltoside [Naeczet al. (1985).Biochim. Biophys. Acta 808, 259–272].     

Effects of N,N′-dicyclohexylcarbodiimide on isolated and reconstituted cytochrome b-c1 complex from bovine heart mitochondria

N,N′-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstitued mitochondrial cytochrome b-c₁ complex. DCCD inhibits equally electron flow and proton translocation (i.e., the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzymic activity and the cross-linking, suggesting that the two phenomena may be coupled. Binding of [¹⁴C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity.     

Structural and functional alterations induced in the mitochondrial cytochrome b-c1 complex by N,N'-dicyclohexylcarbodiimide

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstituted mitochondrial cytochrome b-c1 complex. In proteoliposomes containing b-c1 complex DCCD inhibits equally electron flow and proton translocation catalyzed by the enzyme. In both isolated and reconstituted systems the inhibitory effect is accompanied by structural alterations in the polypeptide pattern of the enzyme consistent with cross-linking between subunits V and VII. The kinetics of inhibition of enzymic activity correlates with that of the cross-linking, suggesting that the two phenomena may be coupled. Binding of [14C] DCCD to both isolated and reconstituted enzyme was also observed, though it was not correlated kinetically with the inhibition.     

Quantitative analysis of the combined effects of temperature, evaporative demand and light on leaf elongation rate in well-watered field and laboratory-grown maize plants

The respective effects of meristem temperature, vapour pressuredeficit (VPD) and photosynthetic photon flux density (PPFD)on leaf elongation rate (LER) of maize, in the absence of waterdeficit in the soil have been quantified. This analysis wascarried out in a series of field experiments in northern andsouthern France over several seasons and years, and in growthchamber experiments. LER was measured with 10 min steps, togetherwith meristem temperature, VPD and PPFD at leaf level in threetypes of experiments: in growth chamber experiments with stepsin PPFD or VPD at constant meristem temperature, in growth chamberexperiments with several combinations of constant, but contrasting,PPFDs, VPDs and meristem temperatures, and in the field withfluctuating conditions, (i) When evaporative demand was low(night or day with low air VPD), LER was only linked to meristemtemperature, regardless of other climatic conditions, (ii) Lighthad no effect per se on LER in the range from 0 to 1500 molm^–2 s^–1 for time-scales longer than 2 h, providedthat its indirect effects on meristem temperature and on evaporativedemand were corrected (in the growth chamber) or taken intoaccount (in the field), and provided that cumulated PPFD overa weekly time-scale was compatible with field conditions, (iii)Evaporative demand sensed by growing leaves, as estimated bymeristem-to-air vapour pressure difference, markedly affectedLER in the range from 1–4 kPa, at all time-scales understudy, with a unique relationship in the growth chamber (constantconditions) and in the field (fluctuating conditions). Thiseffect was only observed when PPFD was high enough for stomatato open. The negative effect of evaporative demand on LER wasprobably not due to long distance root-to-shoot signalling,since soil was wet, calculated root water potential remainedclose to 0 MPa and concentration of ABA in the xylem sap wasvery low. Therefore, it is proposed to model maize LER witha two-step process, involving the calculation of the maximumLER at a given meristem temperature and then the calculationof the reduction in LER due to evaporative demand. Joint analysisof the whole set of data by using the two equations yieldeda r² of 0.75. This two-step process would be more accurate thanthe provision of LER from temperature only in cases where airVPD frequently exceeds 2 kPa. Key words: Leaf growth, light, evaporative demand, temperature, thermal time, water deficit, ABA, Zea mays L.