Mitochondrial genome distribution in histochemically cytochrome c oxidase-negative muscle fibres in patients with a mixture of deleted and wild type mitochondrial DNA.

In situ hybridization studies were performed on a series of chronic progressive external ophthalmoplegia patients harbouring large mitochondrial DNA deletions, using intra- and extra-deletional probes. Clear differences in the distribution of wild type and deleted mitochondrial genomes were seen in both ragged-red and non-ragged red, cytochrome c oxidase-negative fibres, with an accumulation of deleted genomes in the subsarcolemmal zone. Wild type genome content was normal or decreased in the cytochrome c oxidase-negative regions of one case, but in two patients, wild type mtDNA content in cytochrome c oxidase-negative regions was either normal (most fibres) or increased (occasional fibres). The latter observation suggests there may be a stage in the natural history of ragged-red fibre evolution where wild type genomes are transiently increased. The significance of this finding is discussed.     

Use of acyclovir in the prevention of herpes infections after allogenic bone marrow grafts

In a double-bind controlled study, oral Acyclovir has been compared to a placebo in a series of 39 consecutive patients undergoing bone marrow transplantation. A dose of 200 mg was given every 6 h from day 8 to day 35 after transplantation. Pharmacokinetic studies have shown the good absorption of the drug despite intestinal damage related to chemoradiotherapy or gut graft-versus-host disease (GVHD), there was no sign of toxicity. The protection against herpes simplex virus (HSV) infection was complete in the treated group when compared to the control group even in patients with high anti-HSV antibody titres. The same protection was observed against cytomegalovirus (CMV) infection. The incidence of HSV and CMV was the same in both groups after treatment ended. This study confirms the efficacy of Acyclovir against HSV infection and possibly against CMV infection when it is given prophylactically after bone marrow transplantation.     

Effects of suppression of eggshell calcification and of 1,25(OH)2D3 on Mg2+, Ca2+ and Mg2+HCO-3 ATPase, alkaline phosphatase, carbonic anhydrase and CaBP levels--I. The laying hen uterus

Stimulation of Mg2+, Ca2+ and Mg2+HCO-3 dependent ATPase activity in mitochondrial and microsomal fractions from the uteri of laying hens is demonstrated. ATPase activity was greatest with 5 mM concentrations of Mg2+ at pH 8.5, and at pH 7.4-7.8 following the addition of bicarbonate. Suppression of eggshell calcification, induced by insertion of a thread into the uterus, did not alter Mg2+, Ca2+ and Mg2+HCO-3 ATPase activities. Alkaline phosphatase activity was generally low, and was unaffected by suppression of eggshell calcification. Levels of carbonic anhydrase and calcium binding protein were lower in the uteri of hens laying shell-less eggs. Injections of 1,25(OH)2D3 in hens laying shell-less eggs did not alter CaBP levels or enzyme activities. It is concluded that factors other than 1,25(OH)2D3 and gonadal hormones are involved in the regulation of uterine CaBP levels.     

Heterologous expression, purification, and biochemistry of the oligomycin sensitivity conferring protein (OSCP) from yeast.

Yeast Saccharomyces cerevisiae oligomycin sensitivity conferring proteins (OSCP) have been expressed in Escherichia coli. Heterologous expression results in production of a protein that is identical to yeast mature OSCP, including the absence of the initiating methionine residue. Yeast OSCP expressed in E. coli has been purified to homogeneity and it is able to reconstitute oligomycin-sensitive ATPase using purified F1- and F1/OSCP-depleted membranes (electron transport particles (ETP). Binding of F1 to ETP is dependent on the addition of OSCP. Binding studies using 35S-OSCP indicated that OSCP binds to ETP with a Kd of 200 nM and a capacity of 420 pmol/mg particle protein, whereas OSCP does not interact with F1 in the absence of ETP. These data indicate that yeast OSCP must first form a specific complex with F0, which then binds F1 forming the functional complex. To identify functional domains in yeast OSCP, two deletion mutants have been made. Antibodies directed to these deletion products do not inhibit OSCP-dependent binding of F1 to ETP. However, antibodies directed against the last one-third of OSCP greatly reduce the oligomycin sensitivity of the reconstituted ATPase. These data suggest that OSCP is involved in a functional role in energy transduction or proton translocation and serves a structural role in the yeast mitochondrial ATP synthase.     

Characterization of the oxidative 3alpha-hydroxysteroid dehydrogenase activity of human recombinant 11-cis-retinol dehydrogenase.

11-cis-Retinol dehydrogenase catalyzes the oxidation of cis-retinols, a rate-limiting step in the biosynthesis of 9-cis-retinoic acid. It is also active toward 3alpha-hydroxysteroids, and thus might be involved in steroid metabolism. To better understand the role of this enzyme, we produced stable transfectants expressing 11-cis-retinol dehydrogenase in human embryonic kidney 293 cells. In vitro enzymatic assays have demonstrated that, with an appropriate exogenous cofactor, the enzyme catalyzes the interconversion of 5alpha-androstane-3alpha,17beta-diol and dihydrotestosterone and that of androsterone and androstanedione. However, using intact transfected cells, we found that the enzyme catalyzes reactions only in the oxidative direction. Thus, it is possible that 5alpha-androstane-3alpha,17beta-diol (an inactive androgen) can be converted into dihydrotestosterone, the most potent androgen, by the action of 11-cis-retinol dehydrogenase. This reaction could constitute a non-classical pathway of production of active androgens in the peripheral tissues. We also showed that all-trans-, 9-cis- and 13-cis-retinol inhibit the oxidative 3alpha-hydroxysteroid steroid activity of 11-cis-retinol dehydrogenase with similar K(i) values. Since all-trans-retinol is a precursor of cis-retinols, its inhibitory effect on the activity suggests that it could play an important role in modulating the formation of 9-cis-retinoic acid. In addition, we examined the effect of several known enzyme modulators, namely carbenoxolone, phenylarsine oxide and phosphatidylcholine, on 11-cis-retinol dehydrogenase activity. Taken together, our results suggest that, in humans, this enzyme might play a role in the biosynthesis of both 9-cis-retinoic acid and dihydrotestosterone.     

Molecular cytogenetic characterization of Roegneria ciliaris chromosome additions in common wheat

The development of alien addition lines is important both for transferring useful genes from related species into common wheat and for studying the relationship between alien chromosomes and those of wheat. Roegneria ciliaris (2n=4x=28, S^cS^cY^cY^c) is reported to be a potential source of resistance to wheat scab, which may be useful in wheat improvement. The amphiploid common wheat-R. ciliaris and BC₁F₇ or BC₂F₆ derivatives were screened by C-banding, genomic in situ hybridization (GISH), fluorescent in situ hybridization (FISH) and restriction fragment length polymorphism (RFLP) for the presence of R. ciliaris chromatin introgressed into wheat. Six lines were identified as disomic chromosome additions (DA), one as a ditelosomic addition (Dt), two as double disomic additions (dDA) and one as a monosomic chromosome addition (MA). RFLP analysis using wheat homoeologous group-specific clones indicated that the R. ciliaris chromosomes involved in these lines belong to groups 1, 2, 3, 5 and 7. The genomic affinities of the added R. ciliaris chromosomes were determined by FISH analysis using the repetitive sequence pCbTaq4.14 as a probe. These data suggest that the R. ciliaris chromosomes in five lines belong to the S^c genome. Based on the molecular cytogenetic data, the lines are designated as DA2S^c#1, Dt2S^c#1L, DA3S^c#1, dDA1S^c#2+5Y^c#1, DA5Y^c#1, DA7S^c#1, DA7Y^c#1 and MA?Y^c#1. Based on the present and previous work, 8 of the 14 chromosomes of R. ciliaris have been transferred into wheat.