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Biomedical subjects

R J Wiesner

Publications and source records attributed to R J Wiesner.

At least 19 recordsLinked to original sources
PubMed1996

Biogenesis of thermogenic mitochondria in brown adipose tissue of Djungarian hamsters during cold adaptation.

After cold exposure, cytochrome c oxidase (COX) activity increased about 2.5-fold within 2 weeks in the brown adipose tissue (BAT) of Djungarian hamsters. The mRNAs for COX subunits I and III and the 12 S rRNA, encoded on mitochondrial DNA (mtDNA), as well as mRNAs for COX subunits IV, Va and mitochondrial transcription factor A, encoded in the nucleus, were unchanged when expressed per unit of total tissue RNA. However, since total tissue RNA doubled per BAT depot, while total DNA remained unchanged, the actual levels of these transcripts were increased within BAT cells. In contrast, the abundance of mRNA for uncoupling protein was increased 10-fold, indicating specific activation of this gene. In addition, the maximal rate of protein synthesis analysed in a faithful in organello system was increased 2.5-fold in mitochondria isolated from BAT after 7 days of cold exposure. We conclude from these data that the biogenesis of thermogenic mitochondria in BAT following cold adaptation is achieved by increasing the overall capacity for synthesis of mitochondrial proteins in both compartments, by increasing their mRNAs as well as the ribosomes needed for their translation. In addition, the translational rate for COX subunits as well as all other proteins encoded on mtDNA is increased. Thus the pool of subunits encoded on mtDNA required for assembly of respiratory chain complexes is provided. By comparison with other models of increased mitochondrial biogenesis, we propose that thyroid hormone (generated within BAT cells by 5'-deiodinase, and induced upon sympathetic stimulation), which is a well known regulator of the biogenesis of mitochondria in many tissues, is also the major effector of these adaptive changes in BAT.

Acclimatization↗
PubMed1995

Ca2+ sensitizing effects of EMD 53998 after troponin replacement in skinned fibres from porcine atria and ventricles.

Skinned fibres from porcine ventricles exhibited a higher Ca2+ sensitivity (pCa50, i.e. -log10 Ca2+ concentration required for half-maximal activation, for force generation) than atrial fibres. The thiadiazinone derivative EMD 53998 increased Ca2+ sensitivity and Ca2+ efficacy in both preparations. The drug effect depended on the isoform of troponin (Tn). Using the vanadate method TnI and TnC could be partly extracted and replaced by foreign tropin or by the TnI subunit of added foreign troponins. We investigated the relationship between pCa and force development before and after replacement of TnI with foreign troponin (bovine ventricular troponin, cTn, or rabbit skeletal muscle troponin, sTn) in the presence and absence of EMD 53998. Substitution with bovine cTn increased Ca2+ sensitivity to a value characteristic of bovine ventricular skinned fibres (pCa50 = 5.4) and was further increased by EMD 53998. Substitution with sTn also increased Ca2+ sensitivity, but subsequent addition of EMD 53998 caused little further increase in Ca2+ sensitivity. Following extraction of TnI with vanadate, skinned fibres contracted in a Ca(2+)-independent manner and failed to relax at a pCa of 8. Relaxation could be induced, however, by bovine ventricular TnI and rabbit skeletal muscle recombinant TnI. This relaxation could be reversed by EMD 53998 (100 microM). The Ca(2+)-independent force of contracted fibres could also be depressed by a TnI inhibitory peptide, (cTnI 137-148) and, in addition, this effect was antagonized by EMD 53998.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Stoichiometry of mitochondrial transcripts and regulation of gene expression by mitochondrial transcription factor A.

The steady state concentration of cytochrome c oxidase subunit I mRNA and 12 S rRNA, respectively, measured by a quantitative reverse transcription/polymerase chain reaction method, was 4 and 15 molecules per molecule of mt DNA in rat liver and 2 and 9 molecules in rat muscle, respectively. These results imply that in the mitochondrial compartment, the molar concentration of all thirteen mRNAs by far exceeds the concentration of ribosomes, a situation fundamentally different from the cytosolic compartment. Following thyroid hormone treatment, both mitochondrial transcripts increased, in parallel with the mRNA encoding mitochondrial transcription factor A. We conclude that this transcription factor might be the rate limiting factor for mitochondrial transcription in vivo, at least under these conditions.

Animals↗
PubMed1994

Coordination of nuclear and mitochondrial gene expression during the development of cardiac hypertrophy in rats.

We studied the coordination of nuclear and mitochondrial gene expression during cardiac hypertrophy following aortic stenosis or thyroid hormone treatment in rats. We measured mRNA levels for representative subunits of cytochrome-c oxidase, two encoded by mitochondrial DNA and two encoded by the nucleus, as well as the levels of one mitochondrial rRNA. In both models of hypertrophy, an increase of total tissue RNA, reflecting mainly cytosolic ribosomes, accompanied the increase in ventricular weight. Relative levels of mitochondrial rRNA remained unchanged, indicating a net synthesis of mitochondrial ribosomes as well. In both models, cytochrome-c oxidase activity and nuclear-encoded mRNAs remained fairly constant, whereas levels of mitochondrial mRNAs were transiently decreased 24 h after the growth stimulus. We conclude that, in the initial phase of hypertrophy, the signal regulating the synthesis of mitochondrial rRNA is synchronized with nuclear gene expression, whereas the signal regulating mitochondrial mRNA synthesis is not. We postulate that differential regulation of mitochondrial transcription and premature termination of the polycistronic transcript (the latter giving rise to the mitochondrial rRNAs) account for the observed results.

Animals↗
PubMed1993

Increased expression of F1ATP synthase subunits in yeast strains carrying point mutations which destabilize the beta subunit.

In yeast strains (S. cerevisiae) carrying a point mutation of the ATP2 gene, which destabilizes the beta subunit of F1 ATP synthase in vitro, the growth rate was reduced significantly, demonstrating that the mutation is also deleterious in vivo. Immunoblots showed that levels of the mutated beta, but also of the wild-type alpha subunit were increased in the mutated strains, together with levels of the corresponding mRNAs (approximately 1.6-fold). Northern analysis showed that this was due to both the appearance of new transcript species as well as upregulation of the cognate transcripts, strongly indicating that the increase was probably due to activation of transcription. Levels of other mitochondrial proteins, e.g. cytochrome c oxidase, were unaffected. We conclude that a specific signal communicates the actual performance of the ATP synthase inside the mitochondria to the nuclear genes encoding its subunits.

Blotting, Northern↗
PubMed1993

Denervated chicken breast muscle displays discoordinate regulation and differential patterns of expression of alpha f and beta tropomyosin genes.

The expression of the alpha fast (alpha f) and beta tropomyosin (TM) genes has been analysed with muscle-specific and common cDNA probes after unilateral nerve section of the pectoralis major muscle (PM) in 4-week-old chickens. The following were observed in denervated muscles. (1) The beta TM mRNA, which was repressed during development, reaccumulates in a biphasic curve with the increase in the beta TM protein lagging behind the changes in its mRNA. Accordingly, no beta TM is seen in products translated in vitro from total and polyA+ RNA obtained 1 week after denervation. No such translation block is seen with RNA obtained from control or muscles denervated for 6 weeks. (2) No changes in the alpha fTM mRNA and corresponding protein are observed. (3) RNA processing of the two genes is not changed. (4) In the contralateral muscles, transitory increases in alpha f and beta TM mRNAs are observed while the corresponding proteins remain unchanged. Our data suggest that muscle fibres display early and long-term responses to the loss of neural input which might result from a combination of changes produced by regenerative processes and reprogramming of existing fibres. Moreover, in contrast to normal development, no reciprocal changes of alpha f and beta TM expression are seen in denervated muscles.

Animals↗
PubMed1992

Counting target molecules by exponential polymerase chain reaction: copy number of mitochondrial DNA in rat tissues.

In this report, we show that the actual number of target molecules of the polymerase chain reaction can be determined by measuring the concentration of product accumulating in consecutive cycles. The equation describing product accumulation, log Nn = log eff x n + log N0, can be analyzed by linear regression and the molar concentration of target at cycle zero, N0, is obtained. Using this new approach, the actual content of mitochondrial DNA was determined in rat tissues and ranged from 116 x 10(9) molecules/g in fast-twitch skeletal muscle to 743 x 10(9) molecules/g in liver. Using morphometric data from the literature, mitochondria were found to contain 1 to 3 DNA molecules. There was no relation between the oxidative capacity of a tissue and its content of mitochondrial DNA, indicating that transcriptional and posttranscriptional mechanisms rather than gene dosage, as postulated by others, determine to what extent the mitochondrial genome is expressed.

Animals↗
PubMed1992

Expression of the junD proto-oncogene in the rat spinal cord and skin following noxious cutaneous ultraviolet irradiation.

Noxious peripheral stimulation induces the expression of various proto-oncogenes in rat spinal neurons. However, proto-oncogene expression seems to differ depending on the mode of the stimulus. Here, we report that noxious cutaneous ultraviolet (UV) irradiation results in a nearly 8-fold increase in junD mRNA levels in the rat lumbar spinal cord. RNA slot-blotting and hybridization techniques revealed a transcriptional activation of the junD proto-oncogene after 6 h, but not 1 h following UV exposure. These results suggest that low-frequency ongoing afferent impulse discharge is reflected by an accumulation in junD transcripts.

Afferent Pathways↗
PubMed1992

Regulation by thyroid hormone of nuclear and mitochondrial genes encoding subunits of cytochrome-c oxidase in rat liver and skeletal muscle.

Biogenesis of mitochondria involves the expression of genes located on nuclear chromosomes as well as on mitochondrial DNA. We studied the coordination of the two genomes by measuring transcript levels for nuclear (IV, Va, and VIc) and mitochondrial (II and III) subunits of cytochrome-c oxidase after altering the mitochondrial content of rat muscle and liver by altering the thyroid state of the animals. Tissue levels of these mRNAs were generally decreased in hypothyroid animals and were up-regulated again after thyroid hormone (T3) treatment. However, significant increases in the levels of all nuclear transcripts were observed in the liver 24 h after T3 treatment, but were delayed or remained unaltered (VIc) in muscle. In contrast, levels of mitochondrial transcripts were elevated early in muscle and late in liver. The abundance of the corresponding polypeptides, which were analyzed by immunoblotting, changed in direction and magnitude according to the changes in their mRNAs, indicating pretranslational control. We conclude that the two genomes are regulated by T3 not through a common coordinating mechanism, but via two separate pathways, which respond to T3 with tissue-specific kinetics. S1-nuclease protection analysis showed that probably only one transcript for subunit VIc is present in both tissues, thus excluding the possibility that the tissue-specific response is due to the expression of two isogenes. The abundance of mitochondrial DNA was unaltered despite the observed changes in mitochondrial transcripts, indicating that mitochondrial gene expression is regulated by transcriptional mechanisms and not by gene dosage as has been postulated by others.

Animals↗
PubMed1991

Correlations between a nuclear and a mitochondrial mRNA of cytochrome c oxidase subunits, enzymatic activity and total mRNA content, in rat tissues.

Cytochrome c oxidase (COX), like other multi-subunit components of the respiratory chain, is controlled by both the nuclear and the mitochondrial genome. In order to find wether there is a close relationship between mRNAs encoded by the nucleus and by the mitochondrion, and between these mRNAs and enzyme activity, we compared six rat tissues (ventricle, liver, m. soleus, m. plantaris, and the white and red portions of m. gastrocnemius). We found a tenfold range for COX activity, a tenfold range for the contents of mRNA III (mitochondrial) and mRNA VIc (nuclear), a threefold range for total [poly(A)+] mRNA content and a sevenfold range for total RNA content in these tissues. The ratio of mRNA III to mRNA VIc was equal in each tissue, indicating the presence of a mechanism that coordinates the two genomes. There was a good correlation between mRNA content and COX activity (r = 0.78 for VIc, r = 0.77 for III; p less than 0.0001), demonstrating that the expression of this enzyme is mainly under pretranslational control.

Animals↗
PubMed1991

Purification of mitochondrial DNA from total cellular DNA of small tissue samples.

A method is presented for the isolation of highly purified mitochondrial (mt)DNA from a crude DNA extract, making use of the different mobilities of covalently closed circular mtDNA vs. endonuclease-digested nuclear DNA in agarose gels. The preparation is virtually free of any contaminating linear DNA, as judged from its electron microscopic appearance, and can be used for further procedures such as polymerase chain reaction (PCR). Since isolation of mitochondria is not a prerequisite for this method, it can be applied to tissue samples in the mg range. In principle, the method can be applied to every eukaryotic species, provided a molecular hybridization probe is available which permits the position of mtDNA to be located in an agarose gel. This probe can be a cDNA, a DNA fragment generated by PCR, or mtDNA itself, if only the approximate size of the genome is known.

Animals↗
PubMed1991

Quantitative approaches for studying gene expression.

The methods currently available for measuring mRNAs and proteins are reviewed, with a special emphasis on their application to physiological questions. The article focuses on the quantitative determination of cellular contents, but also on assessment of rates of synthesis and degradation, and turnover.

Animals↗
PubMed1989

Glutamate degradation in the ischemic dog heart: contribution to anaerobic energy production.

The present study investigated the conversion of amino acids to succinate and the contribution of this pathway to anaerobic energy production during regional ischemia in the dog heart in situ. The relation between regional myocardial blood flow, estimated by the tracer microsphere technique, and myocardial contents of metabolites (glutamate, alanine, succinate, lactate) as well as their local arterio-venous differences (A-V) were determined. During 30 min of coronary artery occlusion, myocardial glutamate decreased from 2.3 mumol/g wet wt in control tissue to 1.2 mumol/g wet wt in severely ischemic areas, while aspartate was unaffected. Myocardial alanine increased in a 1: 1 stoichiometry compared to glutamate, and succinate accumulated. During control perfusion (118 mmHg), A-V of lactate, succinate and glutamate were +470, -0.7 and -3.9 nmol/ml, respectively. Stepwise reduction of perfusion pressure led to the release of lactate and succinate from the underperfused area; extraction of glutamate occurred at the lowest perfusion pressure investigated (34 mmHg; A-V: -500, -10.4 and +4.2 nmol/ml, respectively). The data indicate that during regional ischemia in vivo, succinate is synthetized exclusively from glutamate via 2-oxo-glutarate, following transamination with glycolytic pyruvate yielding alanine, while the contribution of aspartate is negligible. Using tissue levels of glutamate and lactate together with the local arterio-venous concentration differences of these compounds, it can be estimated that degradation of glutamate delivers 20% of the ATP generated by substrate level phosphorylation reactions. Thus energy production by the glutamate degradation pathway is significant in vivo under conditions of flow deprivation.

Adenosine Triphosphate↗
PubMed1988

Subcellular distribution of malate-aspartate cycle intermediates during normoxia and anoxia in the heart.

The subcellular distribution of adenine nucleotides, phosphocreatine and intermediates of the malate-aspartate cycle was investigated in adult rat heart myocytes under normoxia and anoxia. Cytosolic and mitochondrial concentrations of metabolites were determined by a fractionation method using digitonin. Under normoxia, cytosolic/mitochondrial gradients were found for ATP (c/m = 4), AMP (c/m less than 0.01), citrate (c/m = 0.5), aspartate (c/m = 3), glutamate (c/m = 2), while phosphocreatine and glutamine were confined to the cytosolic space. No gradients were found for malate and 2-oxoglutarate. The results show that the transport of electrons from the cytosol into the mitochondria is supported by the glutamate gradient and by a high glutamate/aspartate ratio inside the mitochondria (Glu/Asp = 15) which is maintained by the energy-dependent Glu-Asp exchange across the mitochondrial membrane. Under anoxia, cytosolic glutamate is transaminated with pyruvate, yielding alanine and 2-oxoglutarate, which is oxidized to succinate inside the mitochondria and leaves the cell. The data indicate that stimulation of transamination is caused by a mass action effect following a decrease in cytosolic 2-oxoglutarate which may be due to succinate-2-oxoglutarate exchange across the mitochondrial membrane. Inhibition of the energy-dependent inward transport of glutamate may support this process.

Adenine Nucleotides↗
PubMed1988

Pathways of succinate formation and their contribution to improvement of cardiac function in the hypoxic rat heart.

Hypoxia led to a dramatic acceleration of amino acid breakdown together with succinate synthesis in the rat heart. Our data do not confirm the simultaneous conversion of aspartate and glutamate to succinate, which has been repeatedly assumed in the literature (7, 8, 21, 28-30), but rather suggest that different pathways are involved during developing hypoxia and that glutamate is the sole source for anaerobic succinate production from endogenous sources in the glucose-perfused heart. Perfusion of hypoxic rat hearts with 2-oxoglutarate, malate, and fumarate (5 mM each) increased succinate formation three- to fourfold. The beneficial effects of these substances on left ventricular systolic pressure, end diastolic pressure, and time of recovery may be due to the elevated content of ATP in these hearts compared to hypoxic controls with glucose as the sole substrate. However, the maintenance of a high rate of anaerobic glycolysis in hearts perfused with 2-oxoglutarate, malate, and fumarate and not the small stimulation of succinate synthesis is considered to be the most important mechanism of cardiac protection. A proposed pathway assumes that malate, after dehydration to fumarate, may serve as an alternative electron acceptor for cytosolic NADH during conditions of oxygen deficiency, thereby cancelling glycolytic inhibition.

Amino Acids↗