Direct quantification of picomolar concentrations of mRNAs by mathematical analysis of a reverse transcription/exponential polymerase chain reaction assay.
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Animals↗
Biomedical subjects
R J Wiesner
Publications and source records attributed to R J Wiesner.
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↗
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↗
Regulation of mitochondrial gene expression: transcription versus replication.
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Animals↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
The anaerobic heart: succinate formation and mechanical performance of cat papillary muscle.
Quiescent or paced, isometrically contracting right ventricular papillary muscles of cat heart were incubated under oxygenated or hypoxic (Po2 less than 10 Torr) conditions in order to investigate lactate and succinate formation. Both compounds were produced and released into the incubation medium during oxygen deficiency. Mechanical performance stimulated synthesis of both compounds under hypoxic as well as oxygenated conditions. Pacing led to the accumulation of large amounts of glutamine both under oxygenated and hypoxic conditions. Hypoxia resulted in a marked depletion of high-energy phosphates and concomitantly mechanical performance was impaired, i.e. developed tension fell rapidly and relaxation rate decreased. Supplying hypoxic, contracting muscles with aspartate (2 mM) resulted in maintenance of muscular function to some extent and led to augmented release of succinate and lactate. The data indicate that anaerobic succinate formation is correlated to the energy requiring processes of the myocardium. Maintenance of myocardial function by the supply of amino acids may be related to their conversion into succinate and to the stimulation of glycolysis.
Alanine↗