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J A Enríquez

Publications and source records attributed to J A Enríquez.

At least 19 recordsLinked to original sources
PubMed2001

Association between seminal plasma carnitine and sperm mitochondrial enzymatic activities.

Cellular parameters of the seminogram have been previously shown to correlate with L-carnitine concentration in the seminal fluid. Carnitine is involved in a variety of metabolic processes playing an important role in maintaining an active oxidative phosphorylation (OXPHOS). Recently, we have found a significant association between the specific activities of the respiratory chain complexes and the seminogram parameters and here we have studied the relationship between the spermatozoa OXPHOS activities and L-carnitine concentration in the seminal plasma. Carnitine, but not prostatic secretions, positive and significantly correlate with mitochondrial respiratory complex activities and the citric acid cycle enzymes citrate synthase and succinate dehydrogenase. It is remarkable that the ratios of the respiratory chain complexes to citrate synthase or succinate dehydrogenase, significant but negatively correlated with L-carnitine concentration. As carnitine in seminal plasma is secreted from the epididymis our results strongly suggest that relationships between carnitine secretion, seminal quality and OXPHOS activities could be because of a parallel response to the same regulatory event.

Carnitine↗
PubMed2000

Human mtDNA haplogroups associated with high or reduced spermatozoa motility.

A variety of mtDNA mutations responsible for human diseases have been associated with molecular defects in the OXPHOS system. It has been proposed that mtDNA genetic alterations can also be responsible for sperm dysfunction. In addition, it was suggested that if sperm dysfunction is the main phenotypic consequence, these mutations could be fixed as stable mtDNA variants, because mtDNA is maternally inherited. To test this possibility, we have performed an extensive analysis of the distribution of mtDNA haplogroups in white men having fertility problems. We have found that asthenozoospermia, but not oligozoospermia, is associated with mtDNA haplogroups in whites. Thus, haplogroups H and T are significantly more abundant in nonasthenozoospermic and asthenozoospermic populations, respectively, and show significant differences in their OXPHOS performance.

Base Sequence↗
PubMed2000

Very rare complementation between mitochondria carrying different mitochondrial DNA mutations points to intrinsic genetic autonomy of the organelles in cultured human cells.

In the present work, a large scale investigation was done regarding the capacity of cultured human cell lines (carrying in homoplasmic form either the mitochondrial tRNA(Lys) A8344G mutation associated with the myoclonic epilepsy and ragged red fiber (MERRF) encephalomyopathy or a frameshift mutation, isolated in vitro, in the gene for the ND4 subunit of NADH dehydrogenase) to undergo transcomplementation of their recessive mitochondrial DNA (mtDNA) mutations after cell fusion. The presence of appropriate nuclear drug resistance markers in the two cell lines allowed measurements of the frequency of cell fusion in glucose-containing medium, non-selective for respiratory capacity, whereas the frequency of transcomplementation of the two mtDNA mutations was determined by growing the same cell fusion mixture in galactose-containing medium, selective for respiratory competence. Transcomplementation of the two mutations was revealed by the re-establishment of normal mitochondrial protein synthesis and respiratory activity and by the relative rates synthesis of two isoforms of the ND3 subunit of NADH dehydrogenase. The results of several experiments showed a cell fusion frequency between 1.4 and 3.4% and an absolute transcomplementation frequency that varied between 1.2 x 10(-5) and 5.5 x 10(-4). Thus, only 0.3-1.6% of the fusion products exhibited transcomplementation of the two mutations. These rare transcomplementing clones were very sluggish in developing, grew very slowly thereafter, and showed a substantial rate of cell death (22-28%). The present results strongly support the conclusion that the capacity of mitochondria to fuse and mix their contents is not a general intrinsic property of these organelles in mammalian cells, although it may become activated in some developmental or physiological situations.

Aging↗
PubMed2000

Seminal quality correlates with mitochondrial functionality.

Oligozoospermia is an important manifestation of male subfertility and very little attention has been paid to study a possible relationship between the total number of ejaculated spermatozoa and mitochondrial functionality. In this work we report a direct correlation between spectrophotometrically measured mitochondrial enzyme activities (citrate synthase and respiratory complex I, II, I+III, II+III and IV) and seminogram parameters (sperm motility, vitality and cell concentration). In addition, total ejaculated spermatozoa correlate much better with the nuclear-encoded citrate synthase and complex II than with the mitochondrial-encoded complex I, III and IV activities. Furthermore, total number of spermatozoa has a significant but negative correlation with the ratios of complex I, complex III and complex IV to complex II (and citrate synthase). These ratios are significantly higher in aged subjects emphasizing the physiological relevance of this observation. These results suggest that the simultaneous increase of the number of ejaculated spermatozoa and the mitochondrial enrichment of citrate synthase and complex II are both parallel responses to the same regulatory events.

Humans↗
PubMed1999

Direct regulation of mitochondrial RNA synthesis by thyroid hormone.

We have analyzed the influence of in vivo treatment and in vitro addition of thyroid hormone on in organello mitochondrial DNA (mtDNA) transcription and, in parallel, on the in organello footprinting patterns at the mtDNA regions involved in the regulation of transcription. We found that thyroid hormone modulates mitochondrial RNA levels and the mRNA/rRNA ratio by influencing the transcriptional rate. In addition, we found conspicuous differences between the mtDNA dimethyl sulfate footprinting patterns of mitochondria derived from euthyroid and hypothyroid rats at the transcription initiation sites but not at the mitochondrial transcription termination factor (mTERF) binding region. Furthermore, direct addition of thyroid hormone to the incubation medium of mitochondria isolated from hypothyroid rats restored the mRNA/rRNA ratio found in euthyroid rats as well as the mtDNA footprinting patterns at the transcription initiation area. Therefore, we conclude that the regulatory effect of thyroid hormone on mitochondrial transcription is partially exerted by a direct influence of the hormone on the mitochondrial transcription machinery. Particularly, the influence on the mRNA/rRNA ratio is achieved by selective modulation of the alternative H-strand transcription initiation sites and does not require the previous activation of nuclear genes. These results provide the first functional demonstration that regulatory signals, such as thyroid hormone, that modify the expression of nuclear genes can also act as primary signals for the transcriptional apparatus of mitochondria.

Animals↗
PubMed1998

[Human mitochondrial genetic system].

The mitochondria are subcellular organelles devoted to energy production in form of ATP that contain their own genetic system. Mitochondrial DNA codify a small, but extremely important, number of polypeptides of the respiratory chain. The other mitochondrial proteins are encoded in the nucleus. Therefore, mitochondrial biogenesis require the coordinated expression of nuclear and mitochondrial genetic systems. The gene arrangement in mitochondrial DNA is extremely compact with the tRNA genes interspersed with the rRNA and protein-coding genes. This organization has its precise counterpart in the mode of expression and distinctive structural features of the RNAs. Both mitochondrial DNA strands are transcribed as a whole in the form of three polycistronic molecules that are later cut by specific enzymes that recognize the 5' and 3' end of the tRNA sequences, to produced the mature rRNA, mRNA and tRNA. The mitochondrial coded mRNAs are translated into proteins by a mitochondrial specific protein-synthesizing machinery. The genetics of the mitochondrial DNA differs from that of the nuclear DNA in several features. In particular, the mitochondrial genome is inherited from the mother that transmit their mitochondrial DNA to all her offsprings. Another characteristic of this genome is its tendency to mutate more frequently than the nuclear DNA. This provides a powerful tool for studying the evolution of man.

DNA, Mitochondrial↗
PubMed1998

[Studies of pathogenicity and characterization of molecular phenotype caused by mutations in human mitochondrial DNA].

Mitochondrial DNA evolves and accumulates mutations more rapidly than nuclear DNA. These nucleotide variation may produce neutral polymorphisms or affect to functional conserved positions being very deleterious. On the other hand the relation between the type of mutation (genotype) and the observed clinical symptoms (phenotype) is nowadays practically unknown. Therefore it is very important to demonstrate clearly that the new described mutations are pathogenic and understanding the molecular mechanisms responsible for the energetic metabolism dysfunction produced by these mutations at cellular level. In the last years several procedures have been developed, including in situ hybridization, single-fiber PCR and the use of patient myoblast, fibroblast and lymphoblast cell culture lines. Specially relevant is the cybrid technology that allow repopulate a cell line depleted of mtDNA with mitochondria obtained from patient fibroblasts, producing transmitochondrial cell lines. The use of these methodology in the last few years has been very important to understand the pathogenic mechanism of some of the classical mutations associated to mitochondrial pathology.

Bone Neoplasms↗
PubMed1996

Evidence for aminoacylation-induced conformational changes in human mitochondrial tRNAs.

Analysis by acid polyacrylamide/urea gel electrophoresis of 14 individual mitochondrial tRNAs (mt-tRNAs) from human cells has revealed a variable decrease in mobility of the aminoacylated relative to the nonacylated form, with the degree of separation of the two forms not being correlated with the mass, polar character, or charge of the amino acid. Separation of the charged and uncharged species has been found to be independent of tRNA denaturation, being observed also in the absence of urea. In another approach, electrophoresis through a perpendicular denaturing gradient gel of several individual mt-tRNAs has shown a progressive unfolding of the tRNA with increasing denaturant concentration, which is consistent with an initial disruption of tertiary interactions, followed by the sequential melting of the four stems of the cloverleaf structure. A detailed analysis of the unfolding process of charged and uncharged tRNALys and tRNALeu(UUR) has revealed that the separation of the two forms of these tRNAs persisted throughout the almost entire range of denaturant concentrations used and was lost upon denaturation of the last helical domain(s), which most likely included the amino acid acceptor stem. These observations strongly suggest that the electrophoretic retardation of the charged species reflects an aminoacylation-induced conformational change of the 3'-end of these mt-tRNAs, with possible significant implications in connection with the known role of the acceptor end in tRNA interactions with the ribosomal peptidyl transferase center and the elongation factor Tu.

Base Sequence↗
PubMed1996

Transient activation of mitochondrial translation regulates the expression of the mitochondrial genome during mammalian mitochondrial differentiation.

Regulation of the expression of the nuclear-encoded beta-subunit of H(+)-ATP synthase (beta-F1-ATPase) gene of oxidative phosphorylation during differentiation of liver mitochondria is mainly exerted at two post-transcriptional levels affecting both the half-life [Izquierdo, Ricart, Ostronoff, Egea and Cuezva (1995) J. Biol. Chem. 270, 10342-10350] and translational efficiency [Luis, Izquierdo, Ostronoff, Salinas, Santarén and Cuezva (1993) J. Biol. Chem. 268, 1868-1875] of the transcript. Herein, we have studied the expression of the mitochondrial (mt) genome during differentiation of rat liver mitochondria in an effort to elucidate the mechanisms of nucleo-mitochondrial cross-talk during biogenesis of the organelle. Estimation of the relative cellular representation of met-DNA in liver reveals a negligible increase in mt-DNA copy number during organelle differentiation. Concurrently, the lack of changes in transcription rates of the mt-DNA "in organello', as well as in steady-state levels of the mt-transcripts, suggests that organelle differentiation is not controlled by an increase in transcription of the mt-genome. However, translation rates in isolated mitochondria revealed a transient 2-fold increase immediately after birth. Interestingly, the transient activation of mitochondrial translation at this stage of liver development is dependent on the synthesis of proteins in cytoplasmic polyribosomes. These findings support the hypothesis that the expression of nuclear and mitochondrial genes during biogenesis of mammalian mitochondria is developmentally regulated by a post-transcriptional mechanism that involves concerted translational control of both genomes.

Aging↗
PubMed1996

The synthesis of mRNA in isolated mitochondria can be maintained for several hours and is inhibited by high levels of ATP.

The dependence for the maintenance of the synthesis and maturation of mitochondrial RNA on the supply of nucleo-cytoplasmic factors has been investigated by a novel in organello RNA synthesis system. We found that mitochondrial DNA transcription can be maintained for several hours in isolated mitochondria. Analysis of the individual mitochondrial RNA species revealed that: the processing of the rRNA precursors and the stability of the mature rRNAs, but not the transcription itself, is severely impaired after short periods of incubation, indicating that these processes are strongly dependent on the mitochondrial interaction with the nucleo-cytoplasmic compartment; the events that lead to the synthesis, processing and turnover of the mitochondrial mRNAs do not require the continuous supply of nucleo-cytoplasmic factors, that are accumulated in excess by mitochondria. Furthermore, we present evidence indicating an inhibition of high ATP levels on the mitochondrial RNA polymerase activity, both in organello and in vitro. Consequently, it is proposed that mitochondrial mRNA synthesis can be regulated in response to changes in intramitochondrial ATP levels. This regulation of mitochondrial mRNA synthesis together with their very rapid turnover described here and elsewhere [Gelfand, R. & Attardi, G. (1981) Mol. Cell Biol. 1, 497-511], could represent a mechanism that would allow each individual mitochondrion to adjust its optimal levels of mRNA, and hence its translation capacity, in response to local energetic demands.

Adenosine Triphosphate↗
PubMed1994

Highly efficient DNA synthesis in isolated mitochondria from rat liver.

We have developed a highly efficient DNA-synthesizing system with isolated intact rat liver mitochondria. The ATP requirements for this in organello DNA synthesis are provided by endogenous synthesis in the presence of exogenous ADP and an oxidizable substrate. In this system, mitochondrial DNA synthesis strikingly proceeds at a constant rate for about 5 h at 37 degrees C. Gel electrophoresis, hybridization and restriction enzyme analyses show that intact mitochondria synthesize nucleic acids with a size of 16.5 kb, that correspond to mitochondrial DNA, and that both DNA strands are replicated. This in organello DNA synthesis requires the supply of dNTPs and decreases at high ADP concentration in the incubation medium.

Adenosine Diphosphate↗
PubMed1993

RNA synthesis in isolated mitochondria from brain cortex, cerebellum and stem: evidence of different transcriptional rates.

1. A system for studying RNA synthesis in isolated sheep brain mitochondria was set up to investigate the transcriptional activity of different brain regions (cortex, cerebellum and brain stem). In this system, mitochondrial DNA is transcribed and RNA processed in a way that faithfully reproduces the in vivo process. 2. The comparison of the electrophoretic patterns of the mitochondrial DNA transcription products showed that although they were qualitatively similar, there were large differences in the rate of mitochondrial DNA transcription of the three regions studied, cerebellum and brain stem showing transcriptional rates which were 34 and 18% respectively of that of cerebral cortex.

Animals↗
PubMed1993

Use of a resolving density gradient created with dextran and poly(ethylene glycol) to purify brain synaptosomes.

Poly(ethylene glycol) and dextran are exclusion agents which when mixed above critical concentrations account for phase separation. In this work, we have used a density gradient created by mixing dextran (denser) and PEG (lighter) solutions to achieve a further purification of synaptosomes by centrifugation. When a brain synaptosomal preparation, containing about 40% of contaminating material, is sedimented in such gradient, two bands of material were obtained. Glutamate decarboxylase activity and glucose-dependent DCIP reduction (an improved marker of the metabolic performance of synaptosomes) were preferentially located in the lower band. These results, together with the electron micrographs of the bands, proved the synaptosomal nature of this lower band. The upper band contained myelin, membranes, vesicles and some synaptosomes. The gradient used shows a high resolution for isopycnic separation of sedimenting material and could be of general interest for subcellular fractionation.

2,6-Dichloroindophenol↗
PubMed1992

Specific increase of a mitochondrial RNA transcript in chronic ethanol-fed rats.

An in vitro transcription system utilizing isolated mitochondria has been used to study the effect of chronic ethanol consumption on liver mitochondrial DNA transcription. The results obtained showed an overall increase of RNA synthesis and a dramatic accumulation of a discrete polyadenylated RNA species. This effect is a consequence of the chronic ethanol consumption since these changes do not occur when isolated control mitochondria are incubated in the presence of ethanol.

Alcoholism↗
PubMedDate not supplied

Autonomous regulation in mammalian mitochondrial DNA transcription.

The regulation of the oxidative phosphorylation system (OXPHOS) biogenesis in eukaryotic cells is unique since it involves the expression of two genomes, the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA). The considerable effort done in collecting information on the factors that influence the expression of the genes encoded in mtDNA and nDNA has revealed that a multiplicity of regulatory options are available in mammalian cells to perform this task. Thus, at least three archetypal situations can be distinguished: mitochondrial proliferation, mitochondrial differentiation, and mitochondrial local tuning (MLT). Each of them seems to be predominantly under the control of specific strategies of regulation, although the description of the detailed molecular mechanisms involved is still in its beginnings. In the present review, we focus on the evidence supporting the existence of mechanisms for autonomous regulation of mtDNA transcription and its role in the integrated regulation of the OXPHOS system biogenesis.

Animals↗