Mitochondria as pharmacological targets in Down syndrome.

Mitochondria play a pivotal role in cellular energy-generating processes and are considered master regulators of cell life and death fate. Mitochondrial function integrates signalling networks in several metabolic pathways controlling neurogenesis and neuroplasticity. Indeed, dysfunctional mitochondria and mitochondrial-dependent activation of intracellular stress cascades are critical initiating events in many human neurodegenerative or neurodevelopmental diseases including Down syndrome (DS). It is well established that trisomy of human chromosome 21 can cause DS. DS is associated with neurodevelopmental delay, intellectual disability and early neurodegeneration. Recently, molecular mechanisms responsible for mitochondrial damage and energy deficits have been identified and characterized in several DS-derived human cells and animal models of DS. Therefore, therapeutic strategies targeting mitochondria could have great potential for new treatment regimens in DS. The purpose of this review is to highlight recent studies concerning mitochondrial impairment in DS, focusing on alterations of the molecular pathways controlling mitochondrial function. We will also discuss the effects and molecular mechanisms of naturally occurring and chemically synthetized drugs that exert neuroprotective effects through modulation of mitochondrial function and attenuation of oxidative stress. These compounds might represent novel therapeutic tools for the modulation of energy deficits in DS.

Estrogen receptor ß activation impairs mitochondrial oxidative metabolism and affects malignant mesothelioma cell growth in vitro and in vivo.

Estrogen receptor (ER)-ß has been shown to possess a tumor suppressive effect, and is a potential target for cancer therapy. Using gene-expression meta-analysis of human malignant pleural mesothelioma, we identified an ESR2 (ERß coding gene) signature. High ESR2 expression was strongly associated with low succinate dehydrogenase B (SDHB) (which encodes a mitochondrial respiratory chain complex II subunit) expression. We demonstrate that SDHB loss induced ESR2 expression, and that activated ERß, by over-expression or by selective agonist stimulation, negatively affected oxidative phosphorylation compromising mitochondrial complex II and IV activity. This resulted in reduced mitochondrial ATP production, increased glycolysis dependence and impaired cell proliferation. The observed in vitro effects were phenocopied in vivo using a selective ERß agonist in a mesothelioma mouse model. On the whole, our data highlight an unforeseen interaction between ERß-mediated tumor suppression and energy metabolism that may be exploited to improve on the therapy for clinical management of malignant mesothelioma.

Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols.

Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.

Helium-Neon laser irradiation of hepatocytes can trigger increase of the mitochondrial membrane potential and can stimulate c-fos expression in a Ca2+-dependent manner.

BACKGROUND AND OBJECTIVE: To gain some insight into the photostimulation of isolated hepatocytes irradiated with Helium-Neon (He-Ne) laser light certain biochemical events were studied with respect to two mechanisms: i) the direct light dependent activation of certain biochemical events investigated in intact cells and isolated mitochondria, ii) the indirect stimulation of processes per se light independent. STUDY DESIGNS/MATERIALS AND METHODS: Irradiation of either isolated hepatocytes or isolated rat liver mitochondria was carried out with He-Ne laser (wavelength, 632.8 nm; fluence, 0.24 J cm-2; fluence rate, 12 mW cm-2). Changes in mitochondrial membrane potential in isolated hepatocytes were monitored using the cationic probe safranine. The c-fos expression was studied by Northern blot and immunoblot analysis. RESULTS: As a result of irradiation, increase of the mitochondrial membrane potential was found to occur in irradiated hepatocytes both in the presence or in the absence of CaCl2. The hyperpolarization of the mitochondrial membrane is assumed to cause an increase in mitochondrial Ca2+ uptake that was measured in isolated mitochondria. Finally, an increase in c-fos expression was found in irradiated hepatocytes when incubated in the presence of CaCl2. CONCLUSION: This paper gives additional information on the mechanism by which He-Ne laser light, either directly or in a cascade-like effect dependent on increase in cell Ca2+, can cause cell stimulation.

Regulation of mRNA and protein levels of beta1 integrin variants in human prostate carcinoma.

Alterations of integrin expression levels in cancer cells correlate with changes in invasiveness, tumor progression, and metastatic potential. The beta1C integrin, an alternatively spliced form of the human beta1 integrin, has been shown to inhibit prostate cell proliferation. Furthermore, beta1C protein levels were found to be abundant in normal prostate glandular epithelium and down-regulated in prostatic adenocarcinoma. To gain further insights into the molecular mechanisms underlying abnormal cancer cell proliferation, we have studied beta1C and beta1 integrin expression at both mRNA and protein levels by Northern and immunoblotting analysis using freshly isolated neoplastic and normal human prostate tissue specimens. Steady-state mRNA levels were evaluated in 38 specimens: 33 prostatic adenocarcinomas exhibiting different Gleason's grade and five normal tissue specimens that did not show any histological manifestation of benign prostatic hypertrophy. Our results demonstrate that beta1C mRNA is expressed in normal prostate and is significantly down-regulated in neoplastic prostate specimens. In addition, using a probe that hybridizes with all beta1 variants, mRNA levels of beta1 are found reduced in neoplastic versus normal prostate tissues. We demonstrate that beta1C mRNA down-regulation does not correlate with either tumor grade or differentiation according to Gleason's grade and TNM system evaluation, and that beta1C mRNA levels are not affected by hormonal therapy. In parallel, beta1C protein levels were analyzed. As expected, beta1C is found to be expressed in normal prostate and dramatically reduced in neoplastic prostate tissues; in contrast, using an antibody to beta1 that recognizes all beta1 variants, the levels of beta1 are comparable in normal and neoplastic prostate, thus indicating a selective down-regulation of the beta1C protein in prostate carcinoma. These results demonstrate for the first time that beta1C and beta1 mRNA expression is down-regulated in prostate carcinoma, whereas only beta1C protein levels are reduced. Our data highlight a selective pressure to reduce the expression levels of beta1C, a very efficient inhibitor of cell proliferation, in prostate malignant transformation.

Kinetic properties and thermal stabilities of mutant forms of mitochondrial aspartate aminotransferase.

Kinetic properties and thermal stabilities of the precursor form of mitochondrial aspartate aminotransferase, the mature form lacking 9 amino acids from the N-terminus, and forms of the mature protein in which cysteine-166 had been mutated to serine or alanine were compared with those of the mature enzyme. The precursor and the cysteine mutants showed moderately impaired catalytic properties consistent with decreased ability to undergo transition from the open to the closed conformation which is an integral part of the mechanism of action of the enzyme. The deletion mutant had a kcat only 2% of that of the mature enzyme but also much reduced Km values for both substrates. In addition it showed enhanced reactivity of cysteine-166 with 5,5'-dithiobis(2-nitrobenzoate), which is characteristic of the closed form of the enzyme, with no enhancement of reactivity in the presence of substrates. This is taken to show that the deletion mutant adopts a conformation that is significantly different from that of the mature enzyme particularly in respect of the small domain. The deletion mutant was found to be more resistant to thermal inactivation over a range of temperatures than were the other forms of the enzyme consistent with its having a more tightly packed small domain.

Active-site Arg --> Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase.

Arg386 and Arg292 of aspartate aminotransferase bind the alpha and the distal carboxylate group, respectively, of dicarboxylic substrates. Their substitution with lysine residues markedly decreased aminotransferase activity. The kcat values with L-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25 degrees C were 0.5, 2.0, and 0.03 s-1 for the R292K, R386K, and R292K/R386K mutations, respectively, kcat of the wild-type enzyme being 220 s-1. Longer dicarboxylic substrates did not compensate for the shorter side chain of the lysine residues. Consistent with the different roles of Arg292 and Arg386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged. Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates. All three mutant enzymes catalyzed as side reactions the beta-decarboxylation of L-aspartate and the racemization of amino acids at faster rates than the wild-type enzyme. The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated L-aspartate to L-alanine 15 times faster (kcat = 0.002 s-1) than the wild-type enzyme. The rates of racemization of L-aspartate, L-glutamate, and L-alanine were 3, 5, and 2 times, respectively, faster than with the wild-type enzyme. Thus, Arg --> Lys substitutions in the active site of aspartate aminotransferase decrease aminotransferase activity but increase other pyridoxal 5'-phosphate-dependent catalytic activities. Apparently, the reaction specificity of pyridoxal 5'-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct.

The irradiation of hepatocytes with He-Ne laser causes an increase of cytosolic free calcium concentration and an increase of cell membrane potential, correlated with it, both increases taking place in an oscillatory manner.

Isolated hepatocytes were irradiated with Helium-Neon laser (fluence: 0.24 Joules x cm-2, fluence rate: 12 mW x cm-2) and changes of both cytosolic free Ca2+ concentration and cell membrane potential were checked by measuring fura-2 and bis-oxonol fluorescence respectively. Irradiation resulted in an enhancement in cytosolic free Ca2+ concentration that requires the presence of Ca2+ in the phase outside hepatocytes; consistently an increase in cell membrane potential was measured correlated with it. Interestingly, the rate of increase of both cytosolic free Ca2+ concentration and cell membrane potential shows special time dependent features similar to those peculiar of oscillatory processes.

Increase in cytosolic and mitochondrial protein synthesis in rat hepatocytes irradiated in vitro by He-Ne laser.

In order to gain an insight into the mechanism of cell photostimulation by laser light, protein synthesis was measured in hepatocytes irradiated with a low-power, continuous-wave He-Ne laser (fluence, 0.24 J cm(-2); fluence rate, 7 and 12 mW cm(-2)). As a result of irradiation, the rate and amount of 35S-methionine incorporated into newly synthesized proteins increased, as demonstrated by gel electrophoresis and quantitative analysis of labelled protein bands. The stimulation of protein synthesis was fluence dependent, with a maximum stimulation at 0.24 J cm(-2) for both fluence rates (12 and 7 mW cm(-2)). Both cytosolic and mitochondrial protein synthesis increased as a result of irradiation, as demonstrated by the measurement of hepatocytes previously treated with chloramphenicol and cycloheximide respectively. An initial investigation showed that stimulation of protein synthesis also occurred in hepatocytes irradiated with a non-coherent radiation source (fluence, 0.24 J cm(-2)).

Substitution of apolar residues in the active site of aspartate aminotransferase by histidine. Effects on reaction and substrate specificity.

In an attempt to change the reaction and substrate specificity of aspartate aminotransferase, several apolar active-site residues were substituted in turn with a histidine residue. Aspartate aminotransferase W140H (of Escherichia coli) racemizes alanine seven times faster (Kcat' = 2.2 x 10(-4) s-1) than the wild-type enzyme, while the aminotransferase activity toward L-alanine was sixfold decreased. X-ray crystallographic analysis showed that the structural changes brought about by the mutation are limited to the immediate environment of H140. In contrast to the tryptophan side chain in the wild-type structure, the imidazole ring of H140 does not form a stacking interaction with the coenzyme pyridine ring. The angle between the two ring planes is about 50 degrees. Pyridoxamine 5'-phosphate dissociates 50 times more rapidly from the W140H mutant than from the wild-type enzyme. A model of the structure of the quinonoid enzyme substrate intermediate indicates that H140 might assist in the reprotonation of C alpha of the amino acid substrate from the re side of the deprotonated coenzyme-substrate adduct in competition with si-side reprotonation by K258. In aspartate aminotransferase I17H (of chicken mitochondria), the substituted residue also lies on the re side of the coenzyme. This mutant enzyme slowly decarboxylates L-aspartate to L-alanine (Kcat' = 8 x 10(-5) s-1). No beta-decarboxylase activity is detectable in the wild-type enzyme. In aspartate aminotransferase V37H (of chicken mitochondria), the mutated residue lies besides the coenzyme in the plane of the pyridine ring; no change in reaction specificity was observed. All three mutations, i.e. W140-->H, I17-->H and V37--H, decreased the aminotransferase activity toward aromatic amino acids by 10-100-fold, while decreasing the activity toward dicarboxylic substrates only moderately to 20%, 20% and 60% of the activity of the wild-type enzymes, respectively. In all three mutant enzymes, the decrease in aspartate aminotransferase activity at pH values lower than 6.5 was more pronounced than in the wild-type enzyme, apparently due to the protonation of the newly introduced histidine residues. The study shows that substitutions of single active-site residues may result in altered reaction and substrate specificities of pyridoxal-5'-phosphate-dependent enzymes.

Increase of both transcription and translation activities following separate irradiation of the in vitro system components with He-Ne laser.

To gain insight into the mechanism by which cell irradiation with low power continuous wave He-Ne laser (632.8 nm) causes a general stimulation of biosynthetic properties, separate components of transcription and translation systems were irradiated (energy dose 2 Joules/cm2; laser power 12 mW) with measurements made of in vitro RNA and protein synthesis. In addition, all tested components were investigated with respect to influence of laser irradiation on their conformation, as spectroscopically monitored. In all cases an increase in both transcription and translation activities was found with a significant change in absorbance/fluorescence spectra.

Activation of mitochondrial DNA replication by He-Ne laser irradiation.

To gain further insight into the effect of He-Ne laser irradiation on the mitochondrial biosynthetic apparatus, DNA synthesis was measured in mitochondria, mitoplasts and mitochondrial matrix fraction irradiated with low power continuous wave He-Ne laser (energy dose 5 Joules/cm2; laser power 12 mW). As a result of irradiation, the amount of alpha-[32P]dATP incorporated in acid insoluble materials was found to increase. Electrophoretic analysis of in vitro synthesised DNA purified from mitochondria, mitoplasts and matrix fraction shows an increase of 50-60% of DNA synthesis in the irradiated samples. These data show that He-Ne laser stimulates the replication of mitochondrial DNA in both intact organelles and soluble matrix fraction, thus suggesting an interaction of laser light with matrix soluble molecule/s.

Import of mutant forms of mitochondrial aspartate aminotransferase into isolated mitochondria.

To gain some insight into the role played by certain protein domains in the import of mitochondrial aspartate aminotransferase in isolated mitochondria, three protein mutants were constructed by using the plasmid pOTS-mAspAT, which contains the nucleotide sequence encoding for the mature form of this enzyme. Two mutant proteins in which Cys-166 was substituted with either serine or alanine and another protein lacking the nine N-terminal amino acids were all synthesized in a cell-free transcription/translation system. Comparison was made among the newly synthesized mutant proteins and the newly synthesized wild type aspartate aminotransferase with respect to their capability to enter mitochondria. All the mutant proteins proved to be able to enter mitochondria even though with a lower efficiency than the wild type enzyme. Interestingly the thiol reagent mersalyl proved to inhibit import of both wild type enzyme and serine mutant, whereas import of alanine mutant was found to be insensitive to mersalyl, thus showing that Cys-166 is the unique -SH group involved in import. Import of mitochondrial aspartate aminotransferase by mitochondria is shown to involve certain protein domains present in the mature protein, two of them being the Cys-166 and the N-terminal regions.