The phosphodiesterase 5 inhibitor sildenafil decreases the proinflammatory chemokine IL-8 in diabetic cardiomyopathy: in vivo and in vitro evidence.

PURPOSE: Interleukin (IL)-8 is a proinflammatory C-X-C chemokine involved in inflammation underling cardiac diseases, primary or in comorbid condition, such diabetic cardiomyopathy (DCM). The phosphodiesterase type 5 inhibitor sildenafil can ameliorate cardiac conditions by counteracting inflammation. The study aim is to evaluate the effect of sildenafil on serum IL-8 in DCM subjects vs. placebo, and on IL-8 release in human endothelial cells (Hfaec) and peripheral blood mononuclear cells (PBMC) under inflammatory stimuli. METHODS: IL-8 was quantified: in sera of (30) DCM subjects before (baseline) and after sildenafil (100 mg/day, 3-months) vs. (16) placebo and (15) healthy subjects, by multiplatform array; in supernatants from inflammation-challenged cells after sildenafil (1 µM), by ELISA. RESULTS: Baseline IL-8 was higher in DCM vs. healthy subjects (149.14 ± 46.89 vs. 16.17 ± 5.38 pg/ml, p < 0.01). Sildenafil, not placebo, significantly reduced serum IL-8 (23.7 ± 5.9 pg/ml, p < 0.05 vs. baseline). Receiver operating characteristic (ROC) curve for IL-8 was 0.945 (95% confidence interval of 0.772 to 1.0, p < 0.01), showing good capacity of discriminating the response in terms of drug-induced IL-8 decrease (sensitivity of 0.93, specificity of 0.90). Sildenafil significantly decreased IL-8 protein release by inflammation-induced Hfaec and PBMC and downregulated IL-8 mRNA in PBMC, without affecting cell number or PDE5 expression. CONCLUSION: Sildenafil might be suggested as potential novel pharmacological tool to control DCM progression through IL-8 targeting at systemic and cellular level.

Testosterone-mediated activation of androgenic signalling sustains in vitro the transformed and radioresistant phenotype of rhabdomyosarcoma cell lines.

PURPOSE: Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in childhood, rarely affects adults, preferring male. RMS expresses the receptor for androgen (AR) and responds to androgen; however, the molecular action of androgens on RMS is unknown. METHODS: Herein, testosterone (T) effects were tested in embryonal (ERMS) and alveolar (ARMS) RMS cell lines, by performing luciferase reporter assay, RT-PCR, and western blotting experiments. RNA interference experiments or bicalutamide treatment was performed to assess the specific role of AR. Radiation treatment was delivered to characterise the effects of T treatment on RMS intrinsic radioresistance. RESULTS: Our study showed that RMS cells respond to sub-physiological levels of T stimulation, finally promoting AR-dependent genomic and non-genomic effects, such as the transcriptional regulation of several oncogenes, the phosphorylation-mediated post-transductional modifications of AR and the activation of ERK, p38 and AKT signal transduction pathway mediators that, by physically complexing or not with AR, participate in regulating its transcriptional activity and the expression of T-targeted genes. T chronic daily treatment, performed as for the hormone circadian rhythm, did not significantly affect RMS cell growth, but improved RMS clonogenic and radioresistant potential and increased AR mRNA both in ERMS and ARMS. AR protein accumulation was evident in ERMS, this further developing an intrinsic T-independent AR activity. CONCLUSIONS: Our results suggest that androgens sustain and improve RMS transformed and radioresistant phenotype, and therefore, their therapeutic application should be avoided in RMS post puberal patients.

Hydrogen peroxide and superoxide anion production during acetic acid-induced yeast programmed cell death.

Hydrogen peroxide production in yeast cells undergoing programmed cell death in response to acetic acid occurred in the majority of live cells 15 min after death induction and was no longer detectable after 60 min. Superoxide anion production was found later, 60 and 90 min after death induction when cells viability was 60 and 30%, respectively. In cells protected from death due to acid stress adaptation neither hydrogen peroxide nor superoxide anion could be observed after acetic acid treatment. The early production of hydrogen peroxide in cells in which survival was 100% could play a major role in acetic acid-induced programmed cell death signaling. Superoxide anion is assumed to be generated in cells already en route to acetic acid-induced programmed cell death.

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.

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.

The molecular basis of phenylketonuria in Lithuania.

We report the spectrum of phenylalanine hydroxylase (PAH) gene mutations in patients with phenylketonuria (PKU) residing in Lithuania. A total of 184 independent chromosomes was investigated. R408W mutation was first analysed through restriction enzyme digestion of exon 12. The remaining uncharacterised PKU chromosomes were analysed by scanning the whole coding sequence of PAH gene by multiplex 'broad range' denaturing gradient gel electrophoresis. Mutations were identified by fluorescent automated sequencing or by restriction enzyme digestion analysis if an abnormal DGGE pattern was recognised. 21 different mutations were identified for 175 PKU chromosomes, with a mutation detection rate of 95%. The most common ones were R408W (73.5% chromosomes) and R158Q (7.0% chromosomes) whereas the remaining mutations appeared to be rare (relative frequencies 0.5%-2%). The high mutation detection rate obtained is an evidence of the efficiency of PAH genetic testing achieved in Lithuania. Moreover, the definition of the PKU mutation profile in the Lithuanian population will allow to perform a genotype-phenotype correlation study thus making feasible genotyped-based prediction of the biochemical phenotype in newborns with hyperphenylalaninemia. This may be useful for refining diagnosis and anticipating dietary requirements.

Characterization of the CYP21 gene 5' flanking region in patients affected by 21-OH deficiency.

In order to test the hypothesis that mutations in the 5' non-coding region of CYP21 gene could contribute to the various spectrum of disease presentation due to 21-OH deficiency, the 400bp nucleotide sequence upstream of the ATG codon of CYP21 gene has been characterized in 28 CAH patients who have previously been genotyped by screening for the ten most frequent CYP21 mutations. Six specific sequence variations (-4C-->T, -73C-->T, -295T-->C, -294A-->C, -283A-->G, -281T-->G) have been identified in this region of CYP21 gene in 3 out of 28 21-OH deficient patients for whom the coding region mutations have been previously identified. Three of these mutations, -295T-->C, -294A-->C, -283A-->G, are apparently generated by a gene-conversion event, thus giving first evidence that this mechanism also applies to the 5' untranslated region of CYP21 gene in 21-OH deficiency. Four other sequence changes, identified at nucleotide position -279, -331, -350 and -353, could be referred to as normal since they are present also in healthy subjects. It may not be excluded that some of the newly-identified single nucleotide changes in the regulatory region could have a modulatory effect on the CYP21 gene transcriptional activity thus affecting the clinical outcome.

The molecular basis of phenylketonuria in Latvia.

Characterization of the molecular basis of phenylketonuria (PKU) in Latvia has been accomplished through the analysis of 96 unrelated chromosomes from 50 Latvian PKU patients. Phenylalanine hydroxylase (PAH) gene mutations have been analyzed through a combined approach in which R158Q, R252W, R261Q, G272X, IVS10-11G>A and R408W mutations were first screened for by PCR or restriction generating PCR amplification of PAH gene exons 5, 7, 11 and 12 followed by digestion with the appropriate diagnostic enzyme. Subsequently 'broad range' denaturing gradient gel electrophoresis analysis of the 13 PAH gene exons has been used to study uncharacterized PKU chromosomes. A mutation detection rate of 98% was achieved. 12 different mutations were found, with the most frequent mutation, R408W, accounting for 76% of Latvian PKU alleles. Six mutations (R408W, E280K, R158Q, A104D, R261Q and P281L) represent 92% of PKU chromosomes. PAH VNTR and STR alleles have been also identified and minihaplotype associations with PKU mutations were also determined.

Characterization of phenylketonuria alleles in the Italian population.

In order to identify the molecular basis of phenylketonuria (PKU) in Italy, we screened the entire coding sequence of the phenylalanine hydroxylase gene in 20 Italian PKU patients, whose origins are scattered throughout Italy. The frequency of each identified mutation and of 5 other European mutations was determined within a panel of 92 Italian PKU patients. This approach allowed us to identify 20 different PKU mutations and characterize 64% of the Italian PKU chromosomes. Eleven mutations (IVS10nt546, L48S, R158Q, R261Q, P281L, R261X, R252W, delta T55, IVS7nt1, IVS12nt1, Y414C) represent 55.4% of the Italian PKU alleles, the most common mutations being IVS10nt546 (12.4%) and L48S (9%). All the other mutations are very rare. These data confirm the great heterogeneity expected from previous RFLP haplotype studies. Genotype/phenotype correlation allowed for assessment of the clinical impact of the 20 identified mutations.

Early release and subsequent caspase-mediated degradation of cytochrome c in apoptotic cerebellar granule cells.

Cytochrome c (cyt c) release was investigated in cerebellar granule cells used as an in vitro neuronal model of apoptosis. We have found that cyt c is released into the cytoplasm as an intact, functionally active protein, that this event occurs early, in the commitment phase of the apoptotic process, and that after accumulation, this protein is progressively degraded. Degradation, but not release, is fully blocked by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylchetone (z-VAD-fmk). On the basis of previous findings obtained in the same neuronal population undergoing excitotoxic death, it is hypothesized that release of cyt c may be part of a cellular attempt to maintain production of ATP via cytochrome oxidase, which is reduced by cytosolic NADH in a cytochrome b5-soluble cyt c-mediated fashion.

Glutamate neurotoxicity, oxidative stress and mitochondria.

The excitatory neurotransmitter glutamate plays a major role in determining certain neurological disorders. This situation, referred to as 'glutamate neurotoxicity' (GNT), is characterized by an increasing damage of cell components, including mitochondria, leading to cell death. In the death process, reactive oxygen species (ROS) are generated. The present study describes the state of art in the field of GNT with a special emphasis on the oxidative stress and mitochondria. In particular, we report how ROS are generated and how they affect mitochondrial function in GNT. The relationship between ROS generation and cytochrome c release is described in detail, with the released cytochrome c playing a role in the cell defense mechanism against neurotoxicity.

Characterization of mitochondrial DNA in primary cardiomyopathies.

With the aim of studying the involvement of the mitochondrial genome in the impairment of heart function, mitochondrial DNA was analyzed by modified primer shift-polymerase chain reaction in a panel of young patients affected by primary cardiomyopathies. Mitochondrial DNA molecules harboring the 7436 bp deletion were specifically found in cardiomyopathic patients as compared with a panel of control subjects. The 4977 bp deletion was commonly detected among the subjects analyzed whereas none of the specific tRNA gene point mutations generally associated with the cardiomyopathic trait were detected. The presence of the 7436 bp deletion as a consequence of a premature aging of the heart muscle, secondary to heart dysfunction, is discussed.

The N-terminal region of mature mitochondrial aspartate aminotransferase can direct cytosolic dihydrofolate reductase into mitochondria in vitro.

Two fused genes were constructed which encode for two chimeric proteins in which either 10 or 191 N-terminal amino acids of mature mitochondrial aspartate aminotransferase had been attached to the entire polypeptide chain of cytosolic dihydrofolate reductase. The precursor and mature form of mitochondrial aspartate aminotransferase, dihydrofolate reductase and both chimeric proteins were synthesized in vitro and their import into isolated mitochondria was studied. Both chimeric proteins were taken up by isolated organelles, where they became protease resistant, thus indicating the ability of the N-terminal portion of the mature moiety of the precursor of mitochondrial aspartate aminotransferase to direct cytosolic dihydrofolate reductase into mitochondria.

Non-radioactive detection of five common microsatellite markers for ATP7B gene in Wilson disease patients.

Haplotype analysis using microsatellite markers is a useful indicator of specific mutations and is often exploited as the first large-scale screening technique to carry out the molecular characterization of the disease gene in probands from a specific population. However, the methodologies available are still cumbersome and require the use of either radioactive compounds or specialized equipment suitable to follow fluorescent dyes. Both these techniques may not be available for newly developing clinical laboratories. We have set up a sensitive and easy-to-use protocol to characterize five closely spaced, highly polymorphic microsatellite polymorphisms (CA repeats) that span the Wilson disease (WD) region, i.e. D13S316, D13S133, D13S301, D13S314, D13S315. The technique described here for the analysis of the WD gene microsatellite system relies on the quick detection method of silver staining, avoiding the use of toxic or sophisticated equipment. This approach could be the method of choice to implement molecular genetic testing in clinical laboratories, even those not especially equipped for DNA analysis and in particular in newly developed molecular genetics centers in countries whose population has not yet been characterized for WD-causing ATP7B gene mutations.

Detection of microsatellites by ethidium bromide staining. The analysis of an STR system in the human phenylalanine hydroxylase gene.

The analysis of short tandem repeat (STR) systems usually relies on polyacrylamide gel electrophoresis analysis followed by visualization with silver staining or autoradiography. Both these techniques may not be suitable for clinical laboratories. We developed a simple procedure based on the visualization of STR alleles by ethidium bromide staining. The 4-bp STR system analysed is located in the human phenylalanine hydroxylase gene. Alleles differing by 4 bp are clearly separated independently of the size of the amplified fragments and homozygous samples are easily identified by comparison of the relative intensity of the electrophoretic bands. This method could be applied to the analysis of other STR systems located in different genetic loci by carefully changing the electrophoretic conditions.

Fumarate permeation in rat liver mitochondria: fumarate/malate and fumarate/phosphate translocators.

Fumarate permeation in isolated rat liver mitochondria was demonstrated by measuring malate and phosphate efflux caused by fumarate added externally to the mitochondrial suspension. The existence of two specific fumarate translocators, fumarate/malate and fumarate/phosphate, is shown here. These carriers are distinguished in the light of different kinetic parameters (Km values are 50 microM and 150 microM, and Vmax values are 17 and 40 nmoles/min X mg mitochondrial protein, respectively) and of differing sensitivity to non-penetrant compounds. Fumarate was found to cause oxaloacetate efflux from mitochondria by means of an indirect process which involves the cooperation of both fumarate/malate and malate/oxaloacetate translocators. Results are discussed in the light of the physiological role played by fumarate translocation in both ureogenesis and aminoacid metabolism.

Cumulative effects of mutations in newly synthesised mitochondrial aspartate aminotransferase on uptake into mitochondria.

Mutant genes were constructed which coded for the precursor form of mitochondrial aspartate aminotransferase in which residue cysteine 166 was mutated to either serine or alanine and for forms of the protein lacking both the presequence and residues 1-9 of the mature protein but carrying the same cysteine mutations. The protein products of all of these mutant genes were imported into mitochondria that had been added to the expression system but with varying degrees of efficiency. The results showed that the effects of mutation of cysteine 166 and of deletion of residues 1-9 of the mature protein on sequestration into mitochondria were essentially cumulative, suggesting that these parts of the protein are involved in distinct steps on the recognition/uptake pathway.

The in vitro-synthesized precursor and mature mitochondrial aspartate aminotransferase share the same import pathway in isolated mitochondria.

Both the precursor and the mature form of mitochondrial aspartate aminotransferase were synthesized in a cell-free coupled transcription/translation system directed by the recombinant expression plasmid pOTS-pmAspAT and pOTS-mAspAT, respectively. Both newly synthesized forms of the protein were imported into isolated mitochondria, with the precursor correctly processed to the mature form. In both cases the import process showed resistance to externally added pronase and was abolished in mitochondria treated with the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Moreover the imported products showed the same intramitochondrial localization as judged by a subfractionation procedure. In both cases import was time dependent and was completed in about 15 min. Finally a competitive inhibition of the import of the precursor of aspartate aminotransferase was found due to externally added purified aspartate aminotransferase.

Expression of cDNAs encoding the precursor and the mature form of chicken mitochondrial aspartate aminotransferase in Escherichia coli.

Both the precursor and the mature form of chicken mitochondrial aspartate aminotransferase were synthesized in Escherichia coli. The precursor was found to sediment quantitatively together with insoluble cell material. In contrast, mature mitochondrial aspartate aminotransferase could be readily extracted from the cells and was indistinguishable from the enzyme isolated from chicken heart in all respects tested: specific activity 230 units mg-1; Mr 2 X 45,000; pI greater than 9; NH2-terminal sequence SSWWSHVEMG, the initiator methionine having been removed by the bacteria. Thus, the polypeptide chain representing mature mitochondrial aspartate aminotransferase is an autonomous folding unit which attains its functional spatial structure independently of the presence of the prepiece, trans-membrane passage, and proteolytic processing.