An Intergenic rs9275596 Polymorphism on Chr. 6p21 Is Associated with Multiple Sclerosis in Latvians.

Background and objectives: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system, leading to demyelination of neurons and potentially debilitating physical and mental symptoms. The disease is more prevalent in women than in men. The major histocompatibility complex (MHC) region has been identified as a major genetic determinant for autoimmune diseases, and its role in some neurological disorders including MS was evaluated. An intergenic single-nucleotide polymorphism (SNP), rs9275596, located between the HLA-DQB1 and HLA-DQA2 genes, is in significant association with various autoimmune diseases according to genome-wide association studies (GWASs). A cumulative effect of this SNP with other polymorphisms from this region was revealed. The aim of the study was to verify the data on rs9275596 association in multiple sclerosis in a case/control study of the Latvian population and to evaluate eventual functional significance of allele substitutions. Materials and Methods: rs9275596 (chr6:32713854; GRCh38.p12) was genotyped in 273 MS patients and 208 controls on main and sex-specific associations. Eventual functional significance of allele substitutions was evaluated in silico using publicly available tools. Results: The rs9275596 rare alleles were identified as a disease susceptibility factor in association with the MS main group and in affected females (p < 0.001 and p < 0.01, respectively). Risk factor genotypes with rare alleles included were associated with the MS common cohort (p < 0.002) and female cohort (odds ratio, OR = 2.24) and were identified as disease susceptible in males (OR = 2.41). It was shown that structural changes of rs9275596 affect the secondary structure of DNA. Functional significance of allele substitutions was evaluated on the eventual sequence affinity to transcription factors (TFs) and splicing signals similarity. A possible impact of the particular polymorphisms on the transcription and splicing efficiency is discussed. Conclusions: Our results suggest susceptibility of rs9275596 to multiple sclerosis in Latvians.

Genetic variations in the PSMA3, PSMA6 and PSMC6 genes are associated with type 1 diabetes in Latvians and with expression level of number of UPS-related and T1DM-susceptible genes in HapMap individuals.

The ubiquitin-proteasome system (UPS), a key player of proteostasis network in the body, was implicated in type 1 diabetes mellitus (T1DM) pathogenesis. Polymorphisms in genes encoding proteasome subunits may potentially affect system efficiency. However, data in this field are still limited. To fulfil this gap, single nucleotide polymorphisms in the PSMB5 (rs11543947), PSMA6 (rs2277460, rs1048990), PSMC6 (rs2295826, rs2295827) and PSMA3 (rs2348071) genes were genotyped on susceptibility to T1DM in Latvians. The rs11543947 was found to be neutral and other loci manifested disease susceptibility, with rs1048990 and rs2348071 being the most significantly associated (P < 0.001; OR 2.042 [1.376-3.032] and OR 2.096 [1.415-3.107], respectively). Risk effect was associated with female phenotype for rs2277460 and family history for rs2277460, rs2295826 and rs2295827. Five-locus genotypes being at risk simultaneously at any two or more loci showed strong (P < 0.0001) T1DM association. The T1DM protective effects (P < 0.001) were shown for five-locus genotype and haplotype homozygous on common alleles and composed of common alleles, respectively. Using SNPexp data set, correlations have been revealed between the rs1048990, rs2295826, rs2295827 and rs2348071 T1DM risk genotypes and expression levels of 14 genes related to the UPS and 42 T1DM-susceptible genes encoding proteins involved in innate and adaptive immunity, antiviral response, insulin signalling, glucose-energy metabolism and other pathways implicated in T1DM pathogenesis. Genotype-phenotype and genotype-genotype clusterings support genotyping results. Our results provide evidence on new T1DM-susceptible loci in the PSMA3, PSMA6 and PSMC6 proteasome genes and give a new insight into the T1DM pathogenesis.

1,4-Dihydropyridine derivatives without Ca2+-antagonist activity up-regulate Psma6 mRNA expression in kidneys of intact and diabetic rats.

Impaired degradation of proteins by the ubiquitin-proteasome system (UPS) is observed in numerous pathologies including diabetes mellitus (DM) and its complications. Dysregulation of proteasomal degradation might be because of altered expression of genes and proteins involved in the UPS. The search for novel compounds able to normalize expression of the UPS appears to be a topical problem. A novel group of 1,4-dihydropyridine (1,4-DHP) derivatives lacking Ca2+-antagonists activities, but capable to produce antidiabetic, antioxidant and DNA repair enhancing effects, were tested for ability to modify Psma6 mRNA expression levels in rat kidneys and blood in healthy animals and in rats with streptozotocin (STZ) induced DM. Psma6 gene was chosen for the study, as polymorphisms of its human analogue are associated with DM and cardiovascular diseases. 1,4-DHP derivatives (metcarbatone, etcarbatone, glutapyrone, J-9-125 and AV-153-Na) were administered per os for three days (0.05 mg/kg and/or 0.5 mg/kg). Psma6 gene expression levels were evaluated by quantitative PCR. Psma6 expression was higher in kidneys compared to blood. Induction of diabetes caused increase of Psma6 expression in kidneys, although it was not changed in blood. Several 1,4-DHP derivatives increased expression of the gene both in kidneys and blood of control and model animals, but greater impact was observed in kidneys. The observed effect might reflect coupling of antioxidant and proteolysis-promoting activities of the compounds.

Evaluation of proteasomal gene polymorphisms in Lithuanian patients with asthma.

OBJECTIVE: To investigate polymorphisms of proteasomal genes PSMA6 (rs1048990 and rs2277460), PSMC6 (rs2295826 and rs2295827) and PSMA3 (rs2348071) in Lithuanian patients with asthma. METHODS: One-hundred forty-six asthma patients and 150 control subjects were studied. DNA was extracted from peripheral blood samples. Five single nucleotide polymorphisms (SNP's) of the three proteasomal genes were analyzed using allele-specific amplification or the cleaved amplified polymorphic sequence method. RESULTS: While certain alleles and genotypes of PSMA6 rs2277460 and rs1048990 and PSMA3 rs2348071 SNP's occurred more frequently in asthma patients than in controls, no statistically significant differences in alleles or genotypes of PSMA6, PSMC6 or PSMA3 were observed between asthma patients and control subjects. However, when male and female study subjects were considered separately, we found that the CG genotype of PSMA6 rs1048990 is significantly more frequent in male asthma patients compared to male control subjects. CONCLUSIONS: We found no significant differences in frequencies of selected five proteasomal gene PSMA6, PSMC6 and PSMA3 SNP's between asthma patients and control subjects overall. Among male subjects, however, the CG PSMA6 rs1048990 genotype was significantly more frequent in asthma patients compared with control subjects.

Changes in glucose transporter expression and nitric oxide production are associated with liver injury in diabetes.

In diabetes mellitus (DM), both hyperglycaemia and hyperlipidaemia can initiate accumulation of fat in the liver, which might be further mediated by inducible nitric oxide synthase. We have studied changes in GLUT1, nitric oxide (NO(·)) concentration and liver damage in two rat DM models. STZ model was induced by strepozotocin 50 mg/kg. HS model was induced by high-fat diet and 30 mg/kg streptozotocin. GLUT1 expression was studied by means of real-time RT-PCR and immunohistochemistry. Production of NO(·) was monitored by means of erythrocyte sedimentation rate spectroscopy of Fe-DETC-NO complex. Liver damage was assessed using histological activity index (HAI). NO(·) concentration was increased in the liver of STZ rats, but it did not change in HS rats (control 36.8 ± 10.3; STZ 142.1 ± 31.1; HS 35.4 ± 9.8 ng/g). Liver HAI was higher in STZ group, 8.6 ± 0.17 versus HS 4.7 ± 0.31, p < 0.05. GLUT1 protein expression was elevated only in STZ group, 16 ± 3 cells/mm(2) versus Control 5 ± 2 cells/mm(2), p = 0.007. Hyperglycaemia sooner causes severe liver damage in rat models of DM, compared with hyperlipidaemia, and is associated with increased NO(·) production. GLUT1 transporter expression might be involved in toxic effects of glucose in the liver. We have obtained novel data about association of GLUT1 expression and NO(·) metabolism in the pathogenesis of liver injury in DM. Increased GLUT1 expression was observed together with overproduction of NO(·) and pronounced liver injury in severely hyperglycaemic rats. On the contrary, moderately hyperglycaemic hyperlipidaemic rats developed only moderate liver steatosis and no increase in GLUT1 and NO(·). GLUT1 overexpression might be implicated in the toxic effects of glucose in the liver. Glycotoxicity is associated with oxidative stress and NO(·) hyperproduction. GLUT1 and NO(·) metabolism might become novel therapeutic targets in liver steatosis.

Functional significance of microsatellite markers.

The review summarizes literature data on the positive results of association studies between the length of microsatellite repeats and predisposition to pathologies. Actually, the data can be classified according to the localization of the microsatellite: in the gene promoter, in the part of exon 1 coding the signal sequence, in gene introns, in the coding areas of genes, and in 3'-untranslated regions. The functional significance of microsatellite length changes can be evaluated in many cases. The authors came up to the conclusion that further studies on microsatellite associations with diseases remain prospective as they reflect changes in the gene functional activity.

Proteasomes and proteasomal gene polymorphism in association with inflammation and various diseases.

A proteasome, a multicatalytic protein complex, is a central particle of the ubiquitin-proteasome proteolytic pathway in all eukaryotic cells. Through the degradation of most intracellular proteins, proteasomes play a significant role in cell processes, such as cell cycle and division, posttranslational protein quality control, cell signaling, and apoptosis. Therefore, the ubiquitin-proteasome system is necessary to ensure the normal functioning of cells and an organism. The associations between alterations in the ubiquitin-proteasome pathway and the development of various autoimmune, neurodegenerative, inflammatory and other diseases in humans have been established. Moreover, the findings of some studies suggest that proteasomes may participate in the pathogenesis of asthma through the regulation of the nuclear factor kappa B signaling pathway. Recently, much attention has been given to the associations between genes encoding the proteasome and their polymorphism, and various diseases. Associations between some proteasomal genes and myocardial infarction, type 2 diabetes mellitus, and other diseases have already been established. However, the results are inconclusive or conflicting and need further clarification.

Comparison of the effects of glibenclamide on metabolic parameters, GLUT1 expression, and liver injury in rats with severe and mild streptozotocin-induced diabetes mellitus.

BACKGROUND AND OBJECTIVE: Glucose transport via GLUT1 protein could be one of additional mechanisms of the antidiabetic action of sulfonylureas. Here, the GLUT1 gene and the protein expression was studied in rats in the course of severe and mild streptozotocin-induced diabetes mellitus and under glibenclamide treatment. MATERIAL AND METHODS: Severe and mild diabetes mellitus was induced using different streptozotocin doses and standard or high fat chow. Rats were treated with glibenclamide (2 mg/kg daily, per os for 6 weeks). The therapeutic effect of glibenclamide was monitored by measuring several metabolic parameters. The GLUT1 mRNA and the protein expression in the kidneys, heart, and liver was studied by means of real-time RT-PCR and immunohistochemistry. RESULTS: The glibenclamide treatment decreased the blood glucose concentration and increased the insulin level in both models of severe and mild diabetes mellitus. Severe diabetes mellitus provoked an increase in both GLUT1 gene and protein expression in the kidneys and the heart, which was nearly normalized by glibenclamide. In the kidneys of mildly diabetic rats, an increase in the GLUT1 gene expression was neither confirmed on the protein level nor influenced by the glibenclamide treatment. In the liver of severely diabetic rats, the heart and the liver of mildly diabetic rats, the GLUT1 gene and the protein expression was changed independently of each other, which might be explained by abortive transcription, and pre- and posttranslational modifications of gene expression. CONCLUSIONS: The GLUT1 expression was found to be affected by the glucose and insulin levels and can be modulated by glibenclamide in severely and mildly diabetic rats. Glibenclamide can prevent the liver damage caused by severe hyperglycemia.

Correction of glycaemia and GLUT1 level by mildronate in rat streptozotocin diabetes mellitus model.

Anti-ischaemic drug mildronate suppresses fatty acid metabolism and increases glucose utilization in myocardium. It was proposed that it could produce a favourable effect on metabolic parameters and glucose transport in diabetic animals. Rats with streptozotocin diabetes mellitus were treated with mildronate (100 mg/kg daily, per os, 6 weeks). Therapeutic effect of mildronate was monitored by measuring animal weight, concentrations of blood glucose, insulin, blood triglycerides, free fatty acids, blood ketone bodies and cholesterol, glycated haemoglobin per cent (HbA1c%) and glucose tolerance. GLUT1 mRNA and protein expression in kidneys, heart, liver and muscles were studied by means of real time RT-PCR and immunohistochemistry correspondingly. In the streptozotocin + mildronate group, mildronate treatment caused a significant decrease in mean blood glucose, cholesterol, free fatty acid and HbA1c concentrations and improved glucose tolerance. Induction of streptozotocin diabetes mellitus provoked increase of both GLUT1 gene and protein expression in kidneys, heart and muscle, mildronate treatment produced normalization of the GLUT1 expression levels. In the liver a similar effect was observed for GLUT1 protein expression, while GLUT1 gene expression was increased by mildronate. Mildronate produces therapeutic effect in streptozotocin diabetes model. Mildronate normalizes the GLUT1 expression up-regulated by streptozotocin diabetes mellitus in kidneys, heart, muscle and liver. Copyright © 2011 John Wiley & Sons, Ltd.

Genetic variations in the PSMA6 and PSMC6 proteasome genes are associated with multiple sclerosis and response to interferon-ß therapy in Latvians.

Several polymorphisms in genes related to the ubiquitin-proteasome system exhibit an association with pathogenesis and prognosis of various human autoimmune diseases. Our previous study reported the association between multiple sclerosis (MS) and the PSMA3-rs2348071 polymorphism in the Latvian population. The current study aimed to evaluate the PSMA6 and PSMC6 genetic variations, their interaction between each other and with the rs2348071, on the susceptibility to MS risk and response to therapy in the Latvian population. PSMA6-rs2277460, -rs1048990 and PSMC6-rs2295826, -rs2295827 were genotyped in the MS case/control study and analysed in terms of genotype-protein correlation network. The possible association with the disease and alleles, single- and multi-locus genotypes and haplotypes of the studied loci was assessed. Response to therapy was evaluated in terms of 'no evidence of disease activity'. To the best of our knowledge, the present study was the first to report that single- and multi-loci variations in the PSMA6, PSMC6 and PSMA3 proteasome genes may have contributed to the risk of MS in the Latvian population. The results of the current study suggested a potential for the PSMA6-rs1048990 to be an independent marker for the prognosis of interferon-ß therapy response. The genotype-phenotype network presented in the current study provided a new insight into the pathogenesis of MS and perspectives for future pharmaceutical interventions.

Tight DNA-protein complexes isolated from barley seedlings are rich in potential guanine quadruplex sequences.

BACKGROUND: The concept of chromatin domains attached to the nuclear matrix is being revisited, with nucleus described as a set of topologically associating domains. The significance of the tightly bound to DNA proteins (TBP), a protein group that remains attached to DNA after its deproteinization should be also revisited, as the existence of these interactions is in good agreement with the concept of the topologically associating domain. The work aimed to characterize the DNA component of TBP isolated from barley seedlings. METHODS: The tight DNA-protein complexes from the first leaves, coleoptiles, and roots of barley seedlings were isolated by purification with chromatography on nitrocellulose or exhaustive digestion of DNA with DNase I. Cloning and transformation were performed using pMOSBBlue Blunt Ended Cloning Kit. Inserts were amplified by PCR, and sequencing was performed on the MegaBace 1000 Sequencing System. The BLAST search was performed using sequence databases at NCBI, CR-EST, and TREP and Ensembl Plants databases. Comparison to MAR/SAR sequences was performed using http://smartdb.bioinf.med.uni-goettingen.de/cgi-bin/SMARtDB/smar.cgi database. The prediction of G quadruplexes (GQ) was performed with the aid of R-studio library pqsfinder. CD spectra were recorded on a Chirascan CS/3D spectrometer. RESULTS: Although the barley genome is AT-rich (43% of GC pairs), most DNA fragments associated with TBP were GC-rich (up to 70% in some fractions). Both fractionation procedures yielded a high proportion of CT-motif sequences presented predominantly by the 16-bp CC(TCTCCC)2 TC fragment present in clones derived from the TBP-bound DNA and absent in free DNA. BLAST analysis revealed alignment with different barley repeats. Some clones, however, aligned with both nuclear and chloroplast structural genes. Alignments with MAR/SAR motifs were very few. The analysis produced by the pqsfinder program revealed numerous potential quadruplex-forming sites in the TBP-bound sequences. A set of oligonucleotides containing sites of possible GQs were designed and ordered. Three of them represented the minus strand of the CT-repeat. Two were derived from sequences of two clones of nitrocellulose retained fraction from leaves and contained GC-rich motifs different from the CT motif. Circular dichroism spectroscopy revealed profound changes in spectra when oligonucleotides were incubated with 100 mM KCl. There was either an increase of positive band in the area of 260 nm or the formation of a positive band at 290 nm. In the former case, changes are typical for parallel G-quadruplexes and, in the latter, 3 + 1 structures. DISCUSSION: The G-quadruplexes anchor proteins are probably involved in the maintenance of the topologically associated domain structure.

Development-dependent changes in the tight DNA-protein complexes of barley on chromosome and gene level.

BACKGROUND: The tightly bound to DNA proteins (TBPs) is a protein group that remains attached to DNA with covalent or non-covalent bonds after its deproteinisation. The functional role of this group is as yet not completely understood. The main goal of this study was to evaluate tissue specific changes in the TBP distribution in barley genes and chromosomes in different phases of shoot and seed development. We have: 1. investigated the TBP distribution along Amy32b and Bmy1 genes encoding low pI alpha-amylase A and endosperm specific beta-amylase correspondingly using oligonucleotide DNA arrays; 2. characterized the polypeptide spectrum of TBP and proteins with affinity to TBP-associated DNA; 3. localized the distribution of DNA complexes with TBP (TBP-DNA) on barley 1H and 7H chromosomes using mapped markers; 4. compared the chromosomal distribution of TBP-DNA complexes to the distribution of the nuclear matrix attachment sites. RESULTS: In the Amy32b gene transition from watery ripe to the milky ripeness stage of seed development was followed by the decrease of TBP binding along the whole gene, especially in the promoter region and intron II. Expression of the Bmy1 gene coupled to ripening was followed by release of the exon III and intron III sequences from complexes with TBPs. Marker analysis revealed changes in the association of chromosome 1H and 7H sites with TBPs between first leaf and coleoptile and at Zadoks 07 and Zadoks 10 stages of barley shoot development. Tight DNA-protein complexes of the nuclear matrix and those detected by NPC-chromatography were revealed as also involved in tissue- and development-dependent transitions, however, in sites different from TBP-DNA interactions. The spectrum of TBPs appeared to be organ and developmental-stage specific. Development of the first leaf and root system (from Zadoks 07 to Zadoks 10 stage) was shown as followed by a drastic increase in the TBP number in contrast to coleoptile, where the TBPs spectrum became poor during senescence. It was demonstrated that a nuclear protein of low molecular weight similar to the described TBPs possessed a high affinity to the DNA involved in TBP-DNA complexes. CONCLUSION: Plant development is followed by redistribution of TBP along individual genes and chromosomes.

Modifications of expression of genes and proteins involved in DNA repair and nitric oxide metabolism by carbatonides [disodium-2,6-dimethyl-1,4-dihydropyridine- 3,5-bis(carbonyloxyacetate) derivatives] in intact and diabetic rats.

Studies on the pathogenesis of diabetes mellitus complications indicate that the compounds reducing free radicals and enhancing DNA repair could be prospective as possible remedies. Carbatonides, the disodium-2,6-dimethyl-1,4- dihydropyridine-3,5-bis(carbonyloxyacetate) derivatives, were tested for these properties. EPR spectroscopy showed that metcarbatone was an effective scavenger of hydroxyl radicals produced in the Fenton reaction, etcarbatone, and propcarbatone were less effective, styrylcarbatone was ineffective. UV/VIS spectroscopy revealed that styrylcarbatone manifested a hyperchromic effect when interacting with DNA, while all other carbatonides showeda hypochromic effect. Rats with streptozotocin induced type 1 DM were treated with metcarbatone, etcarbatone or styrylcarbatone (all compounds at doses 0.05 mg kg-1 or 0.5 mg kg-1) nine days after the DM approval. Gene expression levels in kidneys and blood were evaluated by quantitative RT-PCR; protein expression - immunohistochemically in kidneys, heart, sciatic nerve, and eyes; DNA breakage - by comet assay in nucleated blood cells. Induction of DM induced DNA breaks; metcarbatone and styrylcarbatone (low dose) alleviated this effect. Metcarbatone and etcarbatone up-regulated mRNA and protein of eNOS in kidneys of diabetic animals; etcarbatone also in myocardium. Etcarbatone reduced the expression of increased iNOS protein in myocardium, nerve, and kidneys. iNos gene expression was up-regulated in kidneys by etcarbatone and metcarbatone in diabetic animals. In blood, development of DM increased iNos gene expression; etcarbatone and metcarbatone normalised it. Etcarbatone up-regulated the expression of H2AX in kidneys of diabetic animals but decreased the production of c-PARP1. Taken together, our data indicate that carbatonides might have a potential as drugs intended to treat DM complications.

Effects of an Antimutagenic 1,4-Dihydropyridine AV-153 on Expression of Nitric Oxide Synthases and DNA Repair-related Enzymes and Genes in Kidneys of Rats with a Streptozotocin Model of Diabetes Mellitus.

Development of complications of diabetes mellitus (DM), including diabetic nephropathy, is a complex multi-stage process, dependent on many factors including the modification of nitric oxide (NO) production and an impaired DNA repair. The goal of this work was to study in vivo effects of 1,4-dihydropyridine AV-153, known as antimutagen and DNA binder, on the expression of several genes and proteins involved in NO metabolism and DNA repair in the kidneys of rats with a streptozotocin (STZ)-induced model of DM. Transcription intensity was monitored by means of real-time RT-PCR and the expression of proteins by immunohistochemistry. Development of DM significantly induced PARP1 protein expression, while AV-153 (0.5 mg/kg) administration decreased it. AV-153 increased the expression of Parp1 gene in the kidneys of both intact and diabetic animals. Expression of H2afx mRNA and γH2AX histone protein, a marker of DNA breakage, was not changed in diabetic animals, but AV-153 up-regulated the expression of the gene without any impact on the protein expression. Development of DM was followed by a significant increase in iNOS enzyme expression, while AV-153 down-regulated the enzyme expression up to normal levels. iNos gene expression was also found to be increased in diabetic animals, but unlike the protein, the expression of mRNA was found to be enhanced by AV-153 administration. Expression of both eNOS protein and eNos gene in the kidneys was down-regulated, and the administration of AV-153 normalized the expression level. The effects of the compound in the kidneys of diabetic animals appear to be beneficial, as a trend for the normalization of expression of NO synthases is observed.

New 1,4-Dihydropyridines Down-regulate Nitric Oxide in Animals with Streptozotocin-induced Diabetes Mellitus and Protect Deoxyribonucleic Acid against Peroxynitrite Action.

Diabetes mellitus (DM) and its complications cause numerous health and social problems throughout the world. Pathogenic actions of nitric oxide (NO) are responsible to a large extent for development of complications of DM. Search for compounds regulating NO production in patients with DM is thus important for the development of pharmacological drugs. Dihydropyridines (1,4-DHPs) are prospective compounds from this point of view. The goals of this study were to study the in vivo effects of new DHPs on NO and reactive nitrogen and oxygen species production in a streptozotocin (STZ)-induced model of DM in rats and to study their ability to protect DNA against nocive action of peroxynitrite. STZ-induced diabetes caused an increase in NO production in the liver, kidneys, blood and muscles, but a decrease in NO in adipose tissue of STZ-treated animals. Cerebrocrast treatment was followed by normalization of NO production in the liver, kidneys and blood. Two other DHPs, etaftorone and fenoftorone, were effective in decreasing NO production in kidneys, blood and muscles of diabetic animals. Furthermore, inhibitors of nitric oxide synthase (NOS) and an inhibitor of xanthine oxidoreductase (XOR) decreased NO production in kidneys of diabetic animals. Treatment with etaftorone decreased expression of inducible NOS and XOR in kidneys, whereas it increased the expression of endothelial NOS. In vitro, the studied DHPs did not significantly inhibit the activities of NOS and XOR but affected the reactivity of peroxynitrite with DNA. These new DHPs thus appear of strong interest for treatment of DM complications.

Putative role of nitric oxide synthase isoforms in the changes of nitric oxide concentration in rat brain cortex and cerebellum following sevoflurane and isoflurane anaesthesia.

We have previously observed an increase in nitric oxide (NO) content in rat brain cortex following halothane, sevoflurane or isoflurane anaesthesia. This study was undertaken in order to determine whether isoform-specific nitric oxide synthase (NOS) inhibitors and inducers could modify these increases in NO contents. Rats were subjected to isoflurane and sevoflurane anaesthesia with concomitant administration of neuronal nitric oxide synthase (nNOS) inhibitor 7-Nitro-indazole (7-NI), inducible nitric oxide synthase (iNOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) or lipopolysaccharide. NO concentration in different organs was measured by electron paramagnetic resonance (EPR) spectroscopy. 7-NI significantly decreased NO concentration in cerebellum but not in brain cortex, whereas AMT decreased NO in all the organs studied. Anaesthesia significantly increased NO concentration in brain cortex and decreased that in cerebellum. AMT abolished the NO increase in brain cortex. Anaesthesia enhanced the drastic increase in NO concentration in brain cortex after intraventricular lipopolysaccharide administration. Isoflurane was found to inhibit recombinant nNOS and iNOS activities at high concentrations (EC50=20 mM). Our data suggest a putative role for iNOS in the increase in NO levels produced by isoflurane and sevoflurane, whereas nNOS activity is probably inhibited during anaesthesia.

Identification of an intronic TG repeat polymorphism in the human proteasome core particle PROS-27K gene.

A TG dinucleotide repeat was identified in intron 6 of the human proteasome core particle PROS-27K (IOTA, PSMA6) gene. We present data on the length polymorphism of this repeat in 120 individuals from Latvia and 197 individuals from Finland. A combination of PCR and fluorescent gel electrophoresis was utilized to type the polymorphism. Twelve alleles were observed, varying in length from 10 to 23 TG repeats. Similar allele frequencies were observed in Latvian and Finnish subjects, with 17 and 20 repeats being the most frequent in both populations. We suggest that this TG dinucleotide repeat could be utilized as a prospective marker for genetic linkage and association studies of common diseases.

Juvenile idiopathic arthritis subtype- and sex-specific associations with genetic variants in the PSMA6/PSMC6/PSMA3 gene cluster.

BACKGROUND: The ubiquitin proteasome system plays an exceptional biological role in the antigen processing and immune response and it could potentially be involved in pathogenesis of many immunity-related diseases, including juvenile idiopathic arthritis (JIA). METHODS: The PSMB5 (rs11543947), PSMA6 (rs2277460, rs1048990), PSMC6 (rs2295826, rs2295827), and PSMA3 (rs2348071) proteasomal genes were genotyped on JIA subtype- and sex-specific association; plasma proteasome levels was measured in patients having risk and protective four-locus genotypes and eventual functional significance of allele substitutions was evaluated in silico. RESULTS: Loci rs11543947 and rs1048990 were identified as disease neutral and other loci as disease susceptible (p < 0.05). The rs2277460, rs2295826, and rs2295827 loci had the strongest association with oligoarthritis [odds ratio (OR) = 2.024, 95% confidence interval (CI) 1.101-3.722; OR = 2.371, 95% CI 1.390-4.044; OR = 2.183, 95% CI 1.272-2.737, respectively), but the rs2348071 locus was associated with polyarthritis in females (OR = 3.438, 95% CI 1.626-7.265). A strong (p < 0.001) association was detected between the rs2277460/rs2295826/rs2295827/rs2348071 four-locus genotypes and the healthy phenotype when all loci were homozygous on common alleles (OR 0.439, 95% CI 0.283-0.681) and with the disease phenotype when the rs2348071 and the rs2295826 and/or rs2295827 loci were represented by risk genotypes simultaneously (OR 4.674, 95% CI 2.096-10.425). Rarely observed in controls, the double rs2277460/rs2348071 heterozygotes were rather frequent in affected males and more strongly associated with polyarthritis (p < 0.05). Haplotypes carrying the rare rs2295826/rs2295827 and rs2277460 alleles showed a strong (p < 0.001) association with oligo- and polyarthritis, respectively. The plasma proteasome level was found to be significantly higher in females having four-locus risk genotypes compared with protective genotypes (p < 0.001). Sequence affinity to transcription factors and similarity to splicing signals, microRNAs and/or hairpin precursors potentially depend on allele substitutions in disease susceptible loci. CONCLUSION: We demonstrate for the first time evidence of a sex-specific association of PSMA6/PSMC6/PSMA3 genetic variants with subtypes of JIA and plasma proteasome concentrations. Theoretical models of the functional significance of allele substitutions are discussed.

PSMA6 (rs2277460, rs1048990), PSMC6 (rs2295826, rs2295827) and PSMA3 (rs2348071) genetic diversity in Latvians, Lithuanians and Taiwanese.

PSMA6 (rs2277460, rs1048990), PSMC6 (rs2295826, rs2295827) and PSMA3 (rs2348071) genetic diversity was investigated in 1438 unrelated subjects from Latvia, Lithuania and Taiwan. In general, polymorphism of each individual locus showed tendencies similar to determined previously in HapMap populations. Main differences concern Taiwanese and include presence of rs2277460 rare allele A not found before in Asians and absence of rs2295827 rare alleles homozygotes TT observed in all other human populations. Observed patterns of SNPs and haplotype diversity were compatible with expectation of neutral model of evolution. Linkage disequilibrium between the rs2295826 and rs2295827 was detected to be complete in Latvians and Lithuanians (D´ = 1; r(2) = 1) and slightly disrupted in Taiwanese (D´ = 0.978; r(2) = 0.901). Population differentiation (FST statistics) was estimated from pairwise population comparisons of loci variability, five locus haplotypes and PSMA6 and PSMC6 two locus haplotypes. Latvians were significantly different from all Asians at each of 5 SNPs and from Lithuanians at the rs1048990 and PSMC6 loci. Lithuanian and Asian populations exhibited similarities at the PSMC6 loci and were different at the PSMA6 and PSMA3 SNPs. Considering five locus haplotypes all European populations were significantly different from Asian; Lithuanian population was different from both Latvian and CEU. Allele specific patterns of transcription factor binding sites and splicing signals were predicted in silico and addressed to eventual functionality of nucleotide substitutions and their potential to be involved in human genome evolution and geographical adaptation. Current study represents a novel step toward a systematic analysis of the proteasomal gene genetic diversity in human populations.

Association of obesity with proteasomal gene polymorphisms in children.

The aim of this study was to ascertain possible associations between childhood obesity, its anthropometric and clinical parameters, and three loci of proteasomal genes rs2277460 (PSMA6 c.-110C>A), rs1048990 (PSMA6 c.-8C>G), and rs2348071 (PSMA3 c. 543+138G>A) implicated in obesity-related diseases. Obese subjects included 94 otherwise healthy children in Latvia. Loci were genotyped and then analyzed using polymerase chain reactions, with results compared to those of 191 nonobese controls. PSMA3 SNP frequency differences between obese children and controls, while not reaching significance, suggested a trend. These differences, however, proved highly significant (P < 0.002) in the subset of children reporting a family history of obesity. Among obese children denying such history, PSMA6 c.-8C>G SNP differences, while being nonsignificant, likewise suggested a trend in comparison to the nonobese controls. No PSMA6 c.-110C>A SNP differences were detected in the obese group or its subsets. Finally, PSMA3 SNP differences were significantly associated (P < 0.05) with circulating low-density lipoprotein cholesterol (LDL) levels. Our results clearly implicate the PSMA3 gene locus as an obesity risk factor in those Latvian children with a family history of obesity. While being speculative, the clinical results are suggestive of altered circulatory LDL levels playing a possible role in the etiology of obesity in the young.