The role of common and rare MC4R variants and FTO polymorphisms in extreme form of obesity

Melanocortin 4 receptor (MC4R) is an important regulator of food intake and number of studies report genetic variations influencing the risk of obesity. Here we explored the role of common genetic variation from MC4R locus comparing with SNPs from gene FTO locus, as well as the frequency and functionality of rare MC4R mutations in cohort of 380 severely obese individuals (BMI > 39 kg/m²) and 380 lean subjects from the Genome Database of Latvian Population (LGDB). We found correlation for two SNPs—rs11642015 and rs62048402 in the fat mass and obesity-associated protein (FTO) with obesity but no association was detected for rs17782313 located in the MC4R locus in these severely obese individuals. We sequenced the whole gene MC4R coding region in all study subjects and found five previously known heterozygous non-synonymous substitutions V103I, I121T, S127L, V166I and I251L. Expression in mammalian cells showed that the S127L, V166I and double V103I/S127L mutant receptors had significantly decreased quantity at the cell surface compared to the wild type MC4R. We carried out detailed functional analysis of V166I that demonstrated that, despite low abundance in plasma membrane, the V166I variant has lower EC₅₀ value upon αMSH activation than the wild type receptor, while the level of AGRP inhibition was decreased, implying that V166I cause hyperactive satiety signalling. Overall, this study suggest that S127L may be the most frequent functional MC4R mutation leading to the severe obesity in general population and provides new insight into the functionality of population based variants of the MC4R.     

Different mutations in mucA gene of Pseudomonas aeruginosa mucoid strains in cystic fibrosis patients and their effect on algU gene expression

Alginate biosynthesis in Pseudomonas aeruginosa is a highly regulated process in which algU and mucA genes are key elements. Mutations in mucA gene determine alginate operon overexpression and exopolysaccharide overproduction. In our study, 119 strains of P. aeruginosa were isolated from sputa of 96 cystic fibrosis patients and 84/119 showed nonmucoid phenotype, while 35/119 showed mucoid phenotypes. mucA gene was amplified and sequenced in all strains revealing mutations in 29/35 mucoid strains (82%) and in one non-mucoid strain. 4/29 strains showed mutations never described that generated premature stop and much shorter MucA proteins. In all mutated strains, algU gene expression was analyzed to determine if mutations in mucA, resulting in a strong loss of its protein, could significantly influence its function and subsequently the biosynthetic pathways under algU control. Analysis of algU expression disclosed that the length significantly affects the expression of genes involved in the production of alginate and in the motility and hence survival of P. aeruginosa strains in cystic fibrosis lungs.     

Single nucleotide polymorphisms of the purinergic 1 receptor are not associated with myocardial infarction in a Latvian population

The purinergic 1 receptor (P2RY1) has been implicated in development of heart disease and in individual pharmacodynamic response to anticoagulant therapies. However, the association of polymorphisms in the P2RY1 gene with myocardial infarction (MI), and its associated conditions, has yet to be reported in the literature. We evaluated seven known SNPs in P2RY1 for association with MI in a Latvian population. Seven independent parameters that are related to MI [body mass index (BMI), type 2 diabetes (T2D), angina pectoris, hypertension, hyperlipidemia, atrial fibrillation and heart failure] were investigated. No significant association with MI was observed for any of the polymorphisms. Those SNPs for which the P value was close to significance were located in coding or promoter regions. Intriguingly, carriers of the minor allele in the P2RY1 gene locus showed a tendency towards higher onset age for MI, suggesting a possible protective effect of these SNPs against MI or their contribution in progression as opposed to onset. Finally, a linkage disequilibrium (LD) plot was generated for these polymorphisms in the Latvian population. The results of this study suggest that the role of P2RY1 in individuals from Latvian population is likely to be principally involved in platelet aggregation and thromboembolic diseases, and not as a significant contributing factor to the global metabolic syndrome.     

Does HLA Class II Haplotype play a role in Adult Acute Disseminated Encephalomyelitis? Preliminary Findings from a Southern Italy hospital-based study

Acute Disseminated Encephalomyelitis (ADEM) is an acute, multifocal, monophasic, inflammatory demyelinating disease of the central nervous system that affects predominantly children. Aim of the study was to evaluate the distribution of Human Leukocyte Antigen (HLA) class II haplotype in adult ADEM patients, in order to better characterize this rare clinical entity. Six patients (3 males and 3 females; median age 50 years) with ADEM were retrospectively studied in our Neurology Unit; 29 healthy subjects (8 males and 21 females, mean age 43.4±14.3) were the control group. All the study subjects were molecularly typed for HLA class II haplotype. The frequencies of HLA-DRB1*16 (17% vs 3% in control group; Py=0.02) and HLA-DQB1*05 (42% against 24% in the control group; Py=0.010), as well as the association HLA-DRB1*16/HLA-DQB1*05 were significantly increased in ADEM population compared to the control group. The frequencies of allelic association between 13-04 (P < 0.01) and homozygosis 14 (P < 0.05) alleles at HLA-DRB1* locus and 05-02 (P < 0.05) alleles at HLA-DQB* locus also were increased in ADEM patients. Our preliminary data provide further evidence that the HLA-DR/DQ haplotypes may be involved in susceptibility to immunomediate demyelinating diseases of central nervous system in the Caucasian population.     

Nanog increases focal adhesion kinase (FAK) promoter activity and expression and directly binds to FAK protein to be phosphorylated

Nanog and FAK were shown to be overexpressed in cancer cells. In this report, the Nanog overexpression increased FAK expression in 293, SW480, and SW620 cancer cells. Nanog binds the FAK promoter and up-regulates its activity, whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK promoter contains four Nanog-binding sites. The site-directed mutagenesis of these sites significantly decreased up-regulation of FAK promoter activity by Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by pulldown and immunoprecipitation assays, and proteins co-localize by confocal microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F and Y174F Nanog did not. The wild type Nanog increased cell invasion that was inhibited by the FAK inhibitor and increased by FAK more significantly than with the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased cell growth reversed by FAK overexpression. Thus, these data demonstrate the regulation of the FAK promoter by Nanog, the direct binding of the proteins, the phosphorylation of Nanog by FAK, and the effect of FAK and Nanog cross-regulation on cancer cell morphology, invasion, and growth that plays a significant role in carcinogenesis.     

A comparative study of warheads for design of cysteine protease inhibitors

The effects on potency of cruzain inhibition of replacing a nitrile group with alternative warheads were explored. The oxime was almost an order of magnitude more potent than the corresponding nitrile and has the potential to provide access to the prime side of the catalytic site. Dipeptide aldehydes and azadipeptide nitriles were found to be two orders of magnitude more potent cruzain inhibitors than the corresponding dipeptide nitriles although potency differences were modulated by substitution at P1 and P3. Replacement of the α methylene of a dipeptide aldehyde with cyclopropane led to a loss of potency of almost three orders of magnitude. The vinyl esters and amides that were characterized as reversible inhibitors were less potent than the corresponding nitrile by between one and two orders of magnitude.     

Inhibition of the α-carbonic anhydrase from Vibrio cholerae with amides and sulfonamides incorporating imidazole moieties

We discovered novel and selective sulfonamides/amides acting as inhibitors of the α-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae (VchCA). This Gram-negative bacterium is the causative agent of cholera and colonises the upper small intestine where sodium bicarbonate is present at a high concentration. The secondary sulfonamides and amides investigated here were potent, low nanomolar VchCA inhibitors whereas their inhibition of the human cytosolic isoforms CA I and II was in the micromolar range or higher. The molecules represent an interesting lead for antibacterial agents with a possibly new mechanism of action, although their CA inhibition mechanism is unknown for the moment.     

Changes in ultrastructure and the occurrence of permeability transition in mitochondria during rat liver regeneration.

Mitochondrial bioenergetic impairment has been found in the organelles isolated from rat liver during the prereplicative phase of liver regeneration. To gain insight into the mechanism underlying this impairment, we investigated mitochondrial ultrastructure and membrane permeability properties in the course of liver regeneration after partial hepatectomy, with special interest to the role played by Ca2+ in this process. The results show that during the first day after partial hepatectomy, significant changes in the ultrastructure of mitochondria in situ occur. Mitochondrial swelling and release from mitochondria of both glutamate dehydrogenase and aspartate aminotransferase isoenzymes with an increase in the mitochondrial Ca2+ content were also observed. Cyclosporin-A proved to be able to prevent the changes in mitochondrial membrane permeability properties. At 24 h after partial hepatectomy, despite alteration in mitochondrial membrane permeability properties, no release of cytochrome c was found. The ultrastructure of mitochondria, the membrane permeability properties and the Ca2+ content returned to normal values during the replicative phase of liver regeneration. These results suggest that, during the prereplicative phase of liver regeneration, the changes in mitochondrial ultrastructure observed in liver specimens were correlated with Ca2+-induced permeability transition in mitochondria.     

Different mutations of the RET gene cause different human tumoral diseases.

The RET gene encodes a tyrosine kinase receptor for neurotrophic molecules. RET is a conceptually valuable example of how different mutations of a single gene may cause different diseases. Gene rearrangements activate the oncogenic potential of RET in human thyroid papillary carcinomas. On the other side, different point mutations activate RET in familial multiple endocrine neoplasia syndromes. Finally, inactivating mutations of RET can be present in Hirschsprung's disease patients. The detailed knowledge of the specific RET mutations responsible for human tumors provides relevant tools for the clinical management of these diseases. Moreover, the recent discovery of the growth factors which in vivo stimulate its signaling may shed new light on the role played by RET in the development and differentiation of the central and peripheral nervous system.     

Immunohistochemical localization of carbonic anhydrase isoenzymes II and III in quail kidney

The immunohistochemical localization of carbonic anhydrase isoenzymes has never been investigated in avian renal tissue previously. Enzyme activity has largely been documented by histochemical and physiological reports. In this investigation, specific antisera were used to study the distribution of the cytosolic carbonic anhydrase II and III isoenzymes in the quail kidney. Comparison between the present findings and the corresponding histochemical patterns, previously obtained in the same species by a cobalt phosphate precipitation method, resulted in the bulk of renal carbonic anhydrase activity being attributed to the carbonic anhydrase II isoenzyme. Conversely, moderate carbonic anhydrase III immunostaining appeared to be confined to the smooth muscle cells of ureteral and arteriolar walls. Indirect evidence of the occurrence, in the quail kidney, of a membrane-associated carbonic anhydrase form, antigenically distinct from the II and III isoforms, was inferred.