G-protein beta3 subunit gene (GNB3) variant in causation of essential hypertension

-Essential hypertensives display enhanced signal transduction through pertussis toxin-sensitive G proteins. The T allele of a C825T variant in exon 10 of the G protein beta3 subunit gene (GNB3) induces formation of a splice variant (Gbeta3-s) with enhanced activity. The T allele of GNB3 was shown recently to be associated with hypertension in unselected German patients (frequency=0.31 versus 0.25 in control). To confirm and extend this finding in a different setting, we performed an association study in Australian white hypertensives. This involved an extensively examined cohort of 110 hypertensives, each of whom were the offspring of 2 hypertensive parents, and 189 normotensives whose parents were both normotensive beyond age 50 years. Genotyping was performed by polymerase chain reaction and digestion with BseDI, which either cut (C allele) or did not cut (T allele) the 268-bp polymerase chain reaction product. T allele frequency in the hypertensive group was 0.43 compared with 0.25 in the normotensive group (chi2=22; P=0.00002; odds ratio=2.3; 95% CI=1.7 to 3.3). The T allele tracked with higher pretreatment blood pressure: diastolic=105+/-7, 109+/-16, and 128+/-28 mm Hg (mean+/-SD) for CC, CT, and TT, respectively (P=0.001 by 1-way ANOVA). Blood pressures were higher in female hypertensives with a T allele (P=0.006 for systolic and 0.0003 for diastolic by ANOVA) than they were in male hypertensives. In conclusion, the present study of a group with strong family history supports a role for a genetically determined, physiologically active splice variant of the G protein beta3 subunit gene in the causation of essential hypertension.

Association of a low density lipoprotein receptor microsatellite variant with obesity

OBJECTIVE: To determine whether a microsatellite polymorphism located towards the 3' end of the low density lipoprotein receptor gene (LDLR) is associated with obesity. DESIGN: A cross-sectional case-control study. SUBJECTS: One hundred and seven obese individuals, defined as a body mass index (BMI) > or = 26 kg/m2, and 163 lean individuals, defined as a BMI < 26 kg/m2. MEASUREMENTS: BMI, blood pressure, serum lipids, alleles of LDLR microsatellite (106 bp, 108 bp and 112 bp). RESULTS: There was a significant association between variants of the LDLR microsatellite and obesity, in the overall tested population, due to a contributing effect in females (chi 2 = 12.3, P = 0.002), but not in males (chi 2 = 0.3, P = 0.87). In females, individuals with the 106 bp allele were more likely to be lean, while individuals with the 112 bp and/or 108 bp alleles tended to be obese. CONCLUSIONS: These results suggest that in females, LDLR may play a role in the development of obesity.

Phosphine resistance in India is characterised by a dihydrolipoamide dehydrogenase variant that is otherwise unobserved in eukaryotes

Phosphine (PH3) fumigation is the primary method worldwide for controlling insect pests of stored commodities. Over-reliance on phosphine, however, has led to the emergence of strong resistance. Detailed genetic studies previously identified two loci, rph1 and rph2, that interact synergistically to create a strong resistance phenotype. We compared the genetics of phosphine resistance in strains of Rhyzopertha dominica and Tribolium castaneum from India and Australia, countries having similar pest species but widely differing in pest management practices. Sequencing analysis of the rph2 locus, dihydrolipoamide dehydrogenase (dld), identified two structurally equivalent variants, Proline49>Serine (P49S) in one R. dominica strain and P45S in three strains of T. castaneum from India. These variants of the DLD protein likely affect FAD cofactor interaction with the enzyme. A survey of insects from storage facilities across southern India revealed that the P45/49S variant is distributed throughout the region at very high frequencies, in up to 94% of R. dominica and 97% of T. castaneum in the state of Tamil Nadu. The abundance of the P45/49S variant in insect populations contrasted sharply with the evolutionary record in which the variant was absent from eukaryotic DLD sequences. This suggests that the variant is unlikely to provide a strong selective advantage in the absence of phosphine fumigation.

Structure and Function of Hemoglobin Confined Inside Silica Nanotubes

Investigations on the structure and function of hemoglobin (Hb) confined inside sol-gel template synthesized silica nanotubes (SNTs) have been discussed here. Immobilization of hemoglobin inside SNTs resulted in the enhancement of direct electron transfer during an electrochemical reaction. Extent of influence of nanoconfinement on protein activity is further probed via ligand binding and thermal stability studies. Electrochemical investigations show reversible binding of n-donor liquid ligands, such as pyridine and its derivatives, and predictive variation in their redox potentials suggests an absence of any adverse effect on the structure and function of Hb confined inside nanometer-sized channels of SNTs. Immobilization also resulted in enhanced thermal stability of Hb. The melting or denaturation temperature of Hb immobilized inside SNTs increase by approximately 4 degrees C as compared with that of free Hb in solution.

Application of z-COSY experiment and its variant for accurate chiral discrimination by H-1 NMR

We report the application of z-COSY experiment and a band selected version of it by employing a selective 90 degrees pulse entitled BASE-z-COSY for precise chiral discrimination, quantification of enantiomeric excess and the analyses of the H-1 NMR spectra of chiral molecules aligned in the chiral liquid crystalline solvent poly-gamma-benzyl-L-glutamate (PBLG). We have demonstrated their applicability for obtaining very high resolution in the H-1 NMR spectra of small organic molecules. It is well known that the commonly employed z-COSY experiment disentangles the spectral complexity, provides pure phase spectra with high resolution, aids in the complete spectral analyses, in addition to yielding information on relative signs of the Couplings. The BASE-z-COSY experiment possesses all these properties, permits the measure of enantiomeric excess, in addition to large saving of instrument time.

A rare P2X7 variant Arg307Gln with absent pore formation function protects against neuroinflammation in multiple sclerosis

Multiple sclerosis (MS) is a chronic relapsing-remitting inflammatory disease of the central nervous system characterized by oligodendrocyte damage, demyelination and neuronal death. Genetic association studies have shown a 2-fold or greater prevalence of the HLA-DRB1*1501 allele in the MS population compared with normal Caucasians. In discovery cohorts of Australasian patients with MS (total 2941 patients and 3008 controls), we examined the associations of 12 functional polymorphisms of P2X7, a microglial/macrophage receptor with proinflammatory effects when activated by extracellular adenosine triphosphate (ATP). In discovery cohorts, rs28360457, coding for Arg307Gln was associated with MS and combined analysis showed a 2-fold lower minor allele frequency compared with controls (1.11% for MS and 2.15% for controls, P = 0.0000071). Replication analysis of four independent European MS case–control cohorts (total 2140 cases and 2634 controls) confirmed this association [odds ratio (OR) = 0.69, P = 0.026]. A meta-analysis of all Australasian and European cohorts indicated that Arg307Gln confers a 1.8-fold protective effect on MS risk (OR = 0.57, P = 0.0000024). Fresh human monocytes heterozygous for Arg307Gln have >85% loss of ‘pore’ function of the P2X7 receptor measured by ATP-induced ethidium uptake. Analysis shows Arg307Gln always occurred with 270His suggesting a single 307Gln–270His haplotype that confers dominant negative effects on P2X7 function and protection against MS. Modeling based on the homologous zP2X4 receptor showed Arg307 is located in a region rich in basic residues located only 12 Å from the ligand binding site. Our data show the protective effect against MS of a rare genetic variant of P2RX7 with heterozygotes showing near absent proinflammatory ‘pore’ function.

Action of N-bromosuccinimide on human hemoglobin and its possible bearing on heme-globin linkage

A reaction of N-bromosuccinimide with the heme groups of hemoglobin has been studied spectrophotometrically. The reaction brings about the disappearance of characteristic absorption peaks of hemoglobin and is accompanied by the release of inorganic iron from the heme groups. Urea is obligatory for the reaction to take place at pH 4.0, while it can occur in the absence of urea at pH 7.0. The spectrum of hemoglobin which does not show any peak in the Soret region at pH 4.0 is “normalized” in the presence of urea or sucrose at the same pH. The effect of “normalization” in 8 M urea is apparent over the pH range 3.0–4.5. From the obligatory requirement of urea and sucrose for “normalization” of spectrum and the dependence of the release of inorganic iron on the concentration of urea, it is suggested that heme groups are “buried” within the globin at pH 4.0 and not dissociated from globin as supposed before.

Testis-specific histone H1t is truly a testis-specific variant and not a meiotic-specific variant

Immunoblotting studies using highly specific polyclonal anti-histone H1t-IgG, which was extensively characterized by us previously, did not produce a signal with any of the histone H1 subtypes of either 1-day-old or adult rat ovarian nuclei. The absence of histone H1t in ovarian nuclei was also confirmed by indirect immunofluorescence studies. It is concluded, therefore, that histone H1t is truly a testis-specific histone variant and not a meiotic-specific variant.

Plasmid DNA mediated transfer of the herpes simplex virus thymidine kinase gene to a new bromodeoxyuridine resistant variant of human primary lung carcinoma cells

A human primary lung carcinoma cell line (HPL-R1) established from the tumor biopsy of a lung cancer patient, lacking in cytochrome P1-450 [aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH)], was cloned and used to obtain variants deficient in the expression of thymidine-kinase via treatment with 5-bromo-2'-deoxyuridine, and selection for drug resistance phenotype. The variant cell line, precharacterized for thymidine kinase negative phenotype, was transfected with the thymidine kinase gene bearing p R-tk and px1-tk plasmids. Transfections from both the plasmids, demonstrated a frequency of 5.5 X 10(-5). The transfectants showed a 76-100% retention of the transferred phenotype. These data suggest that transfection in variant human cells can approach significant levels of stability observed with rodent cell recipients.

Characterization of a variant glucose-6-phosphate dehydrogenase from the south Indian population.

Glucose-6-phosphate dehydrogenase (G6PD) is coded by a gene on the X-chromosome. Earlier studies have shown that the South Indian population has a high incidence of this enzyme deficiency. The electrophoretic mobility, pH optimum and the K-m values for G6PD from normal and variant individuals were identical. However, the specific activity of the variant enzyme was 8 times less compared to the value of the normal enzyme. Western blot analysis of partially purified G6PD from normal and variant individuals performed using equal amounts of total protein showed that the variant protein was 3 times less in concentration. Similar analysis performed using protein corresponding to equal enzyme activity units in the normal and variant samples showed that the variant enzyme was 2.25 times less efficient compared to the normal enzyme. RNA dot blot analysis using full length G6PD cDNA probe (PGDT5B, a kind gift from Prof. L Luzzatto) revealed that lymphocytes from normal and variant individuals had equal amounts of G6PD specific mRNA.

Bufferless lysis of erythrocytes for isolation of hemoglobin using modified cellulose acetate membranes

This paper presents a modified cellulose acetate membrane prepared using a dry casting technique that can be used to perform lysis of erythrocytes and isolation of hemoglobin. Isolation of hemoglobin is thus achieved without the use of lysis buffers. Cellulose acetate (CA) membranes are embedded with ammonium chloride (NH4Cl) and potassium bicarbonate (KHCO3), which act as lysing agents. The presence of embedded salts is confirmed using EDS analysis. The pores in the CA membrane act as filters. The average pore size in these membranes is designed to be 1.5 mu M, as characterized by SEM analysis, so that they allow hemoglobin to pass through and block all other cells and unlysed erythrocytes present in blood. When a drop of blood is added to the membrane, the NH4Cl and KHCO3 embedded in the membrane dissolve in plasma and lyse the erythrocytes. The filtered hemoglobin is characterized using UV-Vis Spectroscopy. The results indicate extraction of higher concentration of hemoglobin compared with conventional methods.