Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay

We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu²⁺ in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time–response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample.     

Mutant huntingtin causes metabolic imbalance by disruption of hypothalamic neurocircuits

In Huntington's disease (HD), the mutant huntingtin protein is ubiquitously expressed. The disease was considered to be limited to the basal ganglia, but recent studies have suggested a more widespread pathology involving hypothalamic dysfunction. Here we tested the hypothesis that expression of mutant huntingtin in the hypothalamus causes metabolic abnormalities. First, we showed that bacterial artificial chromosome-mediated transgenic HD (BACHD) mice developed impaired glucose metabolism and pronounced insulin and leptin resistance. Selective hypothalamic expression of a short fragment of mutant huntingtin using adeno-associated viral vectors was sufficient to recapitulate these metabolic disturbances. Finally, selective hypothalamic inactivation of the mutant gene prevented the development of the metabolic phenotype in BACHD mice. Our findings establish a causal link between mutant huntingtin expression in the hypothalamus and metabolic dysfunction, and indicate that metabolic parameters are powerful readouts to assess therapies aimed at correcting dysfunction in HD by silencing huntingtin expression in the brain.     

Some measures of reducing leaching loss of nitrates beyond potential rooting zone

Summary Soil nitrate profiles under seven treatments of an experiment on intercropping in row crops were studied at sowing and the after harvesting of different crops. The estimates of NO₃ ^––N in these profiles indicate that intercropping in the row crops grown during the rainy season considerably reduced leaching loss of nitrates. Where the main crop receives the recommended fertilizer amount and the intercrop a small additional application, intercropping greatly reduced the amount of unutilized nitrates and hence their leaching beyong root zone.     

Genetic Variations of PIP4K2A Confer Vulnerability to Poor Antipsychotic Response in Severely Ill Schizophrenia Patients

Literature suggests that disease severity and neurotransmitter signaling pathway genes can accurately identify antipsychotic response in schizophrenia patients. However, putative role of signaling molecules has not been tested in schizophrenia patients based on severity of illness, despite its biological plausibility. In the present study we investigated the possible association of polymorphisms from five candidate genes RGS4, SLC6A3, PIP4K2A, BDNF, PI4KA with response to antipsychotic in variably ill schizophrenia patients. Thus in present study, a total 53 SNPs on the basis of previous reports and functional grounds were examined for their association with antipsychotic response in 423 schizophrenia patients segregated into low and high severity groups. Additionally, haplotype, diplotype, multivariate logistic regression and multifactor-dimensionality reduction (MDR) analyses were performed. Furthermore, observed associations were investigated in atypical monotherapy (n = 355) and risperidone (n = 260) treated subgroups. All associations were estimated as odds ratio (OR) and 95% confidence interval (CI) and test for multiple corrections was applied. Single locus analysis showed significant association of nine variants from SLC6A3, PIP4K2A and BDNF genes with incomplete antipsychotic response in schizophrenia patients with high severity. We identified significant association of six marker diplotype ATTGCT/ATTGCT (rs746203-rs10828317-rs7094131-rs2296624-rs11013052-rs1409396) of PIP4K2A gene in incomplete responders (corrected p-value = 0.001; adjusted-OR = 3.19, 95%-CI = 1.46–6.98) with high severity. These associations were further observed in atypical monotherapy and risperidone sub-groups. MDR approach identified gene-gene interaction among BDNF_rs7103411-BDNF_rs1491851-SLC6A3_rs40184 in severely ill incomplete responders (OR = 7.91, 95%-CI = 4.08–15.36). While RGS4_rs2842026-SLC6A3_rs2975226 interacted synergistically in incomplete responders with low severity (OR = 4.09, 95%-CI = 2.09–8.02). Our findings provide strong evidence that diplotype ATTGCT/ATTGCT of PIP4K2A gene conferred approximately three-times higher incomplete responsiveness towards antipsychotics in severely ill patients. These results are consistent with the known role of phosphatidyl-inositol-signaling elements in antipsychotic action and outcome. Findings have implication for future molecular genetic studies as well as personalized medicine. However more work is warranted to elucidate underlying causal biological pathway.     

Study of semiconducting nanomaterials under pressure

The pressure induced structural and mechanical properties of nanocrystalline ZnO, ZnS, ZnSe, GaN, CoO, CdSe, CeO(2), SnO(2), SiC, c-BC(2)N, and β-Ga(2)O(3) with different grain sizes have been analyzed under high pressures. The molecular dynamics simulation model has been used to compute isothermal equation of state, volume collapse and bulk modulus of these materials in nano and bulk phases at ambient and high pressures and compared with the experimental data. It is evident from these calculations that the change in particle size affects directly the phase transition pressure and bulk modulus. The values of phase transition pressure and bulk modulus increase with decrease in grain size of the material. The equilibrium cell volume and volume collapse in parent phase is directly proportional to the grain size of the materials. Present results are in good agreement with experimental data. The model is able to explain these thermodynamic properties at varying temperatures and pressures successfully.     

Hexafluoroisopropanol-induced helix–sheet transition of stem bromelain: Correlation to function

Stem bromelain is a proteolytic phytoprotein with a variety of therapeutic effects. Understanding its structural properties could provide insight into the mechanisms underlying its clinical utility. Stem bromelain was evaluated for its conformational and folding properties at the pH conditions it encounters when administered orally. It exists as a partially folded intermediate at pH 2.0. The conformational changes to this intermediate state were evaluated using fluorinated alcohols known to induce changes similar to those seen in vivo. Studies using circular dichroism, fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid and mass spectrometry indicate that treatment with 10–30% hexafluoroisopropanol induces the partially folded intermediate to adopt much of the native protein's secondary structure, but only a rudimentary tertiary structure, characteristic of the molten globule state. Addition of slightly higher concentrations of hexafluoroisopropanol caused transformation from an α-helix to a β-sheet and induced formation of a compact nonnative structure. This nonnative form was more inhibitory of cell survival than either the native or the partially folded intermediate forms, as measured by enhanced suppression of proliferative cues (e.g., extracellular-signal-regulated kinase) and initiation of apoptotic events. The nonnative form also showed better antitumorigenic properties, as evaluated using an induced two-stage mouse skin papilloma model. In contrast, the nonnative state showed only a fraction of the proteolytic activity of the native form. This study demonstrates that hexafluoroisopropanol can induce a conformational change in stem bromelain to a form with potentially useful therapeutic properties different from those of the native protein.     

Combinatorial effect of fish oil (Maxepa) and 1α,25-dihydroxyvitamin D3 in the chemoprevention of DMBA-induced mammary carcinogenesis in rats

The present study demonstrates the anti-tumor effects of combined supplementations of dietary fish oil (Maxepa) and 1α,25-dihydroxyvitamin D₃ (vitamin D₃) on 7,12-dimethylbenz(α)anthracene (DMBA)-induced rat mammary carcinogenesis. Female Sprague–Dawley rats at 50 days of age were treated with 7,12-dimethylbenz(α)anthracene (DMBA; 0.5 mg/100 g body weight) by a single tail vein injection in an oil emulsion. Both fish oil (rich in EPA and DHA) and vitamin D₃ were administered orally at a dose of 0.5 ml/day/rat and 0.3 μg/100 μL propylene glycol twice a week respectively and continued to 35 weeks after DMBA administration. Fish oil in combination with vitamin D₃ resulted in a significant reduction in incidence, multiplicity and volume of mammary tumors. These supplementation also inhibited DMBA-induced mammary 7-methylguanine DNA adducts formation, which was measured by HPLC-fluorescence assay (at four sequential time points; ANOVA, F = 42.56, P < 0.0001). Immunohistochemical analysis revealed that the effect of fish oil and vitamin D₃ occurred through suppression of cell proliferation (BrdU-LI: P < 0.0001). Fish oil and vitamin D₃ together also reduced the mRNA expression of iNOS (84%, P < 0.05). In view of their natural availability, non-toxicity and acceptability; combined supplementation of fish oil and vitamin D₃ might be effective for chemoprevention of mammary carcinogenesis.