Interaction of benzanthrone with cytochrome P450: altered patterns of hepatic xenobiotic metabolism in rats

Benzanthrone, an anthraquinone dye intermediate, is commonly used for the synthesis of a number of polycyclic vat and disperse dyes. Our prior studies have shown that benzanthrone can be metabolized by rat hepatic microsomal cytochrome P450 (P450) (Biochem. Int., 18, 1989, 1237). In this study, the interaction of benzanthrone with rat hepatic microsomal P-450 and its effect on xenobiotic metabolism have been investigated. Parenteral administration of benzanthrone (40 mg/kg body weight) for 3, 7, or 21 days caused no change in the relative body weight or organ weight of rats. The levels of P450 were found to be reduced (33%-50%) in all the benzanthrone-exposed animals at all the time periods. In vitro addition of benzanthrone caused a spectral change with oxidized P450 and concentration-dependent reduction in the carbon monoxide spectrum of dithionite-reduced P450. The addition of benzanthrone to hepatic microsomes prepared from phenobarbital-treated rats resulted in spectral changes characterized by an absorbance maximum at 397 nm indicative of type I binding. In vitro addition of benzanthrone showed a concentration-dependent inhibition of hepatic aminopyrine N-demethylase (APD) and ethoxyresorufin-O-deethylase (ERD) activities with respective I50 values of 9.5 x 10(-4) and 8.0 x 10(-5) M. However, the inhibition of aryl hydrocarbon hydroxylase (AHH) even at the highest concentration of benzanthrone (10(-2) M), was of the order of only 29%. In vivo administration of benzanthrone also led to the inhibition of APD, AHH, and ERD activities at all treatment times although the magnitude of inhibition was of a lower order.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic control of diabetes in general practice clinics: comparison with a hospital clinic.

An assessment was made of the degree of metabolic control achieved in diabetic patients attending mini clinics run by general practitioners compared with that in matched diabetic patients attending a hospital clinic. Patients were grouped according to whether they were being treated with diet alone, an oral hypoglycaemic, insulin once daily, or insulin twice daily. Statistical analysis showed no significant difference between patients attending mini clinics and those attending the hospital clinic in retrospective mean blood glucose, retrospective mean glycosylated haemoglobin (HbA1), or prospective HbA1 concentrations. General practitioners providing diabetic care on an organised basis can achieve a degree of glycaemic control in diabetic patients equal to that reached by a hospital clinic.     

Behavioural alterations in rats induced by single prenatal exposure of haloperidol

Haloperidol (50 mg/kg, i.p.) treatment was given once to two different groups of pregnant Charles Foster rats on gestational day 9 and 14, these being respectively the critical periods of neural morphogenesis and rapid neural cell proliferation in this species. Pregnant control rats were similarly treated with equal volume of vehicle. The pups born were subjected to open-field exploratory behaviour and elevated plus-maze behaviour tests of anxiety and learned helplessness test of depression at 9 weeks of age. The results indicate that prenatal haloperidol treatment on gestational day 14 induces a significant increase in open-field ambulation and faecal droppings whereas haloperidol treatment on gestational day 9 caused significantly decreased rearing and unaltered ambulation in rat offsprings. Rat offsprings treated with haloperidol on gestational day 9 and 14 also displayed significant anxiogenic behaviour pattern on elevated plus-maze. Significantly increased number of escape failures were observed in learned helplessness tests indicating presence of depression in haloperidol treated rat offsprings. These behavioural alterations were found to be more marked in rat offsprings treated with haloperidol on gestational day 14. The results suggest that prenatal single exposure of high dose of haloperidol during critical period of neural cell proliferation leaves a lasting imprint on offsprings resulting in abnormal emotional state.     

Genetic structures of the CIMMYT international yield trial targeted to irrigated environments

International yield trials are assembled by CIMMYT to disseminate promising wheat breeding materials worldwide. To assess the genomic structure and linkage disequilibrium (LD) within this germplasm, wheat lines disseminated during 25 years of the Elite Spring Wheat Yield Trial (ESWYT) targeted for irrigated environments of the world were genotyped with the high-throughput Diversity Arrays Technology (DArT) marker system. Analyses of population structure assigned the ESWYT germplasm into five major sub-populations that are shaped by prominent CIMMYT wheat lines and their descendants. Based on genetic distance, we concluded that a constant level of genetic diversity was maintained over the years of ESWYT dissemination. Genetic distance between the individual ESWYT lines significantly increased when the ESWYT were grouped according to the differences in years of ESWYT dissemination, suggesting a systematic change in allele frequencies over time, most probably due to breeding and directional selection. By means of multiple regression analyses, 78 markers displaying a significant change in allele frequency across years were identified and interpreted as an indicator for constant selection. The markers identified were partly associated with grain yield, leaf, stem, and yellow rust and point to key genomic regions for further investigation. Large numbers of adjacent DArT marker pairs showed significant LD across the ESWYT population and within each of the five sub-populations identified. Sub-population differentiation measured by the fixation index and average genetic distance were highly correlated with LD levels, suggesting that the sub-populations themselves explain much of the LD.     

Molecular characterization of <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek genotypes based on nuclear ribosomal DNA and RAPD polymorphism

Mungbean germplasm characterization, evaluation and improvement are fundamentally based on morpho-agronomic traits. The lack of break-through in mungbean production has been due to non-availability of genetic variability for high yield potential. Forty-four genotypes of mungbean [Vigna radiata (L.)Wilczek] were subjected to random amplified polymorphic DNA (RAPD) analysis to assess the genetic diversity and relationships among the genotypes. Multilocus genotyping by twelve RAPD primers generated 166 markers and detected an average of intraspecific variation amounting to 82% polymorphism in banding patterns. Dendrogram obtained from cluster analysis delineated all the 44 genotypes into six clusters. Higher values of Nei’s gene diversity (h) and Shannon information index (i) and genetic distance analysis validate existence of wide genetic diversity among mungbean genotypes tested. Besides internal transcribed spacer (ITS) length variations, single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELS) were detected at number of sites in nuclear rDNA region and the sequences of representatives of each sub-cluster and all distinct genotypes have been submitted to NCBI database and assigned Gen accession numbers HQ 148136-148147. Multiple sequence alignment revealed further lineages of distinct genotypes with main RAPD clusters. The measures of relative genetic distances among the genotypes of mungbean did not completely correlate the geographical places of their development. The homogeneous phenotypic markers proved insufficient in exhibiting genetic divergence among mungbean genotypes studied. RMG-62, RMG-976, and NDM-56 have been identified as potential source of parents for crop improvement. RAPD primers, OPA-9 and OPA-2 as polymorphic genetic markers and number of pods/plant and number of seeds/plant as dependable phenotypic markers have been identified for improving yield potentials. This genetic diversity will be of significance in developing intraspecific crosses in mungbean crop improvement programme.     

Palmitoyl-protein thioesterase 1 deficiency in Drosophila melanogaster causes accumulation of abnormal storage material and reduced life span

Human neuronal ceroid lipofuscinoses (NCLs) are a group of genetic neurodegenerative diseases characterized by progressive death of neurons in the central nervous system (CNS) and accumulation of abnormal lysosomal storage material. Infantile NCL (INCL), the most severe form of NCL, is caused by mutations in the Ppt1 gene, which encodes the lysosomal enzyme palmitoyl-protein thioesterase 1 (Ppt1). We generated mutations in the Ppt1 ortholog of Drosophila melanogaster to characterize phenotypes caused by Ppt1 deficiency in flies. Ppt1-deficient flies accumulate abnormal autofluorescent storage material predominantly in the adult CNS and have a life span 30% shorter than wild type, phenotypes that generally recapitulate disease-associated phenotypes common to all forms of NCL. In contrast, some phenotypes of Ppt1-deficient flies differed from those observed in human INCL. Storage material in flies appeared as highly laminar spherical deposits in cells of the brain and as curvilinear profiles in cells of the thoracic ganglion. This contrasts with the granular deposits characteristic of human INCL. In addition, the reduced life span of Ppt1-deficient flies is not caused by progressive death of CNS neurons. No changes in brain morphology or increases in apoptotic cell death of CNS neurons were detected in Ppt1-deficient flies, even at advanced ages. Thus, Ppt1-deficient flies accumulate abnormal storage material and have a shortened life span without evidence of concomitant neurodegeneration.     

Bioconversion of grape must into modulated gluconic acid production by Aspergillus niger ORS-4.410

AIMS: Analysis of regulators for modulated gluconic acid production under surface fermentation (SF) condition using grape must as the cheap carbohydrate source, by mutant Aspergillus niger ORS-4.410. Replacement of conventional fermentation condition by solid-state surface fermentation (SSF) for semi-continuous production of gluconic acid by pseudo-immobilization of A. niger ORS-4.410. METHODS AND RESULTS: Grape must after rectification was utilized for gluconic acid production in batch fermentation in SF and SSF processes using mutant strain of A. niger ORS-4.410. Use of rectified grape must led to the improved levels of gluconic acid production (80-85 g l(-1)) in the fermentation medium containing 0.075% (NH4)2HPO4; 0.1% KH2PO4 and 0.015% MgSO4.7H2O at an initial pH 6.6 (+/-0.1) under surface fermentation. Gluconic acid production was modulated by incorporating the 2% soybean oil, 2% starch and 1% H2O2 in fermentation medium at continuously high aeration rate (2.0 l min(-1)). Interestingly, 95.8% yield of gluconic acid was obtained when A. niger ORS-4.410 was pseudo-immobilized on cellulose fibres (bagasse) under SSF. Four consecutive fermentation cycles were achieved with a conversion rate of 0.752-0.804 g g(-1) of substrate into gluconic acid under SSF. CONCLUSIONS: Use of additives modulated the gluconic acid production under SF condition. Semi-continuous production of gluconic acid was achieved with pseudo-immobilized mycelia of A. niger ORS-4.410 having a promising yield (95.8%) under SSF condition. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioconversion of grape must into modulated gluconic acid production under SSF conditions can further be employed in fermentation industries by replacing the conventional carbohydrate sources and expensive, energy consuming fermentation processes.     

A computational approach to design energetic ionic liquids

The present work deals with the theoretical estimation of ion-pair binding energies and the energetic properties of four ion pairs formed by combining the 1-butyl-2,4-dinitro-3-methyl imidazolium ion with nitrate (I), perchlorate (II), dinitramide (III), or 3,5-dinitro-1,2,4-triazolate (IV) anions. The counterpoise-corrected ion-pair binding energies were calculated for each ion pair at the B3LYP/6-311+G(d,p) level of theory. Results show that the cation–anion interaction is strongest for ion pair I and weakest for IV, indicating that the nitrate (I) has a greater tendency to exist as a stable ionic salt whereas the 3,5-dinitro-1,2,4-triazolate (IV) may exist as an ionic liquid. Natural bond orbital (NBO) analysis and electrostatic potential (ESP) mapping revealed that charge transfer occurs in all of the ion pairs, but is greatest (0.25e) for ion pair I and smallest (0.03e) for IV, resulting in ion pair I being the least polarized. A nucleus-independent chemical shift (NICS) study revealed that the aromaticity of the 1-butyl-2,4-dinitro-3-methyl imidazolium ion significantly increases in ion pair IV, indicating that this has the greatest charge delocalization among all of the four ion pairs considered. Studies of thermodynamic and detonation properties showed that ion pair II is the most energetic ion pair in terms of its detonation velocity (D = 7.5 km s^?1) and detonation pressure (P = 23.1 GPa). It is also envisaged that ion pair IV would exist as an energetic azolium azolate type ionic liquid that could be conveniently used as a secondary explosive characterized by detonation parameters D and P of 6.9 km s^?1 and 19.3 GPa, respectively. These values are comparable to those of conventional explosives such as TNT.     

Insulin responsiveness of glucose transporter 4 in 3T3-L1 cells depends on the presence of sortilin

Insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma membrane of fat and skeletal muscle cells plays the key role in postprandial clearance of blood glucose. Glut4 represents the major cell-specific component of the insulin-responsive vesicles (IRVs). It is not clear, however, whether the presence of Glut4 in the IRVs is essential for their ability to respond to insulin stimulation. We prepared two lines of 3T3-L1 cells with low and high expression of myc₇-Glut4 and studied its translocation to the plasma membrane upon insulin stimulation, using fluorescence-assisted cell sorting and cell surface biotinylation. In undifferentiated 3T3-L1 preadipocytes, translocation of myc₇-Glut4 was low regardless of its expression levels. Coexpression of sortilin increased targeting of myc₇-Glut4 to the IRVs, and its insulin responsiveness rose to the maximal levels observed in fully differentiated adipocytes. Sortilin ectopically expressed in undifferentiated cells was translocated to the plasma membrane regardless of the presence or absence of myc₇-Glut4. AS160/TBC1D4 is expressed at low levels in preadipocytes but is induced in differentiation and provides an additional mechanism for the intracellular retention and insulin-stimulated release of Glut4.Adipocytes, skeletal muscle cells, and some neurons respond to insulin stimulation by translocating intracellular glucose transporter 4 (Glut4) to the plasma membrane. In all these cells, the insulin-responsive pool of Glut4 is localized in small membrane vesicles, the insulin-responsive vesicles (IRVs; Kandror and Pilch, 2011 ; Bogan, 2012 ). The protein composition of these vesicles has been largely characterized (Kandror and Pilch, 2011 ; Bogan, 2012 ). The IRVs consist predominantly of Glut4, insulin-responsive aminopeptidase (IRAP), sortilin, low-density-lipoprotein receptor–related protein 1 (LRP1), SCAMPs, and VAMP2. Glut4, IRAP, and sortilin physically interact with each other, which might be important for the biogenesis of the IRVs (Shi and Kandror, 2007 ; Shi et al., 2008 ). In addition, the IRVs compartmentalize recycling receptors, such as the transferrin receptor and the IGF2/mannose 6-phosphate receptor, although it is not clear whether these receptors represent obligatory vesicular components or their presence in the IRVs is explained by mass action (Pilch, 2008 ), inefficient sorting, or other reasons.Deciphering of the protein composition of the IRVs is important because it is likely to explain their unique functional property: translocation to the plasma membrane in response to insulin stimulation. Even if we presume that IRV trafficking is controlled by loosely associated peripheral membrane proteins, the latter should still somehow recognize the core vesicular components that create the “biochemical individuality” of this compartment. In spite of our knowledge of the IRV protein composition, however, the identity of the protein(s) that confer insulin sensitivity to these vesicles is unknown.Insulin responsiveness of the IRVs was associated with either IRAP or Glut4. Thus it was shown that Glut4 interacted with the intracellular anchor TUG (Bogan et al., 2003 , 2012 ), whereas IRAP associated with other proteins implemented in the regulation of Glut4 translocation, such as AS160 (Larance et al., 2005 ; Peck et al., 2006 ), p115 (Hosaka et al., 2005 ), tankyrase (Yeh et al., 2007 ), and several others (reviewed in Bogan, 2012 ). Results of these studies, or at least their interpretations, are not necessarily consistent with each other, as the existence of multiple independent anchors for the IRVs is, although possible, unlikely.Ablation of the individual IRV proteins has also led to controversial data. Thus knockout of IRAP decreases total protein levels of Glut4 but does not affect its translocation in the mouse model (Keller et al., 2002 ). On the contrary, knockdown of IRAP in 3T3-L1 adipocytes has a strong inhibitory effect on translocation of Glut4 (Yeh et al., 2007 ). In yet another study, knockdown of IRAP in 3T3-L1 adipocytes did not affect insulin-stimulated translocation of Glut4 but increased its plasma membrane content under basal conditions (Jordens et al., 2010 ). By the same token, total or partial ablation of Glut4 had various effects on expression levels, intracellular localization, and translocation of IRAP (Jiang et al., 2001 ; Abel et al., 2004 ; Carvalho et al., 2004 ; Gross et al., 2004 ; Yeh et al., 2007 ). Knockdown of either sortilin or LRP1 decreased protein levels of Glut4 (Shi and Kandror, 2005 ; Jedrychowski et al., 2010 ).One model that might explain these complicated and somewhat inconsistent results is that depletion of either major integral protein of the IRVs disrupts the network of interactions between vesicular proteins and thus decreases the efficiency of protein sorting into the IRVs (Kandror and Pilch, 2011 ). Correspondingly, the remaining IRV components that cannot be faithfully compartmentalized in the vesicles are either degraded (Jiang et al., 2001 ; Keller et al., 2002 ; Abel et al., 2004 ; Carvalho et al., 2004 ; Shi and Kandror, 2005 ; Yeh et al., 2007 ; Jedrychowski et al., 2010 ) or mistargeted (Jiang et al., 2001 ; Jordens et al., 2010 ), depending on experimental conditions and types of cells used in these studies. In other words, knockdown of any major IRV component may decrease vesicle formation along with insulin responsiveness. Thus, in spite of a large body of literature, the identity of protein(s) that confer insulin responsiveness to the IRVs is unknown.Here we used a gain-of-function approach to address this question. Specifically, we attempted to “build” functional IRVs in undifferentiated 3T3-L1 preadipocytes by forced expression of the relevant proteins. Undifferentiated preadipocytes do not express Glut4 or sortilin and lack IRVs (ElJack et al., 1999 ; Shi and Kandror, 2005 ; Shi et al., 2008 ). Correspondingly, IRAP, which is expressed in these cells, shows low insulin response (Ross et al., 1998 ; Shi et al., 2008 ). We found that ectopic expression of increasing amounts of Glut4 in undifferentiated preadipocytes does not lead to its marked translocation to the plasma membrane upon insulin stimulation. On the contrary, sortilin expressed in undifferentiated preadipocytes was localized in the IRVs and was translocated to the plasma membrane in response to insulin stimulation. Moreover, upon coexpression with Glut4, sortilin dramatically increased its insulin responsiveness to the levels observed in fully differentiated adipocytes. Thus sortilin may represent the key component of the IRVs, which is responsible not only for the formation of vesicles (Shi and Kandror, 2005 ; Ariga et al., 2008 ; Hatakeyama and Kanzaki, 2011 ), but also for their insulin responsiveness. It is worth noting that sortilin levels are significantly decreased in obese and diabetic humans and mice (Kaddai et al., 2009 ). We thus suggest that sortilin may be a novel and important target in the fight against insulin resistance and diabetes.Our experiments also demonstrate that undifferentiated preadipocytes lack a mechanism for the full intracellular retention of Glut4 that can be achieved by ectopic expression of AS160/TBC1D4.     

Identification of QTL for growth- and grain yield-related traits in rice across nine locations of Asia

Rice double-haploid (DH) lines of an indica and japonica cross were grown at nine different locations across four countries in Asia. Genotype-by-environment (G x E) interaction analysis for 11 growth- and grain yield-related traits in nine locations was estimated by AMMI analysis. Maximum G x E interaction was exhibited for fertility percentage number of spikelets and grain yield. Plant height was least affected by environment, and the AMMI model explained a total of 76.2% of the interaction effect. Mean environment was computed by averaging the nine environments and subsequently analyzed with other environments to map quantitative trait loci (QTL). QTL controlling the 11 traits were detected by interval analysis using mapmaker/qtl. A threshold LOD of >/=3.20 was used to identify significant QTL. A total of 126 QTL were identified for the 11 traits across nine locations. Thirty-four QTL common in more than one environment were identified on ten chromosomes. A maximum of 44 QTL were detected for panicle length, and the maximum number of common QTL were detected for days to heading detected. A single locus for plant height (RZ730-RG810) had QTL common in all ten environments, confirming AMMI results that QTL for plant height were affected the least by environment, indicating the stability of the trait. Two QTL were detected for grain yield and 19 for thousand-grain weight in all DH lines. The number of QTL per trait per location ranged from zero to four. Clustering of the QTL for different traits at the same marker intervals was observed for plant height, panicle number, panicle length and spikelet number suggesting that pleiotropism and or tight linkage of different traits could be the possible reason for the congruence of several QTL. The many QTL detected by the same marker interval across environments indicate that QTL for most traits are stable and not essentially affected by environmental factors.