Gossypium arboreum GHSP26 enhances drought tolerance in Gossypium hirsutum

Heat‐shock proteins (HSP) are molecular chaperones for protein molecules. These proteins play an important role in protein–protein interactions such as, folding and assisting in the establishment of proper protein conformation and prevention of unwanted protein aggregation. A small HSP gene GHSP26 present in Gossypium arboreum responds to dehydration. In the present study, an attempt was made to overcome the problem of drought stress in cotton. A cDNA of GHSP26 was isolated from G. arboreum, cloned in plant expression vector, pCAMBIA‐1301 driven by the cauliflower mosaic virus 35S promoter and introduced into Gossypium hirsutum. The integration and expression studies of putative transgenic plants were performed through GUS assay; PCR from genomic DNA, and quantitative real‐time PCR analysis. Transgenic cotton plants showed an enhanced drought tolerance, suggesting that GHSP26 may play a role in plant responsiveness to drought. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Power calculations for selective genotyping in QTL mapping in backcross mice

Selective genotyping concerns the genotyping of a portion of individuals chosen on the basis of their phenotypic values. Often individuals are selected for genotyping from the high and low extremes of the phenotypic distribution. This procedure yields savings in cost and time by decreasing the total number of individuals genotyped. Previous work by Darvasi et al. (1993) has shown that the power to detect a QTL by genotyping 40-50 % of a population is roughly equivalent to genotyping the entire sample. However, these power studies have not accounted for different strategies of analysing the data when phenotypes of individuals in the middle are excluded, nor have they investigated the genome-wide type I error rate under these different strategies or different selection percentages. Further, these simulation studies have not considered markers over the entire genome. In this paper, we present simulation studies of power for the maximum likelihood approach to QTL mapping by Lander & Botstein (1989) in the context of selective genotyping. We calculate the power of selectively genotyping the individuals from the middle of the phenotypic distribution when performing QTL mapping over the whole mouse genome.     

Isolation of a minireplicon of the virulence plasmid pXO2 of Bacillus anthracis and characterization of the plasmid-encoded RepS replication protein

A minireplicon of plasmid pXO2 of Bacillus anthracis was isolated by molecular cloning in Escherichia coli and shown to replicate in B. anthracis, Bacillus cereus, and Bacillus subtilis. The pXO2 replicon included (i) an open reading frame encoding the putative RepS replication initiation protein and (ii) the putative origin of replication. The RepS protein was expressed as a fusion with the maltose binding protein (MBP) at its amino-terminal end and purified by affinity chromatography. Electrophoretic mobility shift assays showed that the purified MBP-RepS protein bound specifically to a 60-bp region corresponding to the putative origin of replication of pXO2 located immediately downstream of the RepS open reading frame. Competition DNA binding experiments showed that the 5' and central regions of the putative origin were important for RepS binding. MBP-RepS also bound nonspecifically to single-stranded DNA with a lower affinity.     

Single-molecule dynamics reveal an altered conformation for the autoinhibitory domain of plasma membrane Ca(2+)-ATPase bound to oxidatively modified calmodulin

We used single-molecule polarization modulation methods to investigate the activation of the plasma membrane Ca(2+)-ATPase (PMCA) by oxidized calmodulin (CaM). Oxidative modification of methionine residues of CaM to their corresponding sulfoxides is known to inhibit the ability of CaM to activate PMCA. Single-molecule polarization methods were used to measure the orientational mobility of fluorescently labeled oxidized CaM bound to PMCA. We previously identified two distinct populations of PMCA-CaM complexes characterized by high and low orientational mobilities, with the low-mobility population appearing at a subsaturating Ca(2+) concentration [Osborn, K. D., et al. (2004) Biophys. J. 87, 1892-1899]. We proposed that the high-mobility population corresponds to PMCA-CaM complexes with a dissociated (and mobile) autoinhibitory domain, whereas the low-mobility population corresponds to PMCA-CaM complexes where the autoinhibitory domain is not dissociated and therefore the enzyme is not active. In the present experiments, performed with PMCA complexed with oxidatively modified CaM at a saturating Ca(2+) concentration, we found a large population of molecules with an orientationally immobile autoinhibitory domain. In contrast, native CaM bound to PMCA was characterized almost entirely by the more orientationally mobile population at a similar Ca(2+) concentration. The addition of 1 mM ATP to complexes of oxidized CaM with PMCA reduced but did not abolish the low-mobility population. These results indicate that the decline in the ability of oxidized CaM to activate PMCA results at least in part from its reduced ability to induce conformational changes in PMCA that result in dissociation of the autoinhibitory domain after CaM binding.     

Mutations in the AHI1 gene, encoding jouberin, cause Joubert syndrome with cortical polymicrogyria

Joubert syndrome (JS) is an autosomal recessive disorder marked by agenesis of the cerebellar vermis, ataxia, hypotonia, oculomotor apraxia, neonatal breathing abnormalities, and mental retardation. Despite the fact that this condition was described >30 years ago, the molecular basis has remained poorly understood. Here, we identify two frameshift mutations and one missense mutation in the AHI1 gene in three consanguineous families with JS, some with cortical polymicrogyria. AHI1, encoding the Jouberin protein, is an alternatively spliced signaling molecule that contains seven Trp-Asp (WD) repeats, an SH3 domain, and numerous SH3-binding sites. The gene is expressed strongly in embryonic hindbrain and forebrain, and our data suggest that AHI1 is required for both cerebellar and cortical development in humans. The recently described mutations in NPHP1, encoding a protein containing an SH3 domain, in a subset of patients with JS plus nephronophthisis, suggest a shared pathway.     

Single-molecule dynamics of the calcium-dependent activation of plasma-membrane Ca2+-ATPase by calmodulin

The plasma membrane calcium-ATPase (PMCA) helps to control cytosolic calcium levels by pumping out excess Ca2+. PMCA is regulated by the Ca2+ signaling protein calmodulin (CaM), which stimulates PMCA activity by binding to an autoinhibitory domain of PMCA. We used single-molecule polarization methods to investigate the mechanism of regulation of the PMCA by CaM fluorescently labeled with tetramethylrhodamine. The orientational mobility of PMCA-CaM complexes was determined from the extent of modulation of single-molecule fluorescence upon excitation with a rotating polarization. At a high Ca2+ concentration, the distribution of modulation depths reveals that CaM bound to PMCA is orientationally mobile, as expected for a dissociated autoinhibitory domain of PMCA. In contrast, at a reduced Ca2+ concentration a population of PMCA-CaM complexes appears with significantly reduced orientational mobility. This population can be attributed to PMCA-CaM complexes in which the autoinhibitory domain is not dissociated, and thus the PMCA is inactive. The presence of these complexes demonstrates the inadequacy of a two-state model of Ca2+ pump activation and suggests a regulatory role for the low-mobility state of the complex. When ATP is present, only the high-mobility state is detected, revealing an altered interaction between the autoinhibitory and nucleotide-binding domains.     

Oxidative inactivation of purified plasma membrane Ca2+-ATPase by hydrogen peroxide and protection by calmodulin

Calmodulin (CaM)-regulated plasma membrane Ca(2+)-ATPase (PMCA) is critical for the regulation of free intracellular Ca(2+) levels. PMCA activity and levels in neuronal membranes are decreased with aging, possibly due to oxidation-induced inactivation. In the present studies, inhibition of PMCA by H(2)O(2) was characterized in enzyme purified from human erythrocyte membranes. Basal and CaM-stimulated PMCA activities were inhibited by exposure to H(2)O(2) (25-100 microM). However, neither the concentration-dependent enhancement of PMCA activity by CaM nor the binding of CaM to H(2)O(2)-exposed PMCA was disrupted by treatment with H(2)O(2). Rates of inactivation by H(2)O(2) of basal and CaM-stimulated PMCA were nearly identical. The addition of CaM after exposure to H(2)O(2) did not protect enzyme activity, although the binding of CaM to PMCA before exposure to H(2)O(2) protected the enzyme completely, indicating a CaM-induced conformational state resistant to oxidation. H(2)O(2) quenched Trp fluorescence in PMCA, an index of conformational changes, with a rate similar to that observed for enzyme inactivation. H(2)O(2) enhanced the solvent accessibility of Trp residues in PMCA, whereas accessibility of the only Trp residue in the CaM-binding domain peptide was unaltered. Exposure of PMCA to H(2)O(2) led to aggregate formation partially reversible by dithiothreitol (DTT) but not to recovery of activity. Amino acid analysis indicated Cys modification following H(2)O(2) exposure but no Cys oxyacids. Because DTT did not reverse inactivation by H(2)O(2), it appears that the disulfide bond formation led to conformational changes that were not fully reversed when the bonds were reduced. Preincubation of PMCA with CaM protected the enzyme from undergoing this conformational change.     

Pulmonary cytochrome P-450 2J4 is reduced in a rat model of acute Pseudomonas pneumonia

We previously reported that the levels of epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are depressed in microsomes prepared from lungs of rats with acute Pseudomonas pneumonia. We also showed a potential role for cytochrome P-450 (CYP) metabolites of arachidonic acid (AA) in contractile responses of both normal pulmonary arteries and pulmonary arteries from rats with pneumonia. The CYP2J subfamily enzymes (endogenous source of EETs and HETEs) are constitutively expressed in human and rat lungs where they are localized in vascular smooth muscle and endothelium. The purpose of this study was to determine if CYP2J proteins are modified in pneumonia. Pseudomonas organisms were injected via a tracheostomy in the lungs of rats. Later (44 h), lungs were frozen, and microsomes were prepared from pneumonia and control rat lung homogenates. Lung microsomal proteins were then immunoblotted with anti-CYP2B1/2B2, anti-CYP4A, anti-CYP2J9pep2 (which reacts with rat CYP2J3), anti-CYP2J6pep1 (which reacts with rat CYP2J4), anti-CYP2J2pep4, or anti-CYP2J2pep3 (both of which react with all known CYP2J isozymes). Western blotting revealed a prominent 55-kDa band with anti-CYP2J2pep3, anti-CYP2J2pep4, and anti-CYP2J6pep1 (but not anti-CYP2J9pep2) that was reduced in pneumonia compared with control lung microsomes. The CYP2B bands (51-52 kDa) were less prominent and not different between pneumonia and control lungs. CYP4A proteins (20-HETE sources) were not detected in rat lung microsomes. Therefore, rat lung contains a protein with immunological characteristics similar to CYP2J4, and this CYP is reduced after pneumonia. We speculate that CYP2J (but not CYP2B) enzymes and their AA metabolic products (EETs) are involved in the modulation of pulmonary vascular tone in pneumonia in rats.     

Antioxidant,Anti-Skin-Aging,Anti-Inflammatory,and Anti-Acetylcholinesterase Activities of Rourea oligophlebia Extracts

The objective of this study was to evaluate the antioxidant, anti-skin-aging, anti-inflammatory, and anti-acetylcholinesterase activities of the hexane (n-hex), AcOEt, BuOH, MeOH, and aqueous extracts from R. oligophlebia roots. The total phenolic and flavonoid contents (TPC and TFC) were determined using Folin-Ciocalteu and AlCl₃ colorimetric assays. The antioxidant capacity was examined by reducing power (RP), ferric reducing antioxidant power (FRAP), ABTS⋅⁺, and DPPH⋅⁺ radical cation assays. All extracts potentially exhibited antioxidant activity with IC₅₀ values ranging from 2.93 to 5.73 μg/mL for ABTS⋅⁺ and from 5.69 to 7.65 μg/mL for DPPH⋅⁺ except the n-hex extract. The BuOH, MeOH, and aqueous extract possess promising anti-skin-aging activities, as observed by an attenuation of UV-A toxicity on human keratinocytes. We proposed that these anti-skin-aging properties are possibly due to direct scavenging activity against reactive oxygen species and upregulate cellular antioxidant machinery. Moreover, we found that the antioxidant capacity was well correlated with anti-inflammatory capacity against nitric oxide (NO) production in terms of the n-hex, AcOEt, and BuOH extracts with IC₅₀ values from 23.21 to 47.1 μg/mL. In contrast, these activities were found to be poorly correlated with AchE activity. To the best of our knowledge, this is the first report of the antioxidant, anti-skin-aging, anti-inflammatory, and anti-acetylcholinesterase activities of the extracts of R. oligophlebia roots. These findings indicated that this species could be a potential source of natural antioxidant, anti-aging, and anti-inflammatory agents. Consequently, it may be suggested as a medicinal plant that prevents diseases related to oxidative stress and inflammatory responses.     

Polycystic ovary syndrome is linked with the fat mass obesity (FTO) gene variants rs17817449 and rs1421085 in western Saudi Arabia

Polycystic ovary syndrome (PCOS) is characterised by infertility, obesity, insulin resistance and clinical and/or biochemical signs of hyperandrogenism. Obesity is known to be correlated with PCOS causing ovulatory dysfunction and hormone imbalances. Moreover, fat mass and the obesity gene (FTO) were linked with obesity and PCOS. Therefore, it is of interest to determine the genotype and allele frequency for three FTO variants - rs17817449 (G/T), rs1421085 (C/T) and rs8050136 (A/C) -in western Saudi population. 95 PCOS patients and 94 controls were recruited for this study. The genetic variants were assayed using real-time polymerase chain reaction using TaqMan genotyping assays. The chi-squared test was applied to investigate the difference between single nucleotide polymorphisms on PCOS and control subjects, and binary logistic regression was used to determine the association of FTO variants with PCOS symptoms. Variants rs17817449 and rs1421085 were significantly linked with PCOS susceptibility in the study population. Rs17817449 and rs8050136 were significantly associated with hair loss in the PCOS group. Furthermore, rs1421085 and rs8050136 were associated with a high body mass index (BMI>30 kg/m2). Risk alleles in our population associated with hair loss and elevated BMI in women with PCOS were homozygous C for rs8050136. This data will help in defining the genetic predisposition of PCOS among women in western Saudi Arabia.