Site-directed mutagenesis of disulfide bridges in Aspergillus niger NRRL 3135 phytase (PhyA), their expression in Pichia pastoris and catalytic characterization

Earlier studies have established the importance of five disulfide bridges (DBs) in Aspergillus niger phytase. In this study, the relative importance of each of the individual disulfide bridge is determined by its removal by site-directed mutagenesis of specific cysteines in the cloned A. niger phyA gene. Individually, these mutant phytases were expressed in a Pichia expression system and their product purified and characterized. The removal of disulfide bridge 2 yielded a mutant phytase with a complete loss of catalytic activity. The other disulfide mutants displayed a broad array of altered catalytic properties including a lower optimum temperature from 58°C to 53°C for bridge number 1, 37°C for bridge number 3 and 4, and 42°C for bridge number 5. The pH versus activity profile was also modified in the DB mutants. The pH profile of the wild-type phytase was modified by the DB mutations. In bridge number 1, 3, and 4, the second peak at pH 2.5 was abolished, and in bridge number 5, the peak at pH 5.0 was abolished completely leaving only the pH 2.5. While the K _m was not affected drastically, the turnover number was lowered significantly in bridge number 3, 4, and 5.     

Identification of a recurrent insertion mutation in the LDLR gene in a Pakistani family with autosomal dominant hypercholesterolemia

Familial Hypercholesterolemia (FH) results in elevated levels of blood lipids including total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) with normal triglycerides (TG). This disease is one of the major contributors towards an early onset of coronary heart disease (CHD). The aim of the present study was to identify the genes responsible for causing FH in Pakistani population, for this purpose a large consanguineous FH family was selected for genetic analysis. Serum lipid levels, including TC, TG, LDL-C and high density lipoprotein cholesterol (HDL-C), were determined in patients and healthy controls. In order to find the causative mutation in this family, direct sequencing of the low density lipoprotein receptor (LDLR) gene was performed. In addition the part of the Apolipoprotein-B (APOB) gene containing the mutations R3500Q and R3500W was also sequenced. Affected individuals of the family were found to have raised TC and LDL-C levels. Sequencing revealed an insertion mutation (c.2416_2417InsG) in exon 17 of the LDLR gene in all the affected individuals of the family. Common FH causing APOB mutations were not present in this family. Heterozygous individuals had TC levels ranging from ~300–500 mg/dl and the only homozygous individual with typical xanthomas had TC levels exceeding 900 mg/dl. This is the first report of a known LDLR gene mutation causing FH in the Pakistani population. Despite a large heterogeneity of LDLR mutations there are still some common mutations which are responsible for FH throughout the world.     

Plant growth-promoting activity in newly isolated <Emphasis Type="Italic">Bacillus thioparus</Emphasis> (NII-0902) from Western <Emphasis Type="Italic">ghat</Emphasis> forest,India

A Gram-positive rod-shaped bacterium isolated on nutrient agar plates incubated at 28 ± 2°C. The identity of the bacterium was confirmed by sequencing of the 16S rRNA gene and it reveals that it shares highest similarity with Bacillus thioparus CECT 7196^T (99.08%). It was capable of growing at temperatures ranging from 4 to 40°C, but optimum growth was observed at 28 ± 2°C. Strain NII-0902 is endowed with multiple plant growth promotion attributes such as phosphate solubilization, Indole acetic acid (IAA), siderophore and HCN production, which were expressed differentially at sub-optimal temperatures (5–40°C). It was able to solubilize phosphate (17.7 μg ml⁻¹), and produce IAA (139.7 μg ml⁻¹) at 28 ± 2°C. Qualitative detection of siderophore production and HCN were also observed. At 5°C it was found to express all the plant growth promotion attributes except HCN production. The ability to colonize roots is a sine qua non condition for a rhizobacteria to be considered a true plant growth-promoting rhizobacteria (PGPR). Bacillus sp. NII-0902 has a potential ability to colonize roots visualized by transparency, bacterial growth (turbid, milky and narrow zone) along and around roots and truly supported by scanning electron micrograph. Hence, it is proposed that, Bacillus thioparus sp. NII-0902 could be deployed as an inoculant to attain the desired results of bacterization.     

Discovery of novel,orally available benzimidazoles as melanin concentrating hormone receptor 1 (MCHR1) antagonists

Melanin concentrating hormone (MCH) is an important mediator of energy homeostasis and plays role in several disorders such as obesity, stress, depression and anxiety. The synthesis and biological evaluation of novel benzimidazole derivatives as MCHR1 antagonists are described. The in vivo proof of principle for weight loss with a lead compound from this series is exemplified.     

Identification of cultivation condition to produce correctly folded form of a malaria vaccine based on Plasmodium falciparum merozoite surface protein-1 in Escherichia coli

The C-terminal, 19-kDa domain of Plasmodium falciparum merozoite surface protein-1 (PfMSP-1₁₉) is among the leading vaccine candidate for malaria due to its essential role in erythrocyte invasion by the parasite. We designed a synthetic gene for optimal expression of recombinant PfMSP-1₁₉ in Escherichia coli and developed a scalable process to obtain high-quality PfMSP-1₁₉. The synthetic gene construct yielded a fourfold higher expression level of PfMSP-1₁₉ in comparison to the native gene construct. Optimization of cultivation conditions in the bioreactor indicated important role of yeast extract and substrate feeding strategy for obtaining enhanced expression of soluble and correctly folded PfMSP-1₁₉. It was observed that the higher expression level of PfMSP-1₁₉ was essentially associated with the generation of higher level of incorrectly folded PfMSP-1₁₉. A simple purification procedure comprising metal affinity and ion exchange chromatography was developed to purify correctly folded form of PfMSP-1₁₉ from cell lysate. Biochemical and biophysical characterization of purified PfMSP-1₁₉ suggested that it was highly pure, homogeneous, and correctly folded.     

Estradiol enhances cell-associated paraoxonase 1 (PON1) activity in vitro without altering PON1 expression

PON1 is a high density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. In vivo animal and human studies have indicated that estradiol (E2) supplementation enhances serum PON1 activity. In this study, we sought to determine if E2 directly up-regulates cell-associated PON1 activity in vitro and to characterize the mechanism of regulation. In vitro E2 treatment of both the human hepatoma cell line Huh7 and normal rat hepatocytes resulted in a 2- to 3-fold increase in cell-associated PON1 catalytic activity. E2 potently induced PON1 activity with average EC₅₀ values of 15 nM for normal hepatocytes and 68 nM for Huh7. The enhancement of PON1 activity by E2 was blocked by the estrogen receptor (ER) antagonist ICI 182,780 indicating that E2 was acting through the ER. The up-regulation of PON1 activity by E2 did not involve enhancement of PON1 mRNA or protein levels and did not promote secretion of PON1. Thus, E2 can enhance cell-associated PON1 activity in vitro without altering PON1 gene expression or protein level. Our data suggest that E2 may regulate the specific activity and/or stability of cell surface PON1.     

Mental health literacy towards depression among non-medical students at a Malaysian university

Background The aim of the present study was to evaluate the knowledge and perception of depression among students of University Sains Malaysia (USM), in Penang, Peninsular Malaysia.Method Face-to-face interviews were conducted using a pre-validated 21-item questionnaire among students at USM.Results A total of 500 respondents participated in the survey comprising 24.6% (n=123) males and 75.4% (n=377) females. Half (50.0%, n=250) were Malays, followed by Chinese (44.0%, n=220) and Indians (6.0%, n=30). Whilst exploring the respondents' knowledge of the symptoms of depression, it was found that Chinese females had a comparatively better knowledge (P=0.058) of the symptoms of depression in comparison with Malays and Indians. Overall, social issues were attributed as the possible cause of depression. A cursory knowledge level was observed regarding medication for depression. Female students were more inclined towards the use of alternative and traditional medicines. However, with regard to seeking professional help, consultation with a psychiatrist was preferred by the majority.Conclusion Overall, a moderate level of knowledge about the symptoms of depression and a cursory knowledge of its therapy were observed. Those with personal experience of depression had better knowledge of the symptoms and therapy. Alternative treatments and traditional medicines were also favoured. There is a risk that this may affect the ability of Malaysian youths to seek evidence-based mental health care.     

Plant cell walls are enfeebled when attempting to preserve native lignin configuration with poly-p-hydroxycinnamaldehydes: evolutionary implications

The lignin deficient double mutant of cinnamyl alcohol dehydrogenase (CAD, cad-4, cad-5 or cad-c, cad-d) in Arabidopsis thaliana [Sibout, R., Eudes, A., Mouille, G., Pollet, B., Lapierre, C., Jouanin, L., Séguin, A., 2005. Cinnamyl alcohol dehydrogenase-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis. Plant Cell 17, 2059-2076], was comprehensively examined for effects on disruption of native lignin macromolecular configuration; the two genes encode the catalytically most active CAD's for monolignol/lignin formation [Kim, S.-J., Kim, M.-R., Bedgar, D.L., Moinuddin, S.G.A., Cardenas, C.L., Davin, L.B., Kang, C., Lewis, N.G., 2004. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proc. Natl. Acad. Sci., USA 101, 1455-1460]. The inflorescence stems of the double mutant presented a prostrate phenotype with dynamic modulus properties greatly reduced relative to that of the wild type (WT) line due to severe reductions in macromolecular lignin content. Interestingly, initially the overall pattern of phenolic deposition in the mutant was apparently very similar to WT, indicative of comparable assembly processes attempting to be duplicated. However, shortly into the stage involving (monomer cleavable) 8-O-4' linkage formation, deposition was aborted. At this final stage, the double mutant had retained a very limited ability to biosynthesize monolignols as evidenced by cleavage and release of ca. 4% of the monolignol-derived moieties relative to the lignin of the WT line. In addition, while small amounts of cleavable p-hydroxycinnamaldehyde-derived moieties were released, the overall frequency of (monomer cleavable) 8-O-4' inter-unit linkages closely approximated that of WT for the equivalent level of lignin deposition, in spite of the differences in monomer composition. Additionally, 8-5' linked inter-unit structures were clearly evident, albeit as fully aromatized phenylcoumaran-like substructures. The data are interpreted as a small amount of p-hydroxycinnamaldehydes being utilized in highly restricted attempts to preserve native lignin configuration, i.e. through very limited monomer degeneracy during template polymerization which would otherwise afford lignins proper in the cell wall from their precursor monolignols. The defects introduced (e.g. in the vascular integrity) provide important insight as to why p-hydroxycinnamaldehydes never evolved as lignin precursors in the 350,000 or so extant vascular plant species. It is yet unknown at present, however, as to what levels of lignin reduction can be attained in order to maintain the requisite properties for successful agronomic/forestry cultivation. Nor is it known to what extent, if any, such deleterious modulations potentially compromise plant defenses. Finally, prior to investigating lignin primary structure proper, it is essential to initially define the fundamental characteristics of the biopolymer(s) being formed, such as inter-unit frequency and lignin content, in order to design approaches to determine overall sequences of linkages.     

AtPTR3, a wound-induced peptide transporter needed for defence against virulent bacterial pathogens in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Mutation in the wound-induced peptide transporter gene AtPTR3 (At5g46050) of Arabidopsis thaliana has been shown to affect germination on media containing a high salt concentration. The heterologous expression in yeast was utilized to verify that the AtPTR3 protein transports di-and tripeptides. The T-DNA insert in the Atptr3-1 mutant in the Arabidopsis ecotype C24 revealed two T-DNA copies, the whole vector sequence, and the gus marker gene inserted in the second intron of the AtPTR3 gene. An almost identical insertion site was found in the Atptr3-2 mutant of the Col-0 ecotype. The AtPTR3 expression was shown to be regulated by several signalling compounds, most clearly by salicylic acid (SA), but also methyl jasmonate (MeJA) and abscisic acid. Real-time PCR experiments suggested that the wound-induction of the AtPTR3 gene was abolished in the SA and JA signalling mutants. The Atptr3 mutant plants had increased susceptibility to virulent pathogenic bacteria Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tomato, and produced more reactive oxygen species when grown on media containing paraquat or rose bengal. Public microarray data suggest that the AtPTR3 expression was induced by Pseudomonas elicitors and by avirulent P. syringae pathovars and type III secretion mutants. This was verified experimentally for the hrpA mutant with real-time PCR. These results suggest that AtPTR3 is one of the defence-related genes whose expression is reduced by virulent bacterium by type III dependent fashion. Our results suggest that AtPTR3 protects the plant against biotic and abiotic stresses.     

Fasting and glucose effects on pituitary leptin expression: is leptin a local signal for nutrient status?

Leptin, a potent anorexigenic hormone, is found in the anterior pituitary (AP). The aim of this study was to determine whether and how pituitary leptin-bearing cells are regulated by nutritional status. Male rats showed 64% reductions in pituitary leptin mRNA 24 hr after fasting, accompanied by significant (30-50%) reductions in growth hormone (GH), prolactin, and luteinizing hormone (LH), and 70-80% reductions in target cells for gonadotropin-releasing hormone or growth hormone-releasing hormone. There was a 2-fold increase in corticotropes. Subsets (22%) of pituitary cells coexpressed leptin and GH, and <5% coexpressed leptin and LH, prolactin, thyroid-stimulating hormone, or adrenocorticotropic hormone. Fasting resulted in significant (55-75%) losses in cells with leptin proteins or mRNA, and GH or LH. To determine whether restoration of serum glucose could rescue leptin, LH, and GH, additional fasted rats were given 10% glucose water for 24 hr. Restoring serum glucose in fasted rats resulted in pituitary cell populations with normal levels of leptin and GH and LH cells. Similarly, LH and GH cells were restored in vitro after populations from fasted rats were treated for as little as 1 hr in 10-100 pg/ml leptin. These correlative changes in pituitary leptin, LH, and GH, coupled with leptin's rapid restoration of GH and LH in vitro, suggest that pituitary leptin may signal nutritional changes. Collectively, the findings suggest that pituitary leptin expression could be coupled to glucose sensors like glucokinase to facilitate rapid responses by the neuroendocrine system to nutritional cues.