Pcal_1127, a highly stable and efficient ribose-5-phosphate pyrophosphokinase from <Emphasis Type="Italic">Pyrobaculum calidifontis</Emphasis>

Analysis of the genome sequence of Pyrobaculum calidifontis revealed the presence of an open reading frame Pcal_1127 annotated as ribose-5-phosphate pyrophosphokinase. To examine the properties of Pcal_1127 the coding gene was cloned, expressed in Escherichia coli, and the purified gene product was characterized. Pcal_1127 exhibited higher activity when ATP was replaced by dATP as pyrophosphate donor. Phosphate and EDTA activated the enzyme activity and equivalent amount of activity was detected with ATP and dATP in their presence. Recombinant Pcal_1127 could utilize all the four nucleotides as pyrophosphate donors with a marked preference for ATP. Optimum temperature and pH for the enzyme activity were 55 °C and 10.5, respectively. A unique feature of Pcal_1127 was its stability against temperature as well as denaturants. Pcal_1127 exhibited more than 95 % residual activity after heating for 4 h at 90 °C and a half-life of 15 min in the boiling water. The enzyme activity was not affected by the presence of 8 M urea or 4 M guanidinium chloride. Pcal_1127 was a highly efficient enzyme with a catalytic efficiency of 5183 mM^?1 s^?1. These features make Pcal_1127, a novel and unique ribose-5-phosphate pyrophosphokinase.     

Protein tyrosine phosphatase 1B inhibitors isolated from Artemisia roxburghiana

Artemisia roxburghiana is used in traditional medicine for treating various diseases including diabetes. The present study was designed to evaluate the antidiabetic potential of active constituents by using protein tyrosine phosphatase 1B (PTP1B) as a validated target for management of diabetes. Various compounds were isolated as active principles from the crude methanolic extract of aerial parts of A. roxburghiana. All compounds were screened for PTP1B inhibitory activity. Molecular docking simulations were performed to investigate the mechanism behind PTP1B inhibition of the isolated compound and positive control, ursolic acid. Betulinic acid, betulin and taraxeryl acetate were the active PTP1B principles with IC₅₀ values 3.49?±?0.02, 4.17?±?0.03 and 87.52?±?0.03?µM, respectively. Molecular docking studies showed significant molecular interactions of the triterpene inhibitors with Gly220, Cys215, Gly218 and Asp48 inside the active site of PTP1B. The antidiabetic activity of A. roxburghiana could be attributed due to PTP1B inhibition by its triterpene constituents, betulin, betulinic acid and taraxeryl acetate. Computational insights of this study revealed that the C-3 and C-17 positions of the compounds needs extensive optimization for the development of new lead compounds.     

Metal based drugs: design,synthesis and in-vitro antimicrobial screening of Co(II), Ni(II), Cu(II) and Zn(II) complexes with some new carboxamide derived compounds: crystal structures of N-[ethyl(propan-2-yl)carbamothioyl]thiophene-2-carboxamide and its copper(II) complex

A new series of compounds derived from thiophene-2-carboxamide were synthesized and characterized by IR, ¹H-NMR and ¹³C-NMR, mass spectrometry and elemental analysis. These compounds were further used to prepare their Co(II), Ni(II), Cu(II) and Zn(II) metal complexes. All metal(II) complexes were air and moisture stable. Physical, spectral and analytical data have shown the Ni(II) and Cu(II) complexes to exhibit distorted square-planar and Co(II) and Zn(II) complexes tetrahedral geometries. The ligand (L¹) and its Cu(II) complex were characterized by the single-crystal X-ray diffraction method. All the ligands and their metal(II) complexes were screened for their in-vitro antimicrobial activity. The antibacterial and antifungal bioactivity data showed that the metal(II) complexes were found to be more potent than the parent ligands against one or more bacterial and fungal strains.     

Antibiotic Self-Prescribing Trends,Experiences and Attitudes in Upper Respiratory Tract Infection among Pharmacy and Non-Pharmacy Students: A Study from Lahore

Pharmacists are the custodians of drugs; hence their education, training, behaviors and experiences would affect the future use of drugs at community and hospital pharmacies. Therefore, we aimed at evaluating the self-prescribing antibiotic trends, knowledge and attitudes among pharmacy and non-pharmacy students. We found that pharmacy students had higher risks of experiencing URIs related symptoms such as cough (RR; 1.7, p = 0.002), allergy (RR; 2.07, p = 0.03) and running nose (RR; 3.17, p<0.005), compared to non-pharmacy students -resulting in higher probabilities of selecting cough syrups (OR; 2.3, p<0.005), anti-histamines (OR; 1.8, p = 0.036) and anti-inflammatory/anti-pyretic (OR; 2.4, p<0.005) drugs. Likewise, bachelor’s degree pupils (OR; 2, p = 0.045), urban area residents (OR; 2.44; p = 0.002) and pharmacy students (OR; 2.9, p<0.005) exhibited higher propensities of antibiotic self-use–notable classes include, b-lactams (45.9%) followed by macrolides (26.5%) and augmentin (28.94%), respectively. Surprisingly, pharmacy and non-pharmacy students had higher odds of using antibiotics in common cold (OR; 3.2, p<0.005) and pain (OR; 2.37, p = 0.015), respectively. Unlike non-pharmacy students, pharmacy students were likely to select alternative therapy, such as Joshanda (OR; 2.22, p = 0.011) and were well acquainted with antibiotic hazards, with 77% reduction in risk of antibiotics re-use. In conclusion, university students exhibited antibiotic self-prescribing trends in conditions that does not warrant their use, thus are irrational users. The pharmacy education confers very little benefit to rational self-prescribing practices among students, while non-pharmacy students are more vulnerable to repeated antibiotic usage. Thus, the educational and training modules should be designed for university students to disseminate targeted information regarding the potential hazards of antibiotic self-use and importance of consultation with qualified and registered medical doctor/pharmacist before starting with antibiotics.     

Microarray: gateway to unravel the mystery of abiotic stresses in plants

Environmental factors, such as drought, salinity, extreme temperature, ozone poisoning, metal toxicity etc., significantly affect crops. To study these factors and to design a possible remedy, biological experimental data concerning these crops requires the quantification of gene expression and comparative analyses at high throughput level. Development of microarrays is the platform to study the differential expression profiling of the targeted genes. This technology can be applied to gene expression studies, ranging from individual genes to whole genome level. It is now possible to perform the quantification of the differential expression of genes on a glass slide in a single experiment. This review documents recently published reports on the use of microarrays for the identification of genes in different plant species playing their role in different cellular networks under abiotic stresses. The regulation pattern of differentially-expressed genes, individually or in group form, may help us to study different pathways and functions at the cellular and molecular level. These studies can provide us with a lot of useful information to unravel the mystery of abiotic stresses in important crop plants.     

Inhibition of the SERCA Ca2+ pumps by curcumin. Curcumin putatively stabilizes the interaction between the nucleotide-binding and phosphorylation domains in the absence of ATP.

Curcumin is a compound derived from the spice, tumeric. It is a potent inhibitor of the SERCA Ca2+ pumps (all isoforms), inhibiting Ca2+-dependent ATPase activity with IC50 values of between 7 and 15 microm. It also inhibits ATP-dependent Ca2+-uptake in a variety of microsomal membranes, although for cerebellar and platelet microsomes, a stimulation in Ca2+ uptake is observed at low curcumin concentrations (<10 microm). For the skeletal muscle isoform of the Ca2+ pump (SERCA1), the inhibition of curcumin is noncompetitive with respect to Ca2+, and competitive with respect to ATP at high curcumin concentrations ( approximately 10-25 microm). This was confirmed by ATP binding studies that showed inhibition in the presence of curcumin: ATP-dependent phosphorylation was also reduced. Experiments with fluorescein 5'-isothiocyanate (FITC)-labelled ATPase also suggest that curcumin stabilizes the E1 conformational state. The fact that FITC labels the nucleotide binding site of the ATPase (precluding ATP from binding), and the fact that curcumin affects FITC fluorescence indicate that curcumin must be binding to another site within the ATPase that induces a conformational change to prevent ATP from binding. This observation is interpreted, with the aid of recent structural information, as curcumin stabilizing the interaction between the nucleotide-binding and phosphorylation domains, precluding ATP binding.     

Evaluation of the kinetic properties of the folate transport system in intestinal absorptive epithelium during experimental ethanol ingestion

Folate plays a critical role in maintaining normal metabolic, energy, differentiation and growth status of all mammalian cells. The disturbances in body folate homeostasis such as intestinal malabsorption in alcoholism are well-known contributor to folate deficiency associated disorders. The study was sought to delineate the kinetic features of folate transport in intestinal absorptive epithelium that could highlight insights of malabsorption during alcoholism. We studied [³H]-folic acid transport in intestinal brush border membrane (BBM) after 3 months of ethanol administration at 1 g/kg body weight/day to rats. The results showed that the folate transport exhibited saturable kinetics and was pH, Na⁺, temperature, divalent cation sensitive, besides –SH group(s) was/were found important in the folate transport system to be efficiently operative. Importantly, the decreased intestinal BBM folate transport in chronic alcoholism was associated with increased K _m and decreased V _max during alcoholism. In addition, S–S group status of the transporter and presence of Na⁺ at the absorptive site seems to be perturbed during ethanol ingestion. However, H⁺/folate⁻ coupled transport provided the driving force for transport as pH optimum in acidic range was not altered during alcoholism. The inhibition constants of methotrexate and unlabelled folic acid revealed that the two analogues are handled differently by the folate transport system. In addition, the low activity of folate transport system during chronic ethanol exposure was associated with low RBC folate levels. Overall, these findings suggest that the deregulated folate transport kinetics might contribute to intestinal folate malabsorption in alcoholism.     

Activation of TRPC6 calcium channels by diacylglycerol (DAG)-containing arachidonic acid: a comparative study with DAG-containing docosahexaenoic acid

We synthesized a diacylglycerol (DAG)-containing arachidonic acid, i.e., 1-stearoyl-2-arachidonyl-sn-glycerol (SAG), and studied its implication in the modulation of canonical transient receptor potential sub-type 6 (TRPC6) channels in stably-transfected HEK-293 cells. SAG induced the influx of Ca(2+), and also of other bivalent cations like Ba(2+) and Sr(2+), in these cells. SAG-evoked Ca(2+) influx was not due to its metabolites as inhibitors of DAG-lipase (RHC80267) and DAG-kinase (R50922) failed to inhibit the response of the same. To emphasise that SAG exerts its action via its DAG configuration, but not due to the presence of stearic acid at sn-1 position, we synthesized 1-palmitoyl-2-arachidonyl-sn-glycerol (PAG). PAG-induced increases in [Ca(2+)](i) were not significantly different from those induced by SAG. For the comparative studies, we also synthesized the DAG-containing docosahexaenoic acid, i.e., 1-stearoyl-2-docosahexaenoyl-sn-glycerol (SDG). We observed that SDG and 1,2-dioctanoyl-sn-glycerol (DOG), a DAG analogue, also evoked increases in [Ca(2+)](i), which were lesser than those evoked by SAG. However, activation of TRPC6 channels by all the DAG molecular species (SAG, DOG and SDG) required Src kinases as the tyrosine kinase inhibitors, PP2 and SU6656, significantly attenuated the increases in [Ca(2+)](i) evoked by these agents. Moreover, disruption of lipid rafts with methyl-beta-cyclodextrin completely abolished SAG-, DOG- and SDG-induced increases in [Ca(2+)](i). The present study shows that SAG as well as SDG and DOG stimulate Ca(2+) influx through the activation of TRPC6 calcium channels which are regulated by Src kinases and intact lipid raft domains.     

Molecular cloning,characterization, and expression studies of water buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) somatotropin

Cloning, high-level expression, and characterization of the somatotropin (ST) gene of an indigenous Nili-Ravi breed of water buffalo Bubalus bubalis (BbST) are described. Coding, non-coding, and promoter regions of BbST were amplified and sequenced. Sequence analysis revealed several silent and two interesting point mutations on comparison with STs of other vertebrate species. One interesting variation in the BbST sequence was the replacement of a conserved glutamine residue by arginine. A plasmid was also constructed for the production of BbST in Escherichia coli BL21 (RIPL) CodonPlus, under the control of IPTG-inducible T7-lac promoter. High-level expression could be obtained by synthesizing a codon-optimized ST gene and expressing it in the form of inclusion bodies. The inclusion bodies represented over 20% of the E. coli cellular proteins. The biologically active conformation of purified BbST was confirmed by its efficient growth promoting activity in Nb2 cell proliferation assay. The expression system and purification strategy employed promise to be a useful approach to produce BbST for further use in structure—function studies and livestock industry. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 2, pp. 194–202.