Secretory expression and characterization of a highly Ca-activated thermostable L2 lipase

Thermostable lipases are important biocatalysts, showing many interesting properties with industrial applications. Previously, a thermophilic Bacillus sp. strain L2 that produces a thermostable lipase was isolated. In this study, the gene encoding for mature thermostable L2 lipase was cloned into a Pichia pastoris expression vector. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter, the recombinant L2 lipase was secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. After optimization the maximum recombinant lipase activity achieved in shake flasks was 125 U/ml. The recombinant 44.5 kDa L2 lipase was purified 1.8-fold using affinity chromatography with 63.2% yield and a specific activity of 458.1 U/mg. Its activity was maximal at 70 °C and pH 8.0. Lipase activity increased 5-fold in the presence of Ca²⁺. L2 lipase showed a preference for medium to long chain triacylglycerols (C₁₀–C₁₆), corn oil, olive oil, soybean oil, and palm oil. Stabilization at high temperature and alkaline pH as well as its broad substrate specificity offer great potential for application in various industries that require high temperature operations.     

Whole-Genome Characterization of Human and Simian Immunodeficiency Virus Intrahost Diversity by Ultradeep Pyrosequencing

Rapid evolution and high intrahost sequence diversity are hallmarks of human and simian immunodeficiency virus (HIV/SIV) infection. Minor viral variants have important implications for drug resistance, receptor tropism, and immune evasion. Here, we used ultradeep pyrosequencing to sequence complete HIV/SIV genomes, detecting variants present at a frequency as low as 1%. This approach provides a more complete characterization of the viral population than is possible with conventional methods, revealing low-level drug resistance and detecting previously hidden changes in the viral population. While this work applies pyrosequencing to immunodeficiency viruses, this approach could be applied to virtually any viral pathogen.The viral population within each human immunodeficiency virus (HIV)-infected individual is highly diverse and constantly evolving (2, 3). However, our understanding of the viral population is based largely on the consensus sequence of the dominant circulating virus because the full diversity of the viral population is extremely difficult to characterize. One recent study showed that despite viral fitness recovery in vitro, recovery was not correlated with changes observed in the consensus sequence of HIV. Instead, increased fitness correlated with general viral heterogeneity (5). This finding suggests that by limiting our studies to consensus sequences, we are missing many aspects of viral evolution that influence fitness, drug resistance, and immune evasion, among other characteristics. Studies that examined minor viral variants have provided new insights into HIV transmission and pathogenesis, with direct implications for HIV treatment (7, 13). Unfortunately, traditional techniques to identify rare variants, such as molecular cloning, single-genome amplification, or quantitative real-time (qRT)-PCR, are either labor intensive or restricted to the detection of single variants, limiting their widespread use (8, 11, 12, 14).New second-generation technologies have radically altered DNA sequencing. Recent work by our group and others has employed pyrosequencing for targeted ultradeep sequencing of short regions of the viral genome, including CD8⁺ T-lymphocyte epitopes and regions of known drug resistance mutations, demonstrating a practical method to identify extremely low-frequency viral variants (4, 15). While sequencing short regions is appropriate in certain circumstances, the region of interest must be identified in advance, and the effect of mutations in that region on the remaining genome is ignored. Studying the heterogeneity of HIV across the entire genome may provide insights into interactions between minor variants, improve our understanding of HIV evolution, and ultimately provide insights into disease pathogenesis.In this study, we combined pyrosequencing with a transposon-based fragmentation method to allow powerful ultradeep sequencing of the full-length HIV and simian immunodeficiency virus (SIV) genomes, demonstrating a new and highly practical approach to study the complexity of the viral population within a host and identify minor variants on a genome-wide scale. While this study applied pyrosequencing to immunodeficiency viruses, this approach could be applied to any viral pathogen.     

Whitemere, a lake that defies some conventions about nutrients

1. Whitemere, a kettle-hole lake in north-west England (z_m,14 m, area 22.5 ha) has extremely high maximal concentrations (around 1 mg L⁻¹) of total phosphorus (TP) and soluble reactive phosphorus (SRP), and comparatively low maximal concentrations of dissolved inorganic nitrogen (<0.5 mg N L⁻¹). Bioassays indicate that its phytoplankton is nitrogen limited, and it has surface blue-green algal blooms in summer. Palaeoecological investigations have shown that high populations of cyanophytes occurred more than 6000 years ago, even before human settlement of the catchment.
2. The reasons for this combination of features are not immediately clear. This paper attempts to distinguish between two hypotheses: that the high phosphorus concentrations arise from input of phosphate-rich groundwater; and that the high phosphorus concentrations arise from concentration and recycling mechanisms within the lake.
3. The lake is entirely fed by ground water, direct rainfall and surface sheet flow. The concentrations of phosphorus in the ground water are much lower than in the lake water. The lake is stratified and has high phosphorus concentrations in the anaerobic hypolimnion in summer as a result of substantial release from the sediment. Vertical turbulence in the relatively weakly stratified water column may transfer substantial amounts to the epilimnion.There is also substantial phosphorus release from the sediments under the aerobic epilimnion.
4. There is little outflow of water and phosphorus. Most of the phosphorus is returned to the sediment under winter isothermal conditions, to be released again the following summer. The lake has probably maintained these mechanisms for millennia. Recent eutrophication is likely to have been driven by nitrogen inputs from greater intensification of agriculture in the catchment.     

Attenuation of glyceraldehyde-3-phosphate dehydrogenase activity and ATP levels in rat brain synaptosomes by acrylamide

The effect of neurotoxin acrylamide (AC) on energy metabolism has been studied in a purified preparation of the synaptosomes. The synaptosomes were prepared by the flotation technique in a discontinuous Ficoll/sucrose gradient. The purity of the synaptosomes was checked by electron microscopy and by assaying the activity of marker enzymes. By these criterias, free mitochondrial contamination in the synaptosomes was found to be >2%. Incubation of the synaptosomes with different concentrations of AC (2.5, 5.0, and 10mM) produced a concentration-dependent inhibition (15, 35, and 60%, respectively) of glyceraldehyde-3-phosphate dehydrogenase activity. Acrylamide also produced a time-dependent decrease of ATP concentrations in the synaptosomes; about 25% loss of ATP was seen within 1h, while about 60% ATP was lost after 120 min incubation with 10 mM AC. The effect of known inhibitors of glycolysis-iodoacetic acid (IAA), and of oxidative phophorylation-rotenone and antimycin A, was also studied on ATP synthesis by the synaptosomes. IAA was found to be the most potent inhibitor of ATP synthesis, while both rotenone and antimycin A were equally effective in blocking ATP synthesis in the synaptosomes. These studies show that the synaptosome might be used as a suitablein vitro model to study the effect of neurotoxin such as AC on neuronal energy metabolism.Special issue dedicated to Dr. Sidney Ochs.     

Muslims with non-insulin dependent diabetes fasting during Ramadan: treatment with glibenclamide.

OBJECTIVE--To compare the efficacy of two glibenclamide regimens in patients with non-insulin dependent diabetes who were fasting during Ramadan and regular glibenclamide treatment in the non-fasting group. DESIGN--Non-randomised control group of patients who did not fast during Ramadan and two groups of patients who fasted randomised equally to one of two regimens: to take their usual morning dose of glibenclamide in the evening and their usual evening dose before dawn; or to follow this pattern but to reduce the total dose by a quarter. SETTING--Two university hospitals, one private hospital, and two private clinics in Casablanca and Rabat, Morocco. SUBJECTS--591 diabetic patients (198 men, 391 women, two unspecified) with similar duration of diabetes and length and amount of glibenclamide treatment, of whom 542 completed the study. MAIN OUTCOME MEASURES--Serum fructosamine and total glycated haemoglobin concentrations and number of hypoglycaemic events. RESULTS--At the end of Ramadan there were no significant differences between the groups in fructosamine concentration (400 mumol/l in controls and 381 mumol/l and 376 mumol/l in the fasting groups); percentage of glycated haemoglobin (14.7%, 14.0%, and 13.6%); or number of hypoglycaemic events during Ramadan (11, 14, and 10). CONCLUSION--Glibenclamide is effective and safe for patients with non-insulin dependent diabetes who fast during Ramadan. The easiest regimen is to take the normal morning dose (together with any midday dose) at sunset and any evening dose before dawn.     

Inhibition of myotoxic activity of Bothrops asper myotoxin II by the anti-trypanosomal drug suramin

Suramin, a synthetic polysulfonated compound, developed initially for the treatment of African trypanosomiasis and onchocerciasis, is currently used for the treatment of several medically relevant disorders. Suramin, heparin, and other polyanions inhibit the myotoxic activity of Lys49 phospholipase A2 analogues both in vitro and in vivo, and are thus of potential importance as therapeutic agents in the treatment of viperid snake bites. Due to its conformational flexibility around the single bonds that link the central phenyl rings to the secondary amide backbone, the symmetrical suramin molecule binds by an induced-fit mechanism complementing the hydrophobic surfaces of the dimer and adopts a novel conformation that lacks C2 symmetry in the dimeric crystal structure of the suramin-Bothrops asper myotoxin II complex. The simultaneous binding of suramin at the surfaces of the two monomers partially restricts access to the nominal active sites and significantly changes the overall charge of the interfacial recognition face of the protein, resulting in the inhibition of myotoxicity.     

Turkish population data on the factor XIII Val34Leu,glycoprotein (GP)Ibalpha Kozak and P-selectin glycoprotein ligand 1 (PSGL-1) loci

We determined the allele and genotype frequencies of three PCR-based gene polymorphisms factor XIII (FXIII) Val34Leu, glycoprotein (GP) Ibalpha Kozak and P-selectin glycoprotein ligand 1 (PSGL-1) in the Turkish population (n = 126 for FXIII Val34Leu, n = 110 for GPIbalpha Kozak and n = 203 for PSGL-1). To detect these polymorphisms, DNA was extracted from venous blood. Genomic DNA samples were replicated and analysed by a polymerase chain reaction (PCR) method. PCR products were digested by restriction endonuclease enzymes for FXIII Val34Leu and GPIbalpha Kozak. PSGL-1 was analysed by variable number of tandem repeats (VNTR). Allele frequencies of V (Val) and L (Leu) were found to be 0.805 and 0.195 respectively for the FXIII Val34Leu polymorphism. No significant difference was observed between French and Turkish populations for FXIII Val34Leu. Allele frequencies of T and C were calculated to be 0.873 and 0.127 for the GPIbalpha Kozak polymorphism and no significant difference was found between Turkish and French populations. In contrast, the difference between Turkish and Japanese populations was statistically significant (p<0.0001). In the PSGL-1 group, allele frequencies of A, B and C were calculated as 0.818, 0.160, 0.022 respectively. For the PSGL-1, although the difference between Turkish and French populations was not significant, the difference between the Turkish and Japanese was extremely significant (p<0.0001). In conclusion, a Turkish population database has been established for three gene polymorphisms.     

A role for PYK2 in regulation of ERK1/2 MAP kinases and PI 3-kinase by ANG II in vascular smooth muscle

Abnormal vascular smooth muscle cell (VSMC) growth plays a key role in the pathogenesis of hypertension and atherosclerosis. Angiotensin II (ANG II) elicits a hypertrophic growth response characterized by an increase in protein synthesis without cell proliferation. The present study investigated the role of the nonreceptor tyrosine kinase PYK2 in the regulation of ANG II-induced signaling pathways that mediate VSMC growth. Using coimmunoprecipitation analysis, the role of PYK2 as an upstream regulator of both extracellular signal-related kinase (ERK) 1/2 mitogen-activated protein kinase and phosphatidylinositol 3-kinase (PI 3-kinase) pathways was examined in cultured rat aortic VSMC. ANG II (100 nM) promoted the formation of a complex between PYK2 and the ERK1/2 regulators Shc and Grb2. ANG II caused a rapid and Ca(2+)-dependent tyrosine phosphorylation of the adapter molecule p130Cas, which coimmunoprecipitated both PYK2 and PI 3-kinase in ANG II-treated VSMC. Complex formation between PI 3-kinase and p130Cas and PYK2 was associated with a rapid phosphorylation of the ribosomal p70(S6) kinase in a Ca(2+)- and tyrosine kinase-dependent manner. These data suggest that PYK2 is an important regulator of multiple signaling pathways involved in ANG II-induced VSMC growth.