Production and characterization of pharmacologically active recombinant human phosphodiesterase 4B in Dictyostelium discoideum

Phosphodiesterase 4B (PDE4B) is an important therapeutic target for asthma and chronic obstructive pulmonary disease. To identify PDE4 subtype-specific compounds using high-throughput assays, full-length recombinant PDE4 proteins are needed in bulk quantity. In the present study, full-length human PDE4B2 was expressed in the cellular slime mould Dictyostelium discoideum (Dd). A cell density of 2 x 10(7) cells/mL was obtained and up to 1 mg/L recombinant PDE4B2 was purified through Ni-NTA affinity chromatography. The expressed protein was soluble and its activity was comparable to PDE4B2 protein expressed in mammalian cells (K(m)=1.7 microM). The functional significance of the Dd expression system is supported by the demonstration that, in concert with proteins expressed in mammalian systems, there are no major changes in the affinity for PDE4B2 inhibitors and substrates. These findings thus provide the first evidence that Dd can be utilized for the expression and purification of functionally active full-length human PDE4B2 in large amounts required for high-throughput screening of pharmacologically active compounds against this therapeutic target.     

Validity of the work productivity and activity impairment questionnaire - general health version in patients with rheumatoid arthritis

Introduction  

The Work Productivity and Activity Impairment (WPAI) questionnaire is a well validated instrument to measure impairments in work and activities. However, its validation among patients with rheumatoid arthritis (RA) has not been well established. The present study's purpose is to evaluate the construct validity of the WPAI-general health version among RA patients and its ability to differentiate between RA patients with varying health status.     

First Report of bla CTX-M-15-Type ESBL-Producing Klebsiella pneumoniae in Wild Migratory Birds in Pakistan

EcoHealth - We investigated wild migratory birds faecal swabs for extended-spectrum β-lactamases-producing Klebsiella pneumoniae (ESBL-K. pneumoniae) from wetland habitats in Pakistan. ESBL-K....     

An Update on Genetic Modification of Chickpea for Increased Yield and Stress Tolerance

Chickpea is a highly nutritious grain legume crop, widely appreciated as a health food, especially in the Indian subcontinent. The major constraints on chickpea production are biotic (Helicoverpa, bruchid, aphid, ascochyta) and abiotic (drought, heat, salt, cold) stresses, which reduce the yield by up to 90%. Various strategies like conventional breeding, molecular breeding, and modern plant breeding have been used to overcome these problems. Conventionally, breeding programs aim at development of varieties that combine maximum number of traits through inter-specific hybridization, wide hybridization, and hybridization involving more than two parents. Breeding is difficult in this crop because of its self-pollinating nature and limited genetic variation. Recent advances in in vitro culture and gene technologies offer unique opportunities to realize the full potential of chickpea production. However, as of date, no transgenic chickpea variety has been approved for cultivation in the world. In this review, we provide an update on the development of genetically modified chickpea plants, including those resistant to Helicoverpa armigera, Callosobruchus maculatus, Aphis craccivora, as well as to drought and salt stress. The genes utilized for development of resistance against pod borer, bruchid, aphid, drought, and salt tolerance, namely, Bt, alpha amylase inhibitor, ASAL, P5CSF129A, and P5CS, respectively, are discussed.     

Differential effect of ammonium on the induction of nitrate and nitrite reductase activities in roots of barley (Hordeum vulgare) seedlings

The effect of exogenous NH₄⁺ on the induction of nitrate reductase activity (NRA; EC 1.6.6.1) and nitrite reductase activity (NiRA; EC 1.7.7.1) in roots of 8-day-old intact barley (Hordeum vulgare L.) seedlings was studied. Enzyme activities were induced with 0.1, 1 or 10 mM NO₃⁺ in the presence of 0, 1 or 10 mM NH₄⁺, Exogenous NH₄⁺ partially inhibited the induction of NRA when roots were exposed to 0.1 mM, but not to 1 or 10 mM NO₃⁺, In contrast, the induction of NiRA was inhibited by NH₄⁺ at all NO₃⁺ levels. Maximum inhibition of the enzyme activities occurred at 1.0 mM NH₄⁺ Pre-treatment with NH₄⁺ had no effect on the subsequent induction of NRA in the absence of additional NH₄⁺ whereas the induction of NiRA in NH₄⁺-pretreated roots was inhibited in the absence of NH₄⁺ At 10 mM NO₃⁺ L-methionine sulfoximine stimulated the induction of NRA whether or not exogenous NH₄⁺ was present. In contrast, the induction of NiRA was inhibited by L-methionine sulfoximine irrespective of NH₄⁺ supply. During the postinduction phase, exogenous NH₄⁺ decreased NRA in roots supplied with 0.1 mM but not with 1mM NH₃⁺ whereas, NiRA was unaffected by NH₄⁺ at either substrate concentration. The results indicate that exogenous NH₄⁺ regulates the induction of NRA in roots by limiting the availability of NO₃⁺. Conversely, it has a direct effect, independent of the availability of NO₃⁺, on the induction of NiRA. The lack of an NH₄⁺ effect on NiRA during the postinduction phase is apparently due to a slower turnover rate of that enzyme.     

Validation and Comparison of the Therapeutic Efficacy of Boron Neutron Capture Therapy Mediated By Boron-Rich Liposomes in Multiple Murine Tumor Models

Boron neutron capture therapy (BNCT) was performed at the University of Missouri Research Reactor in mice bearing CT26 colon carcinoma flank tumors and the results were compared with previously performed studies with mice bearing EMT6 breast cancer flank tumors. Mice were implanted with CT26 tumors subcutaneously in the caudal flank and were given two separate tail vein injections of unilamellar liposomes composed of cholesterol, 1,2-distearoyl-sn-glycer-3-phosphocholine, and K[nido-7-CH₃(CH₂)₁₅–7,8-C₂B₉H₁₁] in the lipid bilayer and encapsulated Na₃[1-(2`-B₁₀H₉)-2-NH₃B₁₀H₈] within the liposomal core. Mice were irradiated 30 hours after the second injection in a thermal neutron beam for various lengths of time. The tumor size was monitored daily for 72 days. Despite relatively lower tumor boron concentrations, as compared to EMT6 tumors, a 45 minute neutron irradiation BNCT resulted in complete resolution of the tumors in 50% of treated mice, 50% of which never recurred. Median time to tumor volume tripling was 38 days in BNCT treated mice, 17 days in neutron-irradiated mice given no boron compounds, and 4 days in untreated controls. Tumor response in mice with CT26 colon carcinoma was markedly more pronounced than in previous reports of mice with EMT6 tumors, a difference which increased with dose. The slope of the dose response curve of CT26 colon carcinoma tumors is 1.05 times tumor growth delay per Gy compared to 0.09 times tumor growth delay per Gy for EMT6 tumors, indicating that inherent radiosensitivity of tumors plays a role in boron neutron capture therapy and should be considered in the development of clinical applications of BNCT in animals and man.     

Ochratoxin A blood concentration in healthy subjects and bladder cancer cases from Pakistan

Astract  The mycotoxin ochratoxin A (OTA) is a public health issue in many countries. Data on OTA concentrations in foods and in blood are available for several European countries including the Balkan area, as well as for Canada and Japan. Yet, for developing countries such data are scarce. In this study we determined OTA blood levels as biomarker of exposure in bladder cancer patients and in healthy controls from Pakistan. OTA in blood was analyzed after extraction by HPLC with fluorescence detection (limit of detection: <0.03 ng/mL) in 96 patients and in 31 controls. Over 92% of all blood samples (87 patients, 30 controls) contained quantifiable amounts of OTA: The mean OTA concentrations were 0.33 ng/mL (SD 0.42; range: 0.03 to 3.41 ng/mL) in bladder cancer patients, and 0.31 ng/mL (SD 0.29; range: 0.04 to 1.25 ng/mL) in healthy controls. These OTA concentrations are comparable to those reported for the general population in the European Union. Presented at the 27^th Mykotoxin-Workshop, Dortmund Germany, done 13–15, 2005. The IfADo is accredited as WHO Cellaporating Center for Occupational Health.