Use of Bacterial Luciferase as a Reporter Gene in Eukaryotic Systems.

Reporter gene assays are powerful tools for monitoring dynamic molecular changes and for evaluating the responses that occur at the genetic elements within cells in response to exogenous molecules. In general, various protein systems can be used as reporter genes, including luciferases. Here, the present protocol introduces a unique reporter gene system for monitoring molecular events in cells using bacterial luciferase (lux), which can generate blue-green light suitable for gene reporter applications with the highest cost performance. The protocol also guides the assay conditions and necessary components for using of lux gene (lux) as a eukaryotic reporter system. The lux system can be applied to monitor variety of molecular events inside mammalian cellular systems.

Development of a sequential injection-liquid microextraction procedure with GC-FID for analysis of short-chain fatty acids in palm oil mill effluent.

Short-chain fatty acids, such as acetic, propionic, butyric, iso-valeric and valeric acids, play an important role in methanogenesis activity for biogas production processes. Thus, simple and rapid procedures for monitoring the levels of short-chain fatty acids are requisite for sustaining biogas production. This work presents the development of a sequential injection-liquid microextraction (SI-LME) procedure with GC-FID analysis for determination of short-chain fatty acids. GC-FID was employed for detection of the short-chain fatty acids. Calibration curves were linear with good coefficients of determination (r2>0.999), using methacrylic acid as the internal standard. Limits of quantification (LOQ) were in the range of 0.03-0.19mM. The SI-LME procedure employed tert-butyl methyl ether (TBME) as the extracting solvent. Various SI-LME conditions were investigated and optimized to obtain the highest recovery of extraction. With these optimized conditions, an extraction recovery of the five key short-chain fatty acids of 67-90% was obtained, with less than 2% RSD (n=3). The final SI-LME procedure employed two fluidic zones of TBME with a single aqueous fluidic zone of sample sandwiched between the TBME zones, with 5 cycles of flow reversal at a flow rate of 5µL/s for the extraction process. Intra- and inter-day precision values were 0.5-4.0% RSD and 3.3-4.8% RSD, respectively. Accuracy based on percentage of sample recovery were in the range of 69-96, 102-107, and 82-101% (n=4) for acetic, propionic and butyric acids, respectively. The proposed method was applied for the measurement of short-chain fatty acids in palm oil mill effluents used in biogas production in a factory performing palm oil extraction process. The SI-LME method provides improved extraction performance with high precision, and is both simple and rapid with its economical extraction technique. The SI-LME procedure with GC-FID has strong potential for use as a quality control process for monitoring short-chain fatty acid levels in biogas production.

Inhibitors of plasmodial serine hydroxymethyltransferase (SHMT): cocrystal structures of pyrazolopyrans with potent blood- and liver-stage activities.

Several of the enzymes related to the folate cycle are well-known for their role as clinically validated antimalarial targets. Nevertheless for serine hydroxymethyltransferase (SHMT), one of the key enzymes of this cycle, efficient inhibitors have not been described so far. On the basis of plant SHMT inhibitors from an herbicide optimization program, highly potent inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) SHMT with a pyrazolopyran core structure were identified. Cocrystal structures of potent inhibitors with PvSHMT were solved at 2.6 Å resolution. These ligands showed activity (IC50/EC50 values) in the nanomolar range against purified PfSHMT, blood-stage Pf, and liver-stage P. berghei (Pb) cells and a high selectivity when assayed against mammalian cell lines. Pharmacokinetic limitations are the most plausible explanation for lack of significant activity of the inhibitors in the in vivo Pb mouse malaria model.

Characterization of Plasmodium falciparum serine hydroxymethyltransferase-A potential antimalarial target.

Serine hydroxymethyltransferase (SHMT) is a ubiquitous enzyme required for folate recycling and dTMP synthesis. A cDNA encoding Plasmodium falciparum (Pf) SHMT was expressed as a hexa-histidine tagged protein in Escherichia coli BL21-CodonPlus (DE3)-RIL. The protein was purified and the process yielded 3.6 mg protein/l cell culture. Recombinant His(6)-tagged PfSHMT exhibits a visible spectrum characteristic of pyridoxal-5'-phosphate enzyme and catalyzes the reversible conversion of l-serine and tetrahydrofolate (H(4)folate) to glycine and 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate). Steady-state kinetics study indicates that His(6)-tagged PfSHMT catalyzes the reaction by a ternary-complex mechanism. The sequence of substrate binding to the enzyme was also examined by glycine product inhibition. A striking property that is unique for His(6)-tagged PfSHMT is the ability to use D-serine as a substrate in the folate-dependent serine-glycine conversion. Kinetic data in combination with expression result support the proposal of SHMT reaction being a regulatory step for dTMP cycle. This finding suggests that PfSHMT can be a potential target for antimalarial chemotherapy.