Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli

The implementation of efficient technologies for the production of recombinant mammalian proteins remains an outstanding challenge in many structural and functional genomics programs. We have developed a new method for rapid identification of soluble protein expression in E. coli, based on a separation of soluble protein from inclusion bodies by a filtration step at the colony level. The colony filtration (CoFi) blot is very well suited to screen libraries, and in the present work we used it to screen a deletion mutagenesis library.     

Design, synthesis and evaluation of 2,4-diaminoquinazolines as inhibitors of trypanosomal and leishmanial dihydrofolate reductase

This paper describes the design, synthesis and evaluation of a series of 2,4-diaminoquinazolines as inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Compounds were designed by a generating virtual library of compounds and docking them into the enzyme active site. Following their synthesis, they were found to be potent and selective inhibitors of leishmanial dihydrofolate reductase. The compounds were also found to have potent activity against Trypanosoma brucei rhodesiense, a causative organism of African trypanosomiasis and also against Trypanosoma cruzi, the causative organism of Chagas disease. There was significantly lower activity against Leishmania donovani, one of the causative organisms of leishmaniasis.     

Biphenylquinuclidines as inhibitors of squalene synthase and growth of parasitic protozoa

In this paper we describe the preparation of some biphenylquinuclidine derivatives and their evaluation as inhibitors of squalene synthase in order to explore their potential in the treatment of the parasitic diseases leishmaniasis and Chagas disease. The compounds were screened against recombinant Leishmania major squalene synthase and against Leishmania mexicana promastigotes, Leishmania donovani intracellular amastigotes and Trypanosoma cruzi intracellular amastigotes. Compounds that inhibited the enzyme, also reduced the levels of steroids and caused growth inhibition of L. mexicana promastigotes. However there was a lower correlation between inhibition of the enzyme and growth inhibition of the intracellular parasites, possibly due to delivery problems. Some compounds also showed growth inhibition of T. brucei rhodesiense trypomastigotes, although in this case alternative modes of action other than inhibition of SQS are probably involved.     

Cytometric bead array (CBA) for the measurement of cytokines in urine and plasma of patients undergoing renal rejection

Renal rejection is associated with an active immune response regulated by cytokines and in which immunocompetent cells are involved. Previous studies have measured high levels of cytokines in the urine and plasma in various renal dysfunction states. However, some methods used to measured cytokines hinder their use as a diagnostic tool in renal rejection. In this report, cytokine levels were determined in the plasma and urine of kidney transplant patients, with renal rejection and without it, using a cytometric bead array (CBA) technique. Concentrations of six human cytokines (IL-2, IL-4, IL-5, IL-10, TNF-alpha and INF-gamma) were established. Results show that patients who develop renal rejection presented high levels of IL-10 and IFN-gamma cytokines in plasma and urine compared to patients without renal rejection. The CBA technique displayed greater sensitivity in the determination of cytokines in urine than the conventional ELISA technique. Finally, when standard cytokines in plasma and in urine were compared, it was observed that, in plasma, levels of IL-4, IL-5, IL-10, TNF-alpha and IFN-gamma were detected, whereas in urine the levels detected were of IL-4, IL-5, IL-10 and IFN-gamma. These results indicate that the CBA assay is a sensitive method to measure cytokines in urine. In kidney transplant patients undergoing acute renal rejection, the presence of cytokines in urine reflects renal damage and could be a useful method in the diagnosis of renal rejection.     

Toward optimized antibody microarrays: a comparison of current microarray support materials

With the advent of protein and antibody microarray technology several different coatings and protocols have been published, which may be broadly divided into two types: gel-coated surfaces and plain non-gel-coated glass or plastic surfaces, some with chemical groups attached. We have screened 11 different array surfaces of both types and compared them with respect to their detection limit, inter- and intrachip variation, and storage characteristics. Five different antibodies were immobilized onto each type of microarray support, with total protein concentrations ranging from 40 fmol to 25 amol per spot. From these results, it was seen that some antibodies were more suited for use on antibody arrays. All measurements were performed in quadruplicate, and the results revealed high signal uniformity and reproducibility of most plain glass and plastic slides. Lower detection limits were obtained with polyacrylamide-coated slides, making them more suitable for the detection of very low concentrations of antigen. All microarray coatings could be stored for a period of 8 weeks; however, improved results were seen after 2 weeks of storage. In conclusion, the results indicate the need to test each antibody to be used on an antibody array and to select the microarray coating based on experimental requirements.     

Characterization of deoxyuridine 5'-triphosphate nucleotidohydrolase from Trypanosoma cruzi

Inhibition of thrombin by heparin cofactor II (HCII) is accelerated 1000-fold by heparin or dermatan sulfate. To investigate the contribution of basic residues of the A helix of HCII to this activation, we constructed amino acid substitutions (K101Q, R103L, and R106L) by site-directed mutagenesis. K101Q greatly reduced heparin cofactor activity and required a more than 10-fold higher concentration of dermatan sulfate to accelerate thrombin inhibition compared with wild-type recombinant HCII. Thrombin inhibition by R106L was not significantly stimulated by dermatan sulfate. These results provide evidence that basic residues of the A helix of HCII (Lys(101) and Arg(106)) are necessary for heparin- or dermatan sulfate-accelerated thrombin inhibition.