Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase

Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.     

Screening costramid libraries for chromosomal genes: an alternative interspecific hybridization method

Abstract Broad host-range RK2-based cosmid vectors ('costramids') are increasingly used in molecular genetic studies of Gram-negative soil bacteria such as Rhizobium spp. we describe a simple modification of existing methods, whereby a genomic library constructed in a stringently replicated vector can be screened for genes which are undetectable by colony hybridization due to background cross-hybridization. This method allows the use of 'heterologous' probes (interspecies hybridization) to isolate several presumptive genes of interest from a gene bank of Rhizobium sp. NGR234 made in the costramid pRK7813. These are a gene with homology to the citrate synthase gene ( gltA ) or Escherichia coli , the gene encoding δ-aminol evulinic acid synthase ( hemA ), and a gene or genes regulating dicarboxylate transport.     

Genome-wide Screen for Modulation of Hepatic Apolipoprotein A-I (ApoA-I) Secretion

Control of plasma cholesterol levels is a major therapeutic strategy for management of coronary artery disease (CAD). Although reducing LDL cholesterol (LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25–40% reduction in cardiovascular events. Epidemiological studies have shown that a high LDL-c level is not the only risk factor for CAD; low HDL cholesterol (HDL-c) is an independent risk factor for CAD. Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-c that mediates reverse cholesterol transport from tissues to the liver for excretion. Therefore, increasing ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide siRNA screening, targets that regulate hepatocyte ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted enzymes, proteases, G-protein-coupled receptors, metabolic enzymes, ion transporters, and proteins of unknown function. Repression of farnesyltransferase (FNTA) by siRNA and the enzyme inhibitor manumycin A caused elevation of ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.     

长寿老人源双歧杆菌优良菌株的筛选

以人结肠腺癌细胞系HT-29细胞为试材,对来源于广西巴马百岁以上长寿老人肠道的24株双歧杆菌进行了体外黏附试验。结果发现,双歧杆菌均具有一定的黏附能力,其中TTF、Z2、TZ5和J-1菌株具有较高的黏附能力。进一步对4株初筛双歧杆菌耐胃酸、胆汁酸和合成B族维生素能力的试验发现,双歧杆菌TTF菌株不仅能合成较高的B1、B2、B6、B12等多种B族维生素,而且在pH3．0的条件下处理120min存活率达93．11％,同时在2％胆盐浓度下处理24h有较好的存活,具有显著的综合优势。     

Identification of Small Molecule Inhibitors of Jumonji AT-rich Interactive Domain 1B (JARID1B) Histone Demethylase by a Sensitive High Throughput Screen

JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is overexpressed in several cancers, including breast cancer, prostate cancer, and lung cancer. In addition, JARID1B is required for mammary tumor formation in syngeneic or xenograft mouse models. JARID1B-expressing melanoma cells are associated with increased self-renewal character. Therefore, JARID1B represents an attractive target for cancer therapy. Here we characterized JARID1B using a homogeneous luminescence-based demethylase assay. We then conducted a high throughput screen of over 15,000 small molecules to identify inhibitors of JARID1B. From this screen, we identified several known JmjC histone demethylase inhibitors, including 2,4-pyridinedicarboxylic acid and catechols. More importantly, we identified several novel inhibitors, including 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), which inhibits JARID1B with an IC₅₀ of about 3 μm in vitro. Consistent with this, PBIT treatment inhibited removal of H3K4me3 by JARID1B in cells. Furthermore, this compound inhibited proliferation of cells expressing higher levels of JARID1B. These results suggest that this novel small molecule inhibitor is a lead compound that can be further optimized for cancer therapy.     

Small Molecule Inhibitors of Phospholipase C from a Novel High-throughput Screen

Phospholipase C (PLC) isozymes are important signaling molecules, but few small molecule modulators are available to pharmacologically regulate their function. With the goal of developing a general approach for identification of novel PLC inhibitors, we developed a high-throughput assay based on the fluorogenic substrate reporter WH-15. The assay is highly sensitive and reproducible: screening a chemical library of 6280 compounds identified three novel PLC inhibitors that exhibited potent activities in two separate assay formats with purified PLC isozymes in vitro. Two of the three inhibitors also inhibited G protein-coupled receptor-stimulated PLC activity in intact cell systems. These results demonstrate the power of the high-throughput assay for screening large collections of small molecules to identify novel PLC modulators. Potent and selective modulators of PLCs will ultimately be useful for dissecting the roles of PLCs in cellular processes, as well as provide lead compounds for the development of drugs to treat diseases arising from aberrant phospholipase activity.     

Circannual Variation in the Incidence of Uterine Cervix Cancer

The impact of uterine cervix cancer (UCC) can be greatly reduced by regular vaginal examination and other preventive measures. With this aim, UCC screening programs had been developed and applied for several years in Mexico and elsewhere. One point still to be considered in such preventive programs is the possible circannual pattern in the morbidity or mortality of UCC. With the aim of identifying a possible circannual pattern of variation in the incidence of UCC, we analyzed the monthly totals of positive detected cases of UCC in the state of Nuevo León (Mexico) between 1978 and 1987. For eliminating bias due to the seasonal variation in the number of preventive check-ups, data were first expressed in percentage of tests done in the same month. The least-squares fit of a 1 -year cosine curve to the data reveals a statistically significant circannual pattern (p = 0.013), with a maximum of relative incidence detected in February. Results indicate UCC incidence higher than the yearly average during the winter, with secondary peaks in August and November. This pattern of variation is similar if one considers separately for analysis the relative incidence of type III, IV, or V UCC (relating to cases suggesting, highly suggesting, or concluding a malignant alteration, respectively). For evaluating the cost-effectiveness of the UCC screening campaign providing these data, the monthly totals of screening check-ups done over the same period were also analyzed. Results reveal a statistically significant circannual rhythm (p > 0.001), with a maximum detected in June. This timing seems to be related to the incidence of most common diseases in Mexico, such as giardiasis and salmonellosis, but not to the UCC morbidity rates. Health educational and screening campaigns for prevention of UCC and other major conditions should be timed during the year according to morbidity and mortality statistics, for which a circannual pattern of variation is documented herein.     

A 5q12.1–5q12.3 microdeletion in a case with a balanced exceptional complex chromosomal rearrangement

Complex chromosomal rearrangements are very rare chromosomal abnormalities. Individuals with a complex chromosomal rearrangement can be phenotypically normal or display a clinical abnormality. It is believed that these abnormalities are due to either microdeletions or microduplications at the translocation breakpoints or as a result of disruption of the genes located in the breakpoints. In this study we describe a 2-year-old child with mental retardation and developmental delay in whom a de novo apparently balanced exceptional complex chromosomal rearrangement was found through conventional cytogenetic analysis. Using both cytogenetic and FISH analysis, the patient's karyotype was found to be: 46,XY,der(5)t(5;7)(p15.1;7q34),t(5;8)(q13.1;8q24.1)dn. A large, clinically significant deletion which encompassed 887.69 kb was detected at the 5q12.1–5q12.3 (chr5:62.886.523–63.774.210) genomic region using array-CGH. This deleted region includes the HTR1A and RNF180 genes. This is the first report of an individual with an apparently balanced complex chromosomal rearrangement in conjunction with a microdeletion at 5q12.1–5q12.3 in which there are both mental-motor retardation and dysmorphia.     

In vivo phage display — A discovery tool in molecular biomedicine

In vivo phage display is a high-throughput method for identifying target ligands specific for different vascular beds. Targeting is possible due to the heterogeneous expression of receptors and other antigens in a particular vascular bed. Such expression is additionally influenced by the physiological or pathological status of the vasculature. In vivo phage display represents a technique that is usable in both, vascular mapping and targeted drug development. In this review, several important methodological aspects of in vivo phage display experiments are discussed. These include choosing an appropriate phage library, an appropriate animal model and the route of phage library administration. In addition, peptides or antibodies identified by in vivo phage display homing to specific types of vascular beds, including the altered vasculature present in several types of diseases are summarized. Still, confirmation in independent experiments and reproduction of identified sequences are needed for enhancing the clinical applicability of in vivo phage display research.