A bioluminogenic HDAC activity assay: validation and screening

Histone deacetylase (HDAC) enzymes modify the acetylation state of histones and other important proteins. Aberrant HDAC enzyme function has been implicated in many diseases, and the discovery and development of drugs targeting these enzymes is becoming increasingly important. In this article, the authors report the evaluation of homogeneous, single-addition, bioluminogenic HDAC enzyme activity assays that offer less assay interference by compounds in comparison to fluorescence-based formats. The authors assessed the key operational assay properties including sensitivity, scalability, reproducibility, signal stability, robustness (Z'), DMSO tolerance, and pharmacological response to standard inhibitors against HDAC-1, HDAC-3/NcoR2, HDAC-6, and SIRT-1 enzymes. These assays were successfully miniaturized to a 10 μL assay volume, and their suitability for high-throughput screening was tested in validation experiments using 640 drugs approved by the Food and Drug Administration and the Hypha Discovery MycoDiverse natural products library, which is a collection of 10 049 extracts and fractions from fermentations of higher fungi and contains compounds that are of low molecular weight and wide chemical diversity. Both of these screening campaigns confirmed that the bioluminogenic assay was high-throughput screening compatible and yielded acceptable performance in confirmation, counter, and compound/extract and fraction concentration-response assays.     

A potential novel mechanism involving connexin 43 gap junction for control of sertoli cell proliferation by thyroid hormones

There is strong evidence that thyroid hormones through triiodothyronine (T3) regulate Sertoli cell proliferation and differentiation in the neonatal testis. However, the mechanism(s) by which they are able to control Sertoli cell proliferation is unclear. In the present study in vivo approaches (PTU-induced neonatal hypothyroidism known to affect Sertoli cell proliferation) associated with in vitro experiments on a Sertoli cell line were developed to investigate this question. We demonstrated that the inhibitory effect of T3 on Sertoli cell growth, analyzed by evaluating DNA-incorporated [3H] thymidine, was associated with a time and dose-dependent increase in the levels of Cx43, a constitutive protein of gap junctions, known to participate in the control of cell proliferation and the most predominant Cx in the testis. These Cx43 changes were associated with increased gap junction communication measured by gap FRAP. Consistent with these results two specific inhibitors of gap junction coupling, AGA and oleamide, were able to significantly reverse the T3 inhibitory effect on Sertoli cell proliferation. The present data also revealed a nongenomic effect of T3 on Cx43 Sertoli cells that was evidenced by a rapid up-regulation of gap junction plaque number as identified in Cx43-GFP transfected cells exposed to the hormone. This process appears mediated through actin cytoskeleton since incubation of the cells with cytochalasin D totally reversed the T3 stimulatory effect on Cx43-GFP gap junction plaques. Based on these data, we propose a working hypothesis in which Cx43 could be an intermediate target for T3 inhibition of neonatal Sertoli cell proliferation.     

UTILLdb, a Pisum sativum in silico forward and reverse genetics tool

The systematic characterization of gene functions in species recalcitrant to Agrobacterium-based transformation, like Pisum sativum, remains a challenge. To develop a high throughput forward and reverse genetics tool in pea, we have constructed a reference ethylmethane sulfonate mutant population and developed a database, UTILLdb, that contains phenotypic as well as sequence information on mutant genes. UTILLdb can be searched online for TILLING alleles, through the BLAST tool, or for phenotypic information about mutants by keywords.     

Temperature-Sensitive Initiation of Chromosome Replication in a Mutant of Bacillus subtilis

A mutant of Bacillus subtilis Ts37 has been isolated in which deoxyribonucleic acid (DNA) synthesis is inhibited at high temperature. The results presented here indicate that the process of initiation of DNA replication is temperature sensitive in this mutant. After shifting to 45 C, DNA increases 40 to 50% before synthesis ceases; an inhibition of protein synthesis permits an equivalent amount of DNA to be synthesized. A density shift experiment coupled with a marker frequency analysis shows that DNA synthesized at 45 C is highly enriched in the markers situated at the end of the chromosome. Transforming DNA extracted from a culture which has been incubated at 45 C exhibits the relative transforming efficiency for origin and terminus markers characteristic of completed chromosomes. After a shift back from 45 C to 30 C, reinitiation appears to occur always in the same region of the bacterial chromosome; in addition, replication as well as cell division is synchronized.     

Seasonal variations in size and morphology of Acanthocyclops robustus (Copepoda Cyclopoida)

The variations in the body length, swimming legs and some setaeof the freshwater cyclopid Acanthocyclops robustus were analysedduring an annual cycle in the field, and in laboratory experimentsat different culture temperatures. In Lake Créteil. ashallow temperate sand-pit lake, there is a seasonal morphologicalvariation of the setae: the plumose forms were restricted tothe warmer season and the spinose ones to colder periods. Contraryto other years, the specific ‘spine formula’ ofthe swimming legs (3-4-4-4) did not vary during the referenceyear. In laboratory experiments, both sex and post-embryonicdevelopmental temperatures had an effect on the length of adults.A significant parental effect on body length was detected. Pairswith anomalous spine formulae produced offspring either withanomalous spine formulae or typical 3-4-4-4 formulae. No significanteffects of parental culture temperatures, offspring culturetemperatures and sex on the total number of spines were found.Modification of plumose into spinose setae did not occur, whateverthe offspring developmental temperatures. The results presentedhere suggest that the alterations of the appendages of the cyclopidA.robustus could be compared to the cyclomorphosis in otherzooplanktonic groups, and contribute to our understanding ofthe role of phenotypic induction in aquatic biology.     

A new mutation, 3905insT, accounts for 4.8% of 1173 CF chromosomes in Switzerland and causes a severe phenotype

We have analysed 1173 cystic fibrosis (CF) chromosomes from Switzerland for eight mutations in the CF transmembrane conductance regulator (CFTR) gene. This permitted the identification of 88.5% of all mutations present. A novel insertion mutation in exon 20 of the CFTR gene, 3905insT, was discovered. This mutation accounted for 4.8% of CFTR gene mutations in Switzerland and has since been identified in other populations of probable Swiss descent. It is associated with a highly variable clinical phenotype but always with pancreatic insufficiency. Haplotype analysis with three intragenic microsatellites in the CFTR gene showed that the mutation is associated with a haplotype rarely identified on other CFTR alleles and, therefore, that the frequency of the mutation in Switzerland is explained by a founder effect of a relatively recent mutation event. Received: 17 February 1997 / Accepted: 26 March 1977     

The inhibitory effect of polymeric carboxylic amino-acids and urine on calcium oxalate crystallization

A new method has been established to follow the inhibitory effect of some polymeric-dicarboxylic-amino-acids and other poly-anionic derivatives. The technique is based on using calcium specific electrode to measure continuously free calcium ion activity in solution to assess the formation of calcium oxalate precipitate.The retardation effect of Poly-L-glutamic and aspartic acids has been established in 5–100 ppm range and compared to other monomeric and polymeric compounds with inactivated functional groups. The retardation effect is in agreement with previous reports on inhibition effect on crystal growth rate of seed crystals, with the same retardants.The inhibitory effect was also determined with normal urine and compared to pathological urine.     

Influence des conditions préalables de culture sur le métabolisme de l'éthanol et de l'acide acétique par la levure

Résumé Le milieu de croissance préalable semble intervenir sur l'aptitude des levures à métaboliser l'éthanol et l'acide acétique. Dans certains cas, les deux substrats activent l'oxydation des réserves. Ce phénomène se produit avec des levures cultivées sur milieu synthétique contenant du glucose 2% ou de l'éthanol 1%. Il ne se produit pas avec des levures cultivées sur ce même milieu synthétique contenant du glucose 0,5%. Il ne peut cependant être expliqué, ni par une richesse supérieure des cellules en réserves glucidiques, ni par un déséquilibre alimentaire, ni par une différence d'activité respiratoire.

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Influence of culture conditions on ethanol and acetic acid metabolism of yeast

Summary The medium in which yeasts are grown seems to modify their ability to metabolize ethanol and acetic acid. In certain cases, the two substrates activate the oxidation of reserves. The phenomenon is observed with yeasts grown on synthetic medium with glucose 2% or ethanol 1%. It is never observed with yeasts grown on the same medium with glucose 0.5%. It cannot be explained either by an elevated cellular level of carbohydrate reserves, by a nutritional imbalance, or by a difference in respiratory activity.