(2-amino-phenyl)-amides of omega-substituted alkanoic acids as new histone deacetylase inhibitors

A variety of omega-substituted alkanoic acid (2-amino-phenyl)-amides were designed and synthesized. These compounds were shown to inhibit recombinant human histone deacetylases (HDACs) with IC(50) values in the low micromolar range and induce hyperacetylation of histones in whole cells. They induced expression of p21WAF1/Cip1 and caused cell-cycle arrest in human cancer cells. Compounds in this class showed efficacy in human tumor xenograft models.     

Functional genomic approaches using the nematode Caenorhabditis elegans as a model system

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.     

Molecular investigation of two consecutive nosocomial clusters of Candida tropicalis candiduria using pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) typing was applied to the epidemiological investigation of 21 Candida tropicalis isolates collected from urine specimens of 11 patients and one healthcare worker, in an intensive care unit (ICU) over a 4-month period. Seventeen epidemiologically unrelated strains from 14 patients were also tested to determine the discriminatory power of PFGE. PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG), using two restriction enzymes (BssHII and SfiI). The EK pattern was the same in all 38 isolates, while REAG using SfiI separated the isolates into nine types. However, 16 different PFGE types were identified by REAG with BssHII, and the same results were obtained when the results of both REAG tests were combined. In serial urinary isolates from 10 patients, all strains from each patient had the same PFGE pattern. While the epidemiologically unrelated strains from 14 patients consisted of 13 different PFGE types, the 20 isolates from the 11 ICU patients fell into only two PFGE types (types C1 and C2), and these apparently originated from the two different outbreaks. All strains of type C1 (n = 12) were isolated from six patients, between November 1999 and January 2000, and all of the type C2 strains (n=8) were isolated from five patients, during January and February 2000. This study shows two consecutive clusters of C. tropicalis candiduria in an ICU, defined by PFGE typing, and also demonstrates that a PFGE typing method using BssHII is perhaps the most useful method for investigating C. tropicalis candiduria.     

Rhamnolipid production in batch and fed-batch fermentation using<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> BYK-2 KCTC 18012P

The optimization of culture conditions for the bacteriumPseudomonas aeruginosa BYK-2 KCTC 18012P, was performed to increase its rhamnolipid production. The optimum level for carbon, nitrogen sources, temperature and pH, for rhamnolipid production in a flask, were identified as 25 g/L fish oil, 0.01% (w/v) urea, 25 and pH 7.0, respectively. Optimum conditions for batch culture, using a 7-L jar fermentor, were 200 rpm of agitation speed and a 2.0 L/min aeration rate. Under the optimum conditions, on fish oil for 216 h, the final cell and rhamnolipid concentrations were 5.3 g/L and 17.0 g/L respectively. Fed-batch fermentation, with different feeding conditions, was carried out in order to increase, cell growth and rhamnolipid production by thePseudomonas aeruginosa, BYK-2 KCTC 18012P. When 2.5 g of fish oil and 100 mL basal salts medium, containing 0.01% (w/v) urea, were fed intermittently during the fermentation, the final cell and rhamnolipid concentrations at 264 h, were 6.1 and 22.7 g/L respectively. The fed-batch culture resulted in a 1.2-fold increase in the dry cell mass and a 1.3-fold increase in rhamnolipid production, compared to the production of the batch culture. The rhamnolipid production-substrate conversion factor (0.75 g/g) was higher than that of the batch culture (0.68 g/g).     

Modeling the relationship between LVAD support time and gene expression changes in the human heart by penalized partial least squares

MOTIVATION: Heart failure affects more than 20 million people in the world. Heart transplantation is the most effective therapy, but the number of eligible patients far outweighs the number of available donor hearts. The left mechanical ventricular assist device (LVAD) has been developed as a successful substitution therapy that aids the failing ventricle while a patient is waiting for the donor heart. We obtained genomics data from paired human heart samples harvested at the time of LVAD implant and explant. The heart failure patients in our study were supported by the LVAD for various periods of time. The goal of this study is to model the relationship between the time of LVAD support and gene expression changes. RESULTS: To serve the purpose, we propose a novel penalized partial least squares (PPLS) method to build a regression model. Compared with partial least squares and Breiman's random forest method, PPLS gives the best prediction results for the LVAD data.     

Proteomic analysis revealed a strong association of a high level of alpha1-antitrypsin in gastric juice with gastric cancer

To investigate the pathology of gastric disorders, we compared the proteomic patterns of gastric juice from patients with various gastric disorders. In healthy subjects pepsin A, pepsin B and gastric lipase were the major proteins detected by two-dimensional gel electrophoresis. These digestive enzymes were not detected in 60% of gastric cancer cases (18 out of 30 analyzed cases). Interestingly, an extraordinary amount of alpha(1)-antitrypsin was observed in these cases. In contrast to gastric cancer cases, alpha(1)-antitrypsin was detected in only 5% of patients (three out of 56) with chronic atrophic gastritis, and the detection frequency went up as the disease developed (one of four intestinal metaplasia cases, two of seven tubular adenoma cases, a single examined case of hyperplastic polyp and 60% of gastric cancer). Zymography showed that a 60 kDa protease strongly associated with alpha(1)-antitrypsin and mass spectrometric analysis revealed that the gastric alpha(1)-antitrypsin was a protease-cleaved form. Our data suggest that alpha(1)-antitrypsin and 60 kDa protease may serve as good diagnostic and prognostic markers for conditions associated with gastric cancer.     

Effects of transferring in vitro-cultured rabbit embryos to recipient oviducts on mucin coat deposition, implantation and development

A mucin coat is deposited on rabbit embryos during passage through the oviduct; rabbit blastocysts cultured from the 1-cell stage in vitro have no mucin coat. When cultured blastocysts are transferred to recipients, the lack of mucin coat might account in part for subsequent failure of pregnancy. We have investigated the possibility that mucin coat deposition is induced following transfer of in vitro 72 h-cultured blastocysts to oviducts of asynchronous or synchronous recipients. One-cell embryos were collected by flushing oviducts 19-20 h post-coitus and were cultured in vitro for 72 h until they reached the blastocyst stage. The blastocysts were transferred to the oviducts of recipients that were synchronized either with the donors (synchronous) or 1 day later than the donors (asynchronous). They were recovered after 24-48 h and the mucin coat thickness and embryo degeneration rate were measured. The degeneration rate of blastocysts recovered from uteri of synchronous recipients was higher than that from asynchronous recipients (72.2% vs 40.0%). The mucin coats around embryos recovered from oviducts of asynchronous recipients after 48 h were thicker than those from synchronous recipients. More asynchronous recipients were pregnant and gave birth to more pups than synchronous recipients. These results indicate that the oviducts of asynchronous recipients secreted more mucin around the transferred embryos, causing higher rates of implantation of the in vitro-cultured blastocysts.     

Investigations of oocyte in vitro maturation within a mouse model

This study attempted to develop a 'less meiotically competent' murine model for oocyte in vitro maturation (IVM), which could more readily be extrapolated to human clinical assisted reproduction. Oocyte meiotic competence was drastically reduced upon shortening the standard duration of in vivo gonadotrophin stimulation from 48 h to 24 h, and by selecting only naked or partially naked germinal vesicle oocytes, instead of fully cumulus enclosed oocyte complexes. With such a less meiotically competent model, only porcine granulosa coculture significantly enhanced the oocyte maturation rate in vitro, whereas no significant enhancement was observed with macaque and murine granulosa coculture. Increased serum concentrations and the supplementation of gonadotrophins, follicular fluid and extracellular matrix gel within the culture medium did not enhance IVM under either cell-free or coculture conditions. Culture medium conditioned by porcine granulosa also enhanced the maturation rate, and this beneficial effect was not diminished upon freeze-thawing. Enhanced IVM in the presence of porcine granulosa coculture did not, however, translate into improved developmental competence, as assessed by in vitro fertilization and embryo culture to the blastocyst stage.     

Cysteine-321 of human brain GABA transaminase is involved in intersubunit cross-linking

Gamma-aminobutyrate transaminase (GABA-T), a key homodimeric enzyme of the GABA shunt, converts the major inhibitory neurotransmitter GABA to succinic semialdehyde. We previously overexpressed, purified and characterized human brain GABA-T. To identify the structural and functional roles of the cysteinyl residue at position 321, we constructed various GABA-T mutants by site-directed mutagenesis. The purified wild type GABA-T enzyme was enzymatically active, whereas the mutant enzymes were inactive. Reaction of 1.5 sulfhydryl groups per wild type dimer with 5,5 cent-dithiobis-2-nitrobenzoic acid (DTNB) produced about 95% loss of activity. No reactive -SH groups were detected in the mutant enzymes. Wild type GABA-T, but not the mutants, existed as an oligomeric species of Mr = 100,000 that was dissociable by 2-mercaptoethanol. These results suggest that the Cys321 residue is essential for the catalytic function of GABA-T, and that it is involved in the formation of a disulfide link between two monomers of human brain GABA-T.