Masking in Waved‐2 Mice: EGF Receptor Control of Locomotion Questioned

It has been suggested that epidermal growth factors (EGF) are responsible for the inhibition of locomotion by light (i.e., masking) in nocturnal rodents (Kramer et al., ). The poor masking response of waved‐2 (Egfr^wa2) mutant mice, with reduced EGF receptor activity, was adduced in support of this idea. In the present work, we studied the responses to light over a large range in illumination levels, in a variety of tests, with pulses of light and with ultradian light‐dark cycles in Egfr^wa2 mutant mice. No evidence suggested that normal functioning of epidermal growth factor receptors was required, or even involved, in masking.     

Synthesis of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas and evaluation as modulators of the isoforms of nitric oxide synthase

Inhibition of the isoforms of nitric oxide synthase (NOS) has important applications in therapy of several diseases, including cancer. Using 1400 W [N-(3-aminomethylbenzyl)acetamidine], thiocitrulline and N(delta)-(4,5-dihydrothiazol-2-yl)ornithine as lead compounds, series of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas were designed as inhibitors of NOS. Ring-substituted benzyl and phenyl isothiocyanates were synthesised by condensation of the corresponding amines with thiophosgene and addition of ammonia gave the corresponding thioureas in high yields. The substituted 2-amino-4,5-dihydrothiazoles were approached by two routes. Treatment of simple benzylamines with 2-methylthio-4,5-dihydrothiazole at 180 degrees C afforded the corresponding 2-benzylamino-4,5-dihydrothiazoles. For less nucleophilic amines and those carrying more thermally labile substituents, the 4,5-dihydrothiazoles were approached by acid-catalysed cyclisation of N-(2-hydroxyethyl)thioureas. This cyclisation was shown to proceed by an S(N)2-like process. Modest inhibitory activity was shown by most of the thioureas and 4,5-dihydrothiazoles, with N-(3-aminomethylphenyl)thiourea (IC(50)=13 microM vs rat neuronal NOS and IC(50)=23 microM vs rat inducible NOS) and 2-(3-aminomethylphenylamino)-4,5-dihydrothiazole (IC(50)=13 microM vs rat neuronal NOS and IC(50)=19 microM vs human inducible NOS) being the most potent. Several thioureas and 4,5-dihydrothiazoles were found to stimulate the activity of human inducible NOS in a time-dependent manner.     

Bayesian hierarchical modeling for time course microarray experiments

Time course microarray experiments designed to characterize the dynamic regulation of gene expression in biological systems are becoming increasingly important. One critical issue that arises when examining time course microarray data is the identification of genes that show different temporal expression patterns among biological conditions. Here we propose a Bayesian hierarchical model to incorporate important experimental factors and to account for correlated gene expression measurements over time and over different genes. A new gene selection algorithm is also presented with the model to simultaneously identify genes that show changes in expression among biological conditions, in response to time and other experimental factors of interest. The algorithm performs well in terms of the false positive and false negative rates in simulation studies. The methodology is applied to a mouse model time course experiment to correlate temporal changes in azoxymethane-induced gene expression profiles with colorectal cancer susceptibility.     

Inferring missing genotypes in large SNP panels using fast nearest-neighbor searches over sliding windows

MOTIVATION: Typical high-throughput genotyping techniques produce numerous missing calls that confound subsequent analyses, such as disease association studies. Common remedies for this problem include removing affected markers and/or samples or, otherwise, imputing the missing data. On small marker sets imputation is frequently based on a vote of the K-nearest-neighbor (KNN) haplotypes, but this technique is neither practical nor justifiable for large datasets. RESULTS: We describe a data structure that supports efficient KNN queries over arbitrarily sized, sliding haplotype windows, and evaluate its use for genotype imputation. The performance of our method enables exhaustive exploration over all window sizes and known sites in large (150K, 8.3M) SNP panels. We also compare the accuracy and performance of our methods with competing imputation approaches. AVAILABILITY: A free open source software package, NPUTE, is available at http://compgen.unc.edu/software, for non-commercial uses.     

Structure-based design, synthesis and preliminary evaluation of selective inhibitors of dihydrofolate reductase from Mycobacterium tuberculosis

Tuberculosis is an increasing threat, owing to the spread of AIDS and to the development of resistance of the causative organism, Mycobacterium tuberculosis, to the currently available drugs. Dihydrofolate reductase (DHFR) is an important enzyme of the folate cycle; inhibition of DHFR inhibits growth and causes cell death. The crystal structure of M. tuberculosis DHFR revealed a glycerol tightly bound close to the binding site for the substrate dihydrofolate; this glycerol-binding motif is absent from the human enzyme. A series of pyrimidine-2,4-diamines was designed with a two-carbon tether between a glycerol-mimicking triol and the 6-position of the heterocycle; these compounds also carried aryl substituents at the 5-position. These, their diastereoisomers, analogues lacking two hydroxy groups and analogues lacking the two-carbon spacing linker were synthesised by acylation of the anions derived from phenylacetonitriles with ethyl (4S,5R)-4-benzyloxymethyl-2,2-dimethyl-1,3-dioxolane-4-propanoate, ethyl (4S,5S)-4-benzyloxymethyl-2,2-dimethyl-1,3-dioxolane-4-propanoate, tetrahydrooxepin-2-one and 2,3-O-isopropylidene-d-erythronolactone, respectively, to give the corresponding alpha-acylphenylacetonitriles. Formation of the methyl enol ethers, condensation with guanidine and deprotection gave the pyrimidine-2,4-diamines. Preliminary assay of the abilities of these compounds to inhibit the growth of TB5 Saccharomyces cerevisiae carrying the DHFR genes from M. tuberculosis, human and yeast indicated that 5-phenyl-6-((3R,4S)-3,4,5-trihydroxypentyl)pyrimidine-2,4-diamine selectively inhibited M. tuberculosis DHFR and had little effect on the human or yeast enzymes.     

Luteinizing hormone-dependent activation of the epidermal growth factor network is essential for ovulation

In the preovulatory ovarian follicle, mammalian oocytes are maintained in prophase meiotic arrest until the luteinizing hormone (LH) surge induces reentry into the first meiotic division. Dramatic changes in the somatic cells surrounding the oocytes and in the follicular wall are also induced by LH and are necessary for ovulation. Here, we provide genetic evidence that LH-dependent transactivation of the epidermal growth factor receptor (EGFR) is indispensable for oocyte reentry into the meiotic cell cycle, for the synthesis of the extracellular matrix surrounding the oocyte that causes cumulus expansion, and for follicle rupture in vivo. Mice deficient in either amphiregulin or epiregulin, two EGFR ligands, display delayed or reduced oocyte maturation and cumulus expansion. In compound-mutant mice in which loss of one EGFR ligand is associated with decreased signaling from a hypomorphic allele of the EGFR, LH no longer signals oocyte meiotic resumption. Moreover, induction of genes involved in cumulus expansion and follicle rupture is compromised in these mice, resulting in impaired ovulation. Thus, these studies demonstrate that LH induction of epidermal growth factor-like growth factors and EGFR transactivation are essential for the regulation of a critical physiological process such as ovulation and provide new strategies for manipulation of fertility.     

Application of high-performance liquid chromatography to the purification and characterization of ribosomal protein L3 from trichodermin-resistant yeast mutants.

A new h.p.l.c. cation-exchange method has been used to separate proteins from 60S ribosomal subunits prepared from strains of Saccharomyces cerevisiae sensitive or resistant to trichodermin. Ribosomal protein L3 was identified in column eluates by one-dimensional and two-dimensional gel electrophoresis and purified further by reverse-phase h.p.l.c. The protein was cleaved with CNBr and the products were analysed, again by reverse-phase h.p.l.c. A marked difference was observed in the peptide profiles between preparations from trichodermin-sensitive and trichodermin-resistant yeast strains. These results provide the first direct demonstration that, in yeast, mutationally induced resistance to trichodermin can alter the covalent structure of ribosomal protein L3. They convincingly demonstrate the potential of the experimental technique for the rapid and preparative separation of a selected yeast ribosomal protein and its subsequent characterization.     

Synteny mapping in the bovine: genes from human chromosome 4.

Genes homologous to those located on human chromosome 4 (HSA4) were mapped in the bovine to determine regions of syntenic conservation among humans, mice, and cattle. Previous studies have shown that two homologs of genes on HSA4, PGM2 and PEPS, are located in bovine syntenic group U15 (chromosome 6). The homologous mouse genes, Pgm-1 and Pep-7, are on MMU5. Using a panel of bovine x hamster hybrid somatic cells, we have assigned homologs of 11 additional HSA4 loci to their respective bovine syntenic groups. D4S43, D4S10, QDPR, IGJ, ADH2, KIT, and IF were assigned to syntenic group U15. This syntenic arrangement is not conserved in the mouse, where D4s43, D4s10, Qdpr, and Igj are on MMU5 while Adh-2 is on MMU3. IL-2, FGB, FGG, and F11, which also reside on MMU3, were assigned to bovine syntenic group U23. These data suggest that breaks and/or fusions of ancestral chromosomes carrying these genes occurred at different places during the evolution of humans, cattle, and mice.     

The bovine pancreatic spasmolytic polypeptide gene maps to syntenic group U10: implications for the evolution of the human breast cancer estrogen inducible locus.

Recently, homology has been reported for pS2, a protein expressed in many human breast cancers, and a hormonogastric protein known as pancreatic spasmolytic polypeptide (SPP; formerly designated as PSP). The breast cancer estrogen inducible locus (BCEI), which encodes pS2, maps to human chromosome 21 (HSA 21). The SPP locus has not been mapped in humans. Several loci from HSA 21 have been mapped in cattle to syntenic group U10, but a BCEI bovine homolog was not detected. If a bovine BCEI locus does exist, map comparisons predict BCEI will reside on syntenic group U10. The assignment of bovine SPP to syntenic group U10 supports the postulated evolutionary relationship between BCEI and SPP.