Construction of an Integrated Physical and Gene Map of Human Chromosome 20p12 Providing Candidate Genes for Alagille Syndrome

Physical mapping and localization of eSTS markers were used to generate an integrated physical and gene map covering a ∼10-Mb region of human chromosome 20p12 containing the Alagille syndrome (AGS) locus. Seventy-four STSs, 28 of which were derived from cDNA sequences, mapped with an average resolution of 135 kb. The 28 eSTS markers define 20 genes. Six known genes, namely CHGB, BMP2, PLCB1, PLCB4, SNAP, and HJ1, were precisely mapped. Among the genes identified, one maps in the smallest region of overlap of the deletions associated with AGS and could therefore be regarded as a candidate gene for Alagille syndrome.     

Host specialization involving attraction,avoidance and performance,in two phytophagous moth species

Host specialization plays a key role in the extreme diversification of phytophagous insects. Whereas proximate mechanisms of specialization have been studied extensively, their consequences for species divergence remain unclear. Preference for, and performance on hosts are thought to be a major source of divergence in phytophagous insects. We assessed these major components of specialization in two moth species, the European corn borer (ECB) and the Adzuki bean borer (ABB), by testing their oviposition behaviour in different conditions (choice or no‐choice set‐ups) and their performances, by reciprocal transplant at the larval stage on the usual host and an alternative host plant. We demonstrated that both ABB and ECB have a strong preference for their host plants for oviposition, but that relative larval performances on the usual host and an alternative host differed according to the experiment and the trait considered (weight or survival). Finally, we show for the first time that the preference for maize in ECB conceals a strong avoidance of mugwort. The differences in performance, attraction and avoidance between ECB and ABB are discussed in the light of the underlying mechanisms and divergence process.     

Amylase production by three Lactobacillus strains isolated from chicken crop

Three amylolytic Lactobacillus strains designated LEM 220, LEM 207 and LEM 202 were isolated from the chicken crop. They belonged to the subgenus Thermobacterium. Strain LEM 220 resembled Lact. acidophilus. Amylase production was more abundant in cells grown in media containing amylopectin or starch than in media containing glucose or maltose. Optimum pH and temperature of the amylase were 5.5 and 55°C respectively. Hydrolysis of amylopectin gave maltose, maltotriose and small amounts of glucose. Strain LEM 207 also resembled Lact. acidophilus , but differed from strain 220. It had a lower amylase activity. Optimum pH and temperature of the amylase were 6.4 and 40°C, respectively, and hydrolysis of amylopectin gave maltose, maltotriose and carbohydrates higher than maltopentaose. Strain LEM 202 was similar to Lact. vitelinus. It had the lowest amylase activity which was increased only in presence of maltose. Amylase properties were similar to those of LEM 220.     

Methyltrienolone, a specific ligand for cellular androgen receptors.

Methyltrienolone (R 1881), 17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one, a very active androgen, binds specifically to rat prostate cytosol with a higher affinity than androstanolone. Unlike the physiological hormone, however, it is not bound by human sex steroid plasma binding protein, SBP. This specific ligand is thus a useful tool for the detection of elusive androgen receptors and for their study, for instance, in human tumors where interference from plasma contamination has to be circumvented.     

The extraction by micrococcal nuclease of glucocorticoid receptors and mouse mammary tumor virus DNA sequences is dissociated.

Glucocorticoid receptors (RG) and mammary tumor virus (MM-TV) DNA sequences were extracted by micrococcal nuclease digestion from the nuclei of C3H mouse mammary tumor cells in order to specify their relative distribution in chromatin. RG was labelled and translocated into the nuclei by incubating cells with 3H Dexamethasone (3H Dex). The purified nuclei were then treated at 2 degrees C with micrococcal nuclease. Three chromatin fractions were successively obtained: an isotonic extract (ne3H1), ahypotonic extract (ne2) and the residual pellet (P). The Dex-RG complexes were measured by the hydroxyapatite technique. The MMTV DNA sequences were titrated by molecular hybridization with an excess of MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and the MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and MMTV DNA sequences were extracted in a concentration dependent manner while only 10-15% of nucleic acids became soluble in 10% perchloric acid. The extracted 3H Dex-RG complex was found to be partly bound to soluble chromatin and partly free. The free complex displayed similar sedimentation constants (4S, 7S) and DNA binding ability to the cytosol receptor. The 3H Dex-RG complexes were 2 to 8 fold more concentrated in ne1, which is known to be enriched in active chromatin, than in ne2. Conversely, the concentration of MMTV DNA sequences per microgram DNA was the same in the three nuclear fractions. These results suggest that the Dex-RG complexes are concentrated in an active fraction of chromatin. We propose that, among the 20-30 copies of MMTV genes per haploid genome, only a small proportion are transcribed or regulated.     

Association of type II cAMP-dependent protein kinase with p34cdc2 protein kinase in human fibroblasts

Previous independent studies suggested that type II cAMP-dependent protein kinase and the p34cdc2 protein kinase cell cycle regulator co-localize at centrosomes. In order to investigate whether there is an association of type II cAMP-dependent protein kinase with p34cdc2 in human fibroblasts, we used three different approaches. First, the regulatory subunits RI and RII were photoaffinity-labeled with 8-N3-[32P]cAMP, and anti-p34cdc2 immunoprecipitates were screened for the presence of either RI or RII regulatory subunits by one- or two-dimensional gel electrophoresis. Second, anti-RII alpha immunoprecipitates were screened for the presence of p34cdc2 by Western blot using three different affinity-purified antibodies recognizing different domains of human p34cdc2. Conversely, anti-p34cdc2 immunoprecipitates (three different antibodies), as well as the material retained on p13suc1-Sepharose Bio-Beads, which binds specifically p34cdc2, were screened for the presence of RII alpha. Finally, we have looked for cAMP-dependent protein kinase activity specifically inhibited by PKI in immunoprecipitates obtained from extracts treated with different anti-p34cdc2 antibodies. All these experiments gave concordant results and demonstrate that at least at G0/G1, human fibroblasts contain a complex of active type II cAMP-dependent protein kinase associated through its RII alpha subunit with p34cdc2.     

Influence of new hydroxylated triphenylethylene (TPE) derivatives on estradiol binding to uterine cytosol

Twelve homologous triphenyl acrylonitrile derivatives with a p-OH or p-CH3 group on one or more of the phenyl rings were synthesized in order to assess the relative influence of each position on binding to the estrogen receptor (ER) and on inhibition of prostaglandin synthetase (PGS). Their relative binding affinities (RBAs) for [3H]estradiol (E2)-labeled ER were compared at 0 and 25 degrees C in mouse and rat uterus cytosol with those of tamoxifen derivatives, cyclofenil and diethylstilbestrol. RBAs in both species were closely correlated (r = 0.92) although the RBAs were about twice as high in the mouse as in the rat. The unsubstituted skeleton had an RBA of much less than 0.1 (estradiol = 100). An OH-group in R1 or R2 (Fig. 1) engendered very low affinity whereas an OH-group in R gave rise to a compound with an RBA equivalent to that of E2, emphasizing the importance of this position in the interaction with ER. Compounds with an additional OH-group in R1 or R2 were significantly better competitors than E2. No further increase in RBA was noted with the trihydroxy derivative. The effect of the introduction of a hydrophobic CH3-group decreased affinity as expected in R, but also in position R1 unless a second OH-group was present in R2. None of the 12 test-compounds competed significantly for binding to the "anti-estrogen binding site" in rat kidney supernatant. Although polar groups were not necessary for inhibition of PGS, inhibition was enhanced by the presence of a hydroxy group in R or R1 (but not R2). Even greater inhibition was obtained by the further introduction of a CH3-group in R1 or R respectively. The conformations of these derivatives are compared to those of known estrogen ligands and anti-inflammatory agents in order to obtain further information on these protein recognition sites.     

Differences in steroid 5alpha-reductase iso-enzymes expression between normal and pathological human prostate tissue.

We studied the expression level and cell-specific expression patterns of 5alpha-reductase (5alpha-R) types 1 and 2 iso-enzymes in human hyperplastic and malignant prostate tissue by semi-quantitative RT-PCR and in situ hybridisation analyses. In situ hybridisation established that 5alpha-R1 mRNA is preferentially expressed by epithelial cells and little expressed by stromal cells whereas 5alpha-R2 mRNA is expressed by both epithelium and stroma. Semi-quantitative RT-PCR has been performed on total RNA from different zones of normal prostate, BPH tissues and liver. We found that 5alpha-R1 and 5alpha-R2 mRNAs expression was near the same in all zones of normal prostate. In BPH tissue, 5alpha-R1 and 5alpha-R2 mRNAs expression was slightly but significantly increased, when it was compared to the levels recorded for normal prostate. In cancer samples, 5alpha-R1 mRNA expression was higher than in normal and hyperplastic prostate but the level of 5alpha-R2 mRNA was not statistically different from that observed in the different zones of normal prostate. In liver, 5alpha-R2 mRNA level was similar to that measured in BPH but 5alpha-R1 mRNA expression was ten times higher. The increase observed in 5alpha-R isoenzymes expression in BPH tissue could play an important role in the pathogenesis and/or maintenance of the disease.     

High-performance liquid chromatographic assay for the measurement of the novel microtubule inhibitor 1069C85 in biological tissues and fluids

1069C85 is a novel tubulin binder developed to circumvent the resistance associated with the Vinca alkaloids. Cytotoxic activity has been demonstrated in vitro against a variety of tumour cell lines, including a variant of the P388 leukaemia with acquired resistance to vincristine. A phase I clinical trial is planned and an assay suitable for preclinical and clinical pharmacokinetics has been developed. A high-performance liquid chromatographic (HPLC) assay is described which allows measurement of 1069C85 in plasma, urine, and tissue samples. The method uses reversed-phase chromatography with isocratic elution and detection by fluorescence at 406 nm following excitation at 340 nm. The assay is specific, sensitive (limit of sensitivity 0.25 ng/ml) and reproducible (coefficient of variation <5%). The method has been used to study the pharmacokinetics of 1069C85 in Balb C mice following a single oral dose of 1 mg/kg. The maximum plasma concentration was reached 15 min after administration and subsequent elimination was slow with a half life of 6.5 ± 2.2 h. The drug remained detectable in plasma, at 1 ± 0.5 ng/ml, 24 h after this dose. This assay will be used to determine the pharmacokinetic profile of 1069C85 in mice and in a forthcoming phase I clinical trial.