Effects of a GnRH agonist on oocyte number and maturation in mice superovulated with eCG and hCG

The objective was to investigate the effects of a gonadotropin-releasing hormone agonist (GnRH) on ovulation rate and the number and maturation of oocytes in mice superovulated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). Thirty 3-month-old BALB/C female mice (weight: 25-30 g) were assigned to three experimental groups: control, superovulated, and superovulated with GnRH pretreatment (n=10 per group). Control mice received an i.p. injection of 0.1 ml physiological saline solution. Superovulation was induced with 5 IU eCG (i.p.) and 5 IU hCG 48 h later. Mice in the superovulated with GnRH pretreatment group were given GnRH (20 mg/kg Fertirelin, i.m.), 24 h before superovulation. Thirteen hours after hCG administration, mice were sacrificed by cervical dislocation and blood samples were collected to determine serum progesterone concentration (by radioimmunoassay). Ovaries and oviducts were also harvested to enumerate corpora lutea and cumulus-enclosed oocytes. Progesterone concentrations were not significantly different among groups. The oocyte number and the maturation, ovulation rate, and the number of corpora lutea were higher in GnRH-treated mice than both controls and superovulated mice. In conclusion, GnRH given 24 h before superovulation with eCG-hCG increased the number and maturation of oocytes and the rate of ovulation in mice.     

CVT-4325: a potent fatty acid oxidation inhibitor with favorable oral bioavailability

New inhibitors of palmitoyl-CoA oxidation are based on the introduction of nitrogen heterocycles in the ‘Western Portion’ of the molecule. SAR studies led to the discovery of CVT-4325 (shown), a potent FOXi (IC₅₀ = 380 nM rat mitochondria) with favorable PK properties (F = 93%, t_1/2 = 13.6 h, dog).     

A combined allele-specific PCR and RFLP assay to detect the 35delG mutation in the Connexin 26 gene

Mutations in the Connexin-26 (specified GJB2) gene have been shown to be a major cause of nonsyndromic recessive deafness (NSRD), and a single mutation 35delG in the GJB2 gene accounts for the majority of cases of NSRD. For diagnostic analyses and for scientific studies of large numbers of patients, fast and economic assays that can be performed with standard polymerase chain reaction (PCR) instruments are highly desirable. We have developed an allele-specific amplification (ASA)-based restriction fragment length polymorphism (RFLP) assay. We evaluated the multiplex method for its ability to 35delG mutation. Our method is a stable, reproducible and concordend with previously reported PCR-RFLP assays.     

Selecting SNPs for association studies based on population frequencies: a novel interactive tool and its application to polygenic diseases

Common complex polygenic diseases as autoimmune diseases have not been completely understood on a molecular level. While many genes are known to be involved in the pathways responsible for the phenotype, explicit causes for the susceptibility of the disease remain to be elucidated. The susceptibility to disease is thought to be the result of genetic epistatic interactions between common polymorphic genes. This polymorphism is mostly caused by single nucleotide polymorphisms (SNPs). Human subpopulations are known to differ in the susceptibility to the diseases and generally in the distribution of single nucleotide polymorphisms. The here presented approach retrieves SNPs with the most divergent frequencies for selected human subpopulations to help defining properties for the experimental verification of SNPs within defined regions. A web-accessible program implementing this approach was evaluated for multiple sclerosis (MS), a common human polygenic disease. A link to a summary of data from "The SNP Consortium" (TSC) with sex-dependencies of SNPs is available. Associations of SNPs to genes, genetic markers and chromosomal loci are retrieved from the Ensembl project. This tool is recommended to be used in conjunction with microarray analyses or marker association studies that link genes or chromosomal loci to particular diseases.     

A rapid and effective method of extracting fully intact RNA from thermophilic geobacilli that is suitable for gene expression analysis

Extraction of intact RNA is essential for quantitative gene expression analysis. Isolating high quality RNA from gram-positive bacteria is known to be problematic particularly from organisms that have optimal growth temperatures greater than 45 °C. We report a novel extraction protocol for the rapid isolation of fully intact RNA from thermophilic Geobacillus thermoleovorans using a lysing matrix containing a mixture of ceramic and glass beads, triisopropylnaphthalene sulfonic acid (TNS), and p-4-aminosalicyclic acid (PAS). Combining both detergents, TNS and PAS, appeared to increase denaturation of RNases at thermophilic temperatures. Gel electrophoresis revealed that only RNA isolated using the TNS-PAS procedure demonstrated sharp, undegraded 23S, 16S, and 5S ribosomal RNA bands. RNA extracted from geobacilli using commercially available kits was extensively degraded and was not suitable for detecting gene expression. Total RNA yields extracted with the TNS-PAS protocol were greater than eightfold higher than those obtained with available kits. Critically, it was also shown that only RNA isolated with the TNS-PAS-based method was suitable for monitoring thermophile gene expression patterns using RT-PCR analysis.Communicated by G. Antranikian     

Molecular characterization and functional expression of flavonol 6-hydroxylase

Background  

Flavonoids, one of the major groups of secondary metabolites, play important roles in the physiology, ecology and defence of plants. Their wide range of activities is the result of their structural diversity that encompasses a variety of functional group substitutions including hydroxylations. The aromatic hydroxylation at position 6 of flavonols is of particular interest, since it is catalyzed by a 2-oxoglutarate-dependent dioxygenase (ODD), rather than a cytochrome P450-dependent monooxygenase. ODDs catalyze a variety of enzymatic reactions implicated in secondary metabolite biosynthesis.     

4th meeting of the EU research network EUROME: From the identification of genes and cellular networks in murine models of arthritis to novel therapeutic intervention strategies in rheumatoid arthritis,London, UK, 9 March 2004

Rheumatoid arthritis (RA) is a common human disease with a prevalence of about 1% in most parts of the world. At the time of symptom onset it is difficult to predict the severity of subsequent disease course. After 2 years joint erosions are seen in most patients, and most patients become clinically disabled within 20 years. A recent meeting at the Kennedy Institute of Rheumatology (Imperial College, London) brought together representatives from several European centres of excellence, to discuss research funded by the EU Framework 5 Quality of Life Programme. This research network combines gene and protein expression profiling with different animal models of RA to identify cells, genes and pathways contributing to arthritis initiation, progression and chronicity. The studies discussed highlight the reality that collaboration between different research groups is the basis of groundbreaking research and, it is hoped, eventual new therapies for RA.