Comparison of methods for identifying transcription units and transcription map of the Werner syndrome gene region

OBJECTIVE: The audiologic presentation of vestibular schwannoma (VS) associated with neurofibromatosis type 2 (NF2) has not been well characterized. The goal of this study was to investigate the audiologic features of NF2-associated VS and to determine their relationship to the size of the tumor. STUDY DESIGN: A retrospective case review. SETTING: Quaternary governmental medical research institute evaluating patients fitting specific criteria for ongoing clinical studies. PATIENTS: Audiologic and magnetic resonance imaging data were available for 40 patients (25 males, 15 females), with an average age of 32 years, who had been recruited for ongoing clinical and genetic studies of NF2. MAIN OUTCOME MEASURES: The audiologic profile and magnetic resonance imaging characteristic of tumor were retrospectively reviewed. RESULTS: The average size of the tumor at presentation was 7.26 +/- 16.58 cm3 and measured 1.2, 1.6, and 1.1 cm in the anterior/posterior, lateral/medial, and superior/inferior dimensions, respectively. An increase in lateral/medial size of the tumor most significantly correlated with deterioration in mid- (1,000-2,000 Hz) and high- (4,000-8,000 Hz) frequency hearing levels, elevated speech reception threshold, and prolonged auditory brain stem response waves III and V latency. CONCLUSIONS: Patients with NF2 demonstrate a more predictable audiologic profile for a given size tumor than has been previously described with spontaneous or sporadic VS.     

Studies on granuloma formation in Syrian hamsters experimentally infected with Schistosoma mansoni

Juvenile rats are more susceptible to the acute toxicity of the phosphorothionate insecticides parathion and chlorpyrifos than are adult rats. Developmental changes in brain acetylcholinesterase and hepatic aliesterase (carboxylesterase), cytochrome P450, and the P450-mediated metabolism of these two phosphorothionate insecticides were investigated in male Sprague-Dawley rats. Specific activities of acetylcholinesterase in cerebral cortex, but not medulla oblongata, and of liver aliesterases increased with age, indicating the presence of both more target esterases and more protective esterases, respectively, in the adult compared to the juvenile animal. Sensitivity of the brain acetylcholinesterase to inhibition by paraoxon and chlorpyrifosoxon, as measured by IC50 values, did not change significantly with age, whereas the hepatic aliesterase sensitivity to inhibition decreased with age. Progressive increases in activities of P450-mediated activation (desulfuration) (6- to 14-fold) and detoxication (dearylation) (2- to 4-fold), as well as concentrations of P450 (7-fold) and protein (2-fold), were observed between neonate and adult hepatic microsomes. Microsomal pentoxyresorufin O-dealkylase activity followed a developmental pattern similar to desulfuration and dearylation, displaying a 16-fold increase between neonates and adults. However, microsomal ethoxyresorufin O-deethylase activity increased until 21 days of age, displaying a 16-fold increase, then decreased in adulthood to a level 10-fold higher than neonates. These results indicate that target enzyme sensitivity is not responsible for age-related toxicity differences, nor is the potential for hepatic bioactivation, whereas lower levels of hepatic aliesterase-mediated protection and P450-mediated dearylation probably contribute significantly to the greater sensitivity of juveniles to phosphorothionate toxicity.     

Decreased fecal bile acid output in patients with coronary atherosclerosis

In most patients with atherosclerosis, the underlying metabolic derangement remains undefined. Animal experiments have suggested that the ability to produce and excrete large amounts of bile acids may be an adaptation mechanism to cholesterol overload protecting against the atherogenic effects of cholesterol. However, there are very few data on bile acid excretion in human atherosclerosis. In the present study, we have investigated fecal bile acid secretion in subjects with and without coronary artery disease. The target group consisted of 30 patients with proven coronary artery disease and the control group consisted of 27 matched subjects without clinical or laboratory evidence of coronary atherosclerosis. Fecal bile acids were measured by gas-liquid chromatography from 24-hr stool collections under a controlled diet. The patients excreted significantly less bile acids than the controls (325+/-135 vs. 592+/-223 mg/day, respectively, p < 0.0001). The difference was primarily due to a reduced excretion of secondary bile acids. Less than 50% of deoxycholate was excreted by patients (180+/-81 mg/day) as compared to controls (367+/-168 mg/day, p < 0.0002), while lithocholic acid excretion was 111+/-62 mg/day in patients vs. 190 +/-70 mg/day in controls (p < 0.005). The fecal output of the two primary bile acids, cholic and chenodeoxycholic acid, did not differ significantly between patients and controls. The fecal output of total bile acids correlated with that of both secondary bile acids in patients as well as in controls. These findings suggest that patients with coronary heart disease are unable to excrete adequate amounts of bile acids to rid themselves of excess cholesterol, even if they are able to maintain a plasma cholesterol level comparable to that of healthy controls.     

Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.     

Mapping of the ligand-selective domain of the Xenopus laevis corticotropin-releasing factor receptor 1: implications for the ligand-binding site

The nonselective human corticotropin-releasing factor receptor 1 (hCRF-R1) and the ligand-selective Xenopus CRF-R1 (xCRF-R1) were compared. To understand the interactions of sauvagine and ovine CRF, both high-affinity ligands for hCRF-R1 but surprisingly weak ligands for xCRF-R1, chimeric receptors of hCRF-R1 and xCRF-R1 followed by double or multiple point mutations were constructed. Binding studies and cAMP assays demonstrated that the N-terminal domain exhibited the complete ligand selectivity of xCRF-R1. The important region was mapped between amino acids 70 and 89; replacement of amino acids Arg76, Asn81, Gly83, Leu88, and Ala89 in hCRF-R1 with the corresponding amino acids of xCRF-R1 (Gln76, Gly81, Val83, His88, and Leu89) resulted in a receptor that had approximately 30-fold higher affinity for human/rat CRF than for sauvagine. Mutagenesis of these amino acids in xCRF-R1 to the human sequence completely abolished the ligand selectivity of xCRF-R1. Mutagenesis of amino acids 88 and 89 in hCRF-R1 or xCRF-R1 had only a minor (approximately 2.5-fold) effect on the ligand selectivity of the mutant receptor. Substitution of Arg76, Asn81, and Gly83 in hCRF-R1 with the corresponding sequence of xCRF-R1 (Gln76, Gly81, and Val83) resulted in a receptor with approximately 11-fold higher affinity for human/rat CRF compared with ovine CRF or sauvagine. When only two of these three amino acids were mutated, no effect on the ligand selectivity was observed. On the basis of these data, it is suggested that amino acids 70-89 of CRF-R1 are important for the ligand binding site.     

Lesbian mothers who conceived after donor insemination: a follow-up study

This study investigated the development of family relationships in lesbian families who conceived after donor insemination (DI). The main characteristics of this unknown family structure were analysed. An anonymous donor was used to conceive, the family unit consisted of two mothers and a father was non-existent, and both women had a homosexual orientation. A total of 50 lesbian couples who conceived after DI with children aged between 1 and 2 years participated in this study, using a standardized interview created for this study. After the birth of their child, 56% of the lesbian mothers (n = 100) would have wanted the identity of the donor to be registered, while 10% would have done so at the time of the insemination. Opinions differed in 12 of the 50 couples: the biological mother was in favour of identity registration while the social mother was not. Both women considered themselves a parent of the child with equally shared responsibilities. Mothers were open about the special features of their family structure with children and within their immediate social network. Only 30% disclosed their lesbian identity in a broader social environment. The features of these newly created families may influence the psychological development of the children and therefore make long-term follow-up studies indispensable.     

Characterization of nicotinamide mononucleotide adenylyltransferase from thermophilic archaea

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the synthesis of NAD+ and nicotinic acid adenine dinucleotide. It has been purified to homogeneity from cellular extracts of the thermophilic archaeon Sulfolobus solfataricus. Through a database search, a highly significant match was found between its N-terminal sequence and a hypothetical protein coded by the thermophilic archaeon Methanococcus jannaschii MJ0541 open reading frame (GenBank accession no. U67503). The MJ0541 gene was isolated, cloned into a T7-based vector, and expressed in Escherichia coli cells, yielding a high level of thermophilic NMN adenylyltransferase activity. The expressed protein was purified to homogeneity by a single-step chromatographic procedure. Both the subunit molecular mass and the N-terminal sequence of the pure recombinant protein were as expected from the deduced amino acid sequence of the MJ0541 open reading frame-encoded protein. Molecular and kinetic properties of the enzymes from both archaea are reported and compared with those already known for the mesophilic eukaryotic NMN adenylyltransferase.