Distribution of the molossinus allele of Sry, the testis-determining gene, in wild mice

When the Y chromosome of the laboratory inbred mouse strain C57BL/6 (B6) is replaced by the Y of certain strains of Mus musculus domesticus, testis determination fails and all XY fetuses develop either as hermaphrodites or XY females (XY sex reversal). This suggests the presence of at least two alleles of Sry, the male-determining gene on the Y:M. m. domesticus and B6. The B6 Y chromosome is derived from the Japanese house mouse, M. m. molossinus and therefore carries a molossinus Sry allele. As a first step to determine how the molossinus Sry allele evolved, its distribution pattern was determined in wild mice. The cumulative data of 96 M. musculus samples obtained from 58 geographical locations in Europe, North Africa, and Asia show the molossinus Sry allele is restricted to Japan and the neighboring Asian mainland and confirm that Japanese M. m. molossinus mice were derived in part from a race of M. m. musculus from Korea or Manchuria. Sry polymorphisms, as illustrated by the molossinus Sry allele, can serve as molecular markers for studies on the evolution of wild M. musculus populations and can help determine the role sex determination plays in speciation.      

Animal models of fibromyalgia

Animal models of disease states are valuable tools for developing new treatments and investigating underlying mechanisms. They should mimic the symptoms and pathology of the disease and importantly be predictive of effective treatments. Fibromyalgia is characterized by chronic widespread pain with associated co-morbid symptoms that include fatigue, depression, anxiety and sleep dysfunction. In this review, we present different animal models that mimic the signs and symptoms of fibromyalgia. These models are induced by a wide variety of methods that include repeated muscle insults, depletion of biogenic amines, and stress. All potential models produce widespread and long-lasting hyperalgesia without overt peripheral tissue damage and thus mimic the clinical presentation of fibromyalgia. We describe the methods for induction of the model, pathophysiological mechanisms for each model, and treatment profiles.     

Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria

Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have several positive implications with respect to future development of a transformation system for cellulolytic thermophiles. Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280. Received 12 September 2000/ Accepted in revised form 20 November 2000     

Patch-clamp recording of charge movement, Ca(2+) current, and Ca(2+) transients in adult skeletal muscle fibers

Intramembrane charge movement (Q), Ca(2+) conductance (G(m)) through the dihydropyridine-sensitive L-type Ca(2+) channel (DHPR) and intracellular Ca(2+) fluorescence (F) have been recorded simultaneously in flexor digitorum brevis muscle fibers of adult mice, using the whole-cell configuration of the patch-clamp technique. The voltage distribution of Q was fitted to a Boltzmann equation; the Q(max), V(1/2Q), and effective valence (z(Q)) values were 41 +/- 3.1 nC/&mgr;F, -17.6 +/- 0.7 mV, and 2.0 +/- 0.12, respectively. V(1/2G) and z(G) values were -0.3 +/- 0.06 mV and 5.6 +/- 0.34, respectively. Peak Ca(2+) transients did not change significantly after 30 min of recording. F was fit to a Boltzmann equation, and the values for V(F1/2) and z(F) were 6.2 +/- 0.04 mV and 2.4, respectively. F was adequately fit to the fourth power of Q. These results demonstrate that the patch-clamp technique is appropriate for recording Q, G(m), and intracellular [Ca(2+)] simultaneously in mature skeletal muscle fibers and that the voltage distribution of the changes in intracellular Ca(2+) can be predicted by a Hodgkin-Huxley model.     

BioGraph: unsupervised biomedical knowledge discovery via automated hypothesis generation

We present BioGraph, a data integration and data mining platform for the exploration and discovery of biomedical information. The platform offers prioritizations of putative disease genes, supported by functional hypotheses. We show that BioGraph can retrospectively confirm recently discovered disease genes and identify potential susceptibility genes, outperforming existing technologies, without requiring prior domain knowledge. Additionally, BioGraph allows for generic biomedical applications beyond gene discovery. BioGraph is accessible at .     

Secretion of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) by Bacillus subtilis

A fusion of DNA sequences encoding the SPO2 promoter, the alpha-amylase signal sequence from Bacillus amyloliquefaciens, and the mature part of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) was constructed on a Bacillus subtilis multicopy vector. Bacillus cells of the protease-deficient strain DB104 harboring this vector produced and secreted the plant enzyme alpha-galactosidase up to levels of 1,700 U/liter. A growth medium suppressing the residual proteolytic activity of strain DB104 was used to reach these levels in a fermentor. Purification of the secreted product followed by NH2-terminal amino acid sequencing showed that the alpha-amylase signal sequence had been processed correctly. The molecular mass of the product estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was slightly lower than that of the plant purified enzyme, which is most likely due to glycosylation of the latter. The alpha-galactosidase product was active both on the artificial substrate para-nitrophenyl-alpha-D-galactopyranoside and on the galactomannan substrate, guar gum. The activity of this Bacillus sp.-produced enzyme was similar to that of the glycosylated enzyme purified from guar seeds, indicating that glycosylation has no essential function for enzyme activity.     

Comparative study on the production of guar alpha-galactosidase by Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2 in continuous cultures.

Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2, both carrying a multicopy integrated guar alpha-galactosidase, have been cultivated in continuous cultures, using various mixtures of carbon sources and cultivation conditions. Both S. cerevisiae SU50B and H. polymorpha 8/2 are stable and produce high levels of extracellular alpha-galactosidase in continuous cultures for more than 500 h. For these expression systems the strong inducible promoter systems GAL7 and methanol oxidase, respectively, were used. The induction of alpha-galactosidase synthesis by galactose in SU50B is limited by the low galactose uptake. Apart from that, at high dilution rates, the glucose repression is substantial, and a maximal expression level of 28.6 mg of extracellular alpha-galactosidase.g (dry weight) of biomass-1 could be obtained. In H. polymorpha, the induction of alpha-galactosidase synthesis, in addition to methanol oxidase synthesis using formaldehyde, is very effective up to 42 mg of extracellular alpha-galactosidase.g (dry weight) of biomass-1. Productivities in terms of specific production rate enable a good comparison with those of other heterologous expression systems in the literature. The productivities found with S. cerevisiae SU50B and H. polymorpha, 3.25 and 5.5 mg of alpha-galactosidase.g of biomass-1.liter-1.h-1, respectively, rank among the highest reported in the literature. Enzyme production and secretion in H. polymorpha are more complex. A two-peaked optimum is found in enzyme production. No clear explanation of this phenomenon can be given.     

Loss of meiosis in Aspergillus

If strictly mitotic asexual fungi lack recombination, the conventional view predicts that they are recent derivatives from older meiotic lineages. We tested this by inferring phylogenetic relationships among closely related meiotic and strictly mitotic taxa with Aspergillus conidial (mitotic) states. Phylogenies were constructed by using DNA sequences from the mitochondrial small ribosomal subunit, the nuclear ribosomal internal transcribed spacers, and the nuclear 5.8S ribosomal gene. Over 920 bp of sequence was analyzed for each taxon. Phylogenetic analysis of both the mitochondrial and nuclear data sets showed at least four clades that possess both meiotic and strictly mitotic taxa. These results support the hypothesis that strictly mitotic lineages arise frequently from more ancient meiotic lineages with Aspergillus conidial states. Many of the strictly mitotic species examined retained characters that may be vestiges of a meiotic state, including the production of sclerotia, sclerotium-like structures, and hulle cells.