Myosin VIIA mutation screening in 189 Usher syndrome type 1 patients.

Usher syndrome type 1b (USH1B) is an autosomal recessive disorder characterized by congenital profound hearing loss, vestibular abnormalities, and retinitis pigmentosa. The disorder has recently been shown to be caused by mutations in the myosin VIIa gene (MYO7A) located on 11q14. In the current study, a panel of 189 genetically independent Usher I cases were screened for the presence of mutations in the N-terminal coding portion of the motor domain of MYO7A by heteroduplex analysis of 14 exons. Twenty-three mutations were found segregating with the disease in 20 families. Of the 23 mutations, 13 were unique, and 2 of the 13 unique mutations (Arg212His and Arg212Cys) accounted for the greatest percentage of observed mutant alleles (8/23, 31%). Six of the 13 mutations caused premature stop codons, 6 caused changes in the amino acid sequence of the myosin VIIa protein, and 1 resulted in a splicing defect. Three patients were homozygotes or compound heterozygotes for mutant alleles; these three cases were Tyr333Stop/Tyr333Stop, Arg212His-Arg302His/Arg212His-Arg302His, and IVS13nt-8c-->g/Glu450Gln. All the other USH1B mutations observed were simple heterozygotes, and it is presumed that the mutation on the other allele is present in the unscreened regions of the gene. None of the mutations reported here were observed in 96 unrelated control samples, although several polymorphisms were detected. These results add three patients to single case reported previously where mutations have been found in both alleles and raises the total number of unique mutations in MYO7A to 16.     

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.     

Novel members of the mitogen-activated protein kinase activator family in Xenopus laevis.

Mitogen-activated protein (MAP) kinases comprise an evolutionarily conserved family of proteins that includes at least three vertebrate protein kinases (p42, p44, and p55 MAPK) and five yeast protein kinases (SPK1, MPK1, HOG1, FUS3, and KSS1). Members of this family are activated by a variety of extracellular agents that influence cellular proliferation and differentiation. In Saccharomyces cerevisiae, there are multiple physiologically distinct MAP kinase activation pathways composed of structurally related kinases. The recently cloned vertebrate MAP kinase activators are structurally related to MAP kinase activators in these yeast pathways. These similarities suggest that homologous kinase cascades are utilized for signal transduction in many, if not all, eukaryotes. We have identified additional members of the MAP kinase activator family in Xenopus laevis by a polymerase chain reaction-based analysis of embryonic cDNAs. One of the clones identified (XMEK2) encodes a unique predicted protein kinase that is similar to the previously reported activator (MAPKK) in X. laevis. XMEK2, a highly expressed maternal mRNA, is developmentally regulated during embryogenesis and expressed in brain and muscle. Expression of XMEK2 in yeast cells suppressed the growth defect associated with loss of the yeast MAP kinase activator homologs, MKK1 and MKK2. Partial sequence of a second cDNA clone (XMEK3) identified yet another potential MAP kinase activator. The pattern of expression of XMEK3 is distinct from that of p42 MAPK and XMEK2. The high degree of amino acid sequence similarity of XMEK2, XMEK3, and MAPKK suggests that these three are related members of an amphibian family of protein kinases involved in the activation of MAP kinase. Discovery of this family suggests that multiple MAP kinase activation pathways similar to those in yeast cells exist in vertebrates.     

Proportional processing of a visual mate choice signal in the green swordtail,Xiphophorus hellerii

During mate choice, receivers often assess the magnitude (duration, size, etc.) of signals that vary along a continuum and reflect variation in signaller quality. It is generally assumed that receivers assess this variation linearly, meaning each difference in signalling trait between signallers results in a commensurate change in receiver response. However, increasing evidence shows receivers can respond to signals non-linearly, for example through Weber's Law of proportional processing, where discrimination between stimuli is based on proportional, rather than absolute, differences in magnitude. We quantified mate preferences of female green swordtail fish, Xiphophorus hellerii, for pairs of males differing in body size. Preferences for larger males were better predicted by the proportional difference between males (proportional processing) than the absolute difference (linear processing). This demonstration of proportional processing of a visual signal implies that receiver perception may be an important mechanism selecting against the evolution of ever-larger signalling traits.     

Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1^−/D mice). Ckmm-Cre^+/−;Ercc1^−/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre^+/−;Ercc1^−/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre^+/-;Ercc1^−/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre^+/−;Ercc1^−/fl and Ercc1^−/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.     

EXPERIMENTAL STUDIES OF THE EVOLUTIONARY SIGNIFICANCE OF SEXUAL REPRODUCTION. VI. A GREENHOUSE TEST OF THE SIB-COMPETITION HYPOTHESES

This study tests the hypothesis that competition among groups of sexual and asexual siblings generates an advantage for sexual females. Individual tillers of Anthoxanthum odoratum were planted singly, among other siblings from the same family, and among groups of sexual and asexual siblings from different families in pots in an unheated greenhouse. Unlike previous field experiments, there was little difference between the performance of sexual and clonal tillers after two years, despite strong treatment effects and high mortality. The results demonstrate that sib competition does not generate an advantage for sexual reproduction in biotically simple environments.     

Febrile Gastroenteritis Due to Salmonella thompson—Report of an Outbreak

Salmonella thompson, a common pathogen of poultry, has received scant attention as a cause of human gastroenteritis. At least 45 persons were infected with S thompson in Sacramento, California, after eating at a chicken restaurant and 38 became symptomatic. Ten required admission to hospital, and all were treated with antibiotics and improved. In 19 cases cultures of stool specimens for S thompson over a 60-day period showed slower but statistically insignificant differences in salmonellal elimination in 7 patients who received antibiotics when compared with 12 who were untreated. We report this outbreak to increase awareness of the virulence and prevalence of gastroenteritis due to S thompson.     

Effect of noncoliforms on coliform detection in potable groundwater: improved recovery with an anaerobic membrane filter technique

A total of 529 well and distribution potable water samples were analyzed for total coliforms by the most-probable-number and membrane filter (MF) techniques. Standard plate count bacteria and MF noncoliform bacteria were also enumerated. Frequency of coliform detection, turbidity in most-probable-number tubes, and extensive overgrowth by noncoliforms on MF filters were directly proportional to standard plate counts. Recovery of coliforms was greatest by the MF method at low (less than 100 CFU/ml) standard plate count densities and better by the most-probable-number method (confirming gas and turbid tube) at high (greater than 500 CFU/ml) standard plate count densities. In the latter case, overgrowth by noncoliforms on MF filters suppressed sheen development and, in turn, masked coliform detection. Of 341 atypical (no sheen) MF colonies verified by parallel inoculation of lauryl sulfate broth and billiant green-bile broth, 156 were aerogenic in the latter medium. Of atypical isolates, 84% were identified as either Citrobacter or Enterobacter species. A 4.3-fold reduction in numbers of overgrown MF filters and an 2.2-fold increase in numbers of coliforms recovered from 127 water samples was accomplished by anaerobic incubation of MF cultures. This anaerobic modification of the standard MF technique significantly reduced overgrowth and enhanced recovery of coliforms from potable groundwater. This technique is simple, cost effective, and suitable for monitoring of untreated ground water common to some small water systems and private water supplies.     

Comparison of microporous filters for concentration of viruses from wastewater.

The 1-MDS Virosorb filter and the 50S and 30S Zeta-plus filters, all with a net positive charge, were compared with the negatively charged Filterite filter for concentration of naturally occurring coliphages and animal viruses from sewage effluent. When Filterite filters were used, the effluent was adjusted to pH 3.5 and AlCl3 was added before filtration to facilitate virus adsorption. No adjustment was required with the positively charged filters. Sets of each filter type were eluted with 3% beef extract (pH 9.5) or eluted with 0.05 M glycine (pH 11.5). A maximum volume of 19 liters could be passed through 142-mm diameter Filterite filters before clogging, whereas only 11, 11, and 15 liters could be passed through the 1-MDS, 50S, and 30S filters, respectively. For equal volumes passed through the filters, coliphage recoveries were 14, 15, 18, and 37% in primary effluent and 40, 97, 50, and 46% in secondary effluent for the Filterite , 1-MDS, 50S, and 30S filters, respectively. No statistically significant difference was observed in the recovery of animal viruses among the filters from secondary effluent, whereas in the Filterite and 50S filters, higher numbers of viruses from primary effluent were recovered than in the 1-MDS and 30S filters in two of three collections. Glycine was found to be a less-efficient eluent than beef extract in the recovery of naturally occurring viruses.     

Protein synthesis in rat lung. Measurements in vivo based on leucyl-tRNA and rapidly turning-over procollagen I.

The relationships of the specific radioactivities of leucine in serum, leucine acylated to tRNA and leucine in procollagen I, procollagen III and total protein in lungs of unanaesthetized young male rats in vivo were assessed as a function of time during constant intravenous infusion of radiolabelled leucine. The specific radioactivity of free leucine in plasma reached a steady-state plateau value within 30 min of initiation of [3H]leucine infusion. Leucine acylated to tRNA isolated from lungs had the same specific radioactivity as free serum leucine. Leucine in procollagen I rapidly achieved a specific radioactivity equal to that of serum leucine and leucyl-tRNA, indicating that serum leucine and leucyl-tRNA isolated from total lung were in rapid equilibrium with the precursor leucine pool for procollagen I synthesis. On the basis of leucyl-tRNA or free serum leucine as the precursor, half-times of fractional conversion of procollagen I and III were calculated as 9 and 38 min respectively. The incorporation of leucine into mixed lung proteins calculated from the tracer studies was 6.8 mumol/day for the first 30 min of the infusion, after which the calculated rate increased to 15.0 mumol/day. This apparent increase correlated with the appearance of rapidly labelled plasma proteins trapped in the lungs. On the basis of short infusions lasting 30 min or less, followed by vascular perfusion of the lung, the average fractional synthesis rate of mixed pulmonary proteins in young male rats was 20%/day.