Genome-wide examination of myoblast cell cycle withdrawal during differentiation.

Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up-regulation of MyoD, followed by myogenin, and activation of Bcl-2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40% fused myotubes, as levels of p21(WAF1/Cip1) increased and alpha-actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome-wide screen to identify other gene products involved in this process. Using microarrays containing approximately 10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT-PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly-6A, whose expression specifically was up-regulated during cell cycle withdrawal coincident with early myoblast differentiation.     

An open study of auranofin in the treatment of steroid-dependent asthma

To determine the efficacy of oral gold in asthma, 20 patients with steroid-dependent asthma received auranofin at a dose of 3 mg by mouth, twice daily, in a 24-week open clinical trial. Prospective evaluation of bronchial responsiveness to methacholine was determined before and 8 and 16 weeks after initiation of auranofin therapy. Serial spirometry (FEV1 and FVC), lung volumes, and diffusing capacities (single breath carbon monoxide diffusing capacity of the lungs) were measured before and at 10 and 20 weeks after treatment. All subjects were required to record concomitant medications, symptom scores, and morning and evening peak expiratory flow rates. In vitro immunologic studies performed before and after 8 and 20 weeks of auranofin therapy included leukocyte histamine release in response to antihuman IgE, lymphocyte blast transformation in response to concanavalin A and phytohemagglutinin, and leukocyte inhibitory factor activity in response to Candida albicans and tetanus toxoid antigens. In 18 patients evaluated, there were no significant differences between baseline and posttreatment spirometry, single breath carbon monoxide diffusing capacity of the lungs, and lung volumes. At week 16 of treatment, the steroid cumulative dose or the total prednisone dose administered from 7 days before through 10 days after each methacholine test day decreased from a mean of 293 +/- 125 mg at baseline to 192 +/- 115 mg. At week 16, nine of 18 patients (50%) exhibited decreased methacholine responsiveness as defined by a more than one-half log10 increase in the concentration of methacholine causing a 20% decrease in FEV1. A significant correlation (r = 0.60) was observed between the increase in the concentration of methacholine causing a 20% decrease in FEV1 and the decrease in steroid cumulative dose after 16 weeks of treatment. Leukocyte histamine release to anti-IgE exhibited significant reductions from baseline at week 20 to 10(-2) (p less than 0.002) and at 10(-3) (p less than 0.005) dilutions. At week 20, leukocyte inhibitory factor activity in response to Candida increased from baseline at the 0.1 mg per well (p = 0.025) and 1 mg per well (p = 0.05) concentrations; similarly, the responses to tetanus toxoid increased at the 1 mg per well (p less than 0.05) and 0.1 mg per well (p less than 0.01) concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Diagnostic efficiency of carcinoembryonic antigen and CA125 in the cytological evaluation of effusions.

In our previous study, the combination of the concentrations of carcinoembryonic antigen (CEA) and CA125 and the findings from cytological examination in 189 benign and malignant pleural and peritoneal effusions was useful in the diagnosis/classification of malignant effusions. Sensitivity of CEA (level, greater than 5 ng/mL) was 68%; specificity was 99% for the diagnosis of malignant effusions secondary to carcinoma of the lung, breast, gastrointestinal tract, and mucinous carcinoma of the ovary. Sensitivity of CA125 (level, greater than 5000 U/mL) was 85%; specificity was 96% for the diagnosis of malignant effusions in carcinoma of the ovary, fallopian tube, and endometrium. We now expanded the study to include 840 pleural and peritoneal effusions (benign, n = 520; malignant, n = 320) and analyzed the data by the statistical method of Rudolph and colleagues. Based on new cutoff values, ie, CEA level at 6.3 ng/mL and CA125 level at 3652 U/mL, the sensitivities for detection of malignant effusions secondary to carcinomas of the lung, breast, and gastrointestinal tract and mucinous carcinoma of the ovary varied between 75% and 100%; specificity was 98%. Sensitivity of CA125 for detection of malignant effusions from müllerian epithelial carcinoma was 71%; specificity was 99%. The elevated CEA fluid level alone helped to diagnose malignant effusions of the gastrointestinal tract in 54%, breast in 19%, and lung in 16%. The high CA125 fluid level was predictive of müllerian epithelial carcinoma. Adjunctive use of CEA and CA125 levels in fluid enhances the sensitivity of cytological diagnosis and may be predictive of the primary site in patients who present with carcinoma of an unknown primary source.     

Alteration in expression of beta 2 integrins on lamina propria lymphocytes in ulcerative colitis and Crohn's disease

We have previously demonstrated by immunohistochemistry that mucosal expression of beta 2 integrins was enhanced in Crohn's disease and ulcerative colitis as compared to normal controls. We aimed, therefore, to determine whether there was a corresponding alteration in the expression of CD11a/CD18 (LFA-1), the primary lymphocyte beta 2 integrin, among the principal subsets of lamina propria lymphocytes (LPLs). Accordingly, LPLs were extracted from surgical resection specimens derived from patients with Crohn's colitis, ulcerative colitis, and from noninflamed controls. Following immunofluorescent staining, three-color flow-cytometry analysis identified LPLs on the basis of CD45 side scatter gating, which in turn, were further subdivided into CD4(+), CD8(+), and CD19(+) cells to account for the predominant T and B cells in the lamina propria. Expression patterns of CD11a, the alpha-subunit of LFA-1; CD18, the beta-subunit of LFA-1; and alpha d, a novel alpha-subunit of the beta 2 integrin family were assessed for each of these lymphocyte subsets. In Crohn's disease and ulcerative colitis there was an increased mean percentage expression of CD4(+) cells and CD11a(+) cells compared with noninflamed controls. CD11a was more likely to be expressed on CD4(+) cells in both Crohn's disease and ulcerative colitis and compared with controls and less expressed on CD19(+) cells. It is likely that an influx of CD4(+)11a(+) cells into the lamina propria accounted for these changes. These results suggest that although currently there is great interest in harnessing alpha 4 beta 7 in treatment of inflammatory bowel disease, further consideration should be given to the role of CD11a in these disease states.     

Female phenotype and multiple abnormalities in sibs with a Y chromosome and partial X chromosome duplication: H--Y antigen and Xg blood group findings.

A mentally retarded female child with multiple congenital abnormalities had an abnormal X chromosome and a Y chromosome; the karyotype was interpreted as 46,dup(X)(p21 leads to pter)Y. Prenatal chromosome studies in a later pregnancy indicated the same chromosomal abnormality in the fetus. The fetus and proband had normal female genitalia and ovarian tissue. H--Y antigen was virtually absent in both sibs, a finding consistent with the view that testis-determining genes of the Y chromosome may be suppressed by regulatory elements of the X. The abnormal X chromosome was present in the mother, the maternal grandmother, and a female sib: all were phenotypically normal and showed the karyotype 46,Xdup(X)(p21 leads to pter) with non-random inactivation of the abnormal X. Anomalous segregation of the Xga allele suggests that the Xg locus was involved in the inactivation process or that crossing-over at meiosis occurred.     

A limiting factor in the "normalization" of schizophrenic orienting response dysfunction

Forty schizophrenic patients, 40 non-schizophrenic patients, and 40 normal subjects were given 60 each alternating 1000- and 2000-Hz, 1-second tones at 60 dB. Half of each sample, the Press Group (PG), had to press a pedal to the high (low) target tone, ignoring the nontarget tone. The other half, the Nonpress Groups (NPGs), were given no reason to attend. Skin conductance response (SCR), finger pulse volume (FPV), and electroencephalographic (EEG) activity were recorded. NPG schizophrenic subjects were more often nonresponsive in both SCR and FPV than other samples, but less often responsive in EEG only when a 20 percent criterion of alpha blockade was used. Schizophrenic subjects showed greater consistency of OR nonresponsiveness in SCR and FPV, and nonsignificantly greater consistency in criterion alpha block, pointing to a deficit in orienting response (OR) rather than in peripheral response. When the targeted signal was given, schizophrenic subjects showed the same response as other groups in all systems. This was not due to an indiscriminate increase in reactivity, since response increase centered on the targeted signal itself in all groups. As the target signal was repeated, autonomic OR in schizophrenics declined sharply so that they again became underresponsive. Thus, OR "normalization" achieved by targeting significant signals is restricted to relatively early responsiveness. The rapid decline in autonomic OR may help explain differences in schizophrenic subjects between P300 and autonomic ORs to significant stimuli. Schizophrenic subjects were no different from controls in bilateral SCR or FPV asymmetry, but displayed less frequent criterion alpha blockade and reduced background alpha power in the left hemisphere. Each system showed a different pattern of bilateral asymmetry, reflecting complex, not well understood relations among these responses. This was further emphasized by the fact that skin conductance level (SCL) incremented over trials in PG subjects, reflecting sustained activation, while EEG background showed an increase in slower wave power, consistent with reports of increased drowsiness. The only drug effect seen was a lowering of SCL. Neuroleptics were associated with a flexible inhibitory control of SCL, permitting normal-like increment when circumstances required. Depressed patients' data suggested they might show heightened OR nonresponsiveness to innocuous stimuli which might not be subject to "normalization" by manipulation of stimulus significance; hence OR deficit might still differentiate schizophrenic from depressive patients.     

Genetic and transgenic approaches to dissecting muscle development and contractility using the Drosophila model system

Both genetic and transgenic analyses of Drosophila melanogaster, the common fruit fly, are providing important insights into the mechanisms of muscle cell determination and development, myofibril assembly, and muscle contraction. This model system affords tremendous advantages such as ease of isolating mutants defective in these processes, determining the identity of affected genes, and analyzing protein function by transformation with in vitro mutagenized versions of such genes. These approaches have identified a series of proteins that are critical to mesoderm and muscle determination, many of which are likely to serve similar roles in vertebrates. The effects of mutating structural protein genes upon myofibril assembly and function in Drosophila help to define the differential roles of contractile protein isoforms and the importance of proper protein stoichiometry for physiologic function. These studies may also provide insight into the role of structural proteins in vertebrate contractility.