Beta-adrenoceptor-linked protein kinase A (PKA) activity in human fibroblasts from normal subjects and from patients with major depression

Human fibroblasts from normal subjects and from patients with major depression are cultured and their beta-adrenoreceptor-cyclic AMP-protein kinase A (PKA) system characterized. The results indicate that the beta-adrenoreceptor-mediated activation of PKA in the 900 g supernatant fraction of human fibroblasts is mediated via beta-adrenoreceptors. The activation of PKA by isoproterenol is very rapid with maximal stimulation occurring at 5 seconds. The time course of PKA activation by isoproterenol in fibroblasts from patients with major depression is identical to that in fibroblasts from normal subjects but the magnitude of activation is significantly reduced in fibroblasts from patients with major depression. Dose-response curves on cyclic AMP mediated activation of PKA confirmed the previously reported reduction in activation of PKA in patients with major depression but demonstrated that this reduction occurs without a change in the EC50 values of cyclic AMP (approximately 20 nmol/L). The blunted beta-adrenoceptor-linked PKA responses in patients with major depression occur without a change in the expression of the PKA catalytic subunit C alpha. The studies suggest that the beta-adrenoceptor-coupled adenylate cyclase PKA system in human fibroblasts may represent a valid model to explore possible abnormalities in the fine tuning of the beta-adrenergic transduction cascade in patients with affective disorders.     

Clinical evaluation and test-retest reliability of the IHR-McCormick Automated Toy Discrimination Test

The IHR-McCormick Automated Toy Discrimination Test (ATT) measures the minimum sound level at which a child can identify words presented in quiet in the sound field. This 'word-discrimination threshold' provides a direct measure of the ease with which a child can identify speech and a surrogate measure of auditory sensitivity. This paper describes steps taken to maximize the test-retest reliability of the ATT and to enable it to measure word-discrimination thresholds in noise as well as in quiet. It then describes the results of a clinical evaluation of the ATT in which paediatric audiologists measured word-discrimination thresholds in quiet from 215 successive attendees (in the age range 2 to 13 years) at a paediatric audiology clinic presenting over a 2-month period. When children with atypical cognition or delayed development of language were excluded, 72% of the children provided two word-discrimination thresholds and 83% provided at least one word-discrimination threshold. Children who failed to provide word-discrimination thresholds were generally younger than four years of age. Although a few children who could not perform pure-tone or warble-tone audiometry managed to provide word-discrimination thresholds, most children who could perform the ATT could also perform pure-tone audiometry. The average pure-tone threshold in the better-hearing ear could be predicted from the word-discrimination threshold with a 95% confidence interval of +/- 13 dB. The test-retest reliability of the ATT was measured in two ways. First, to enable comparison with published results, the within-subjects standard deviation of word-discrimination thresholds was calculated. It varied as a function of age and degree of impairment, but was never worse than 3.3 dB. Children of four years of age and older displayed the adult reliability of 2.3 dB. Second, the variability of absolute differences between word-discrimination thresholds was calculated. It was such that a change of 7 dB between two runs of the test (e.g. aided and unaided) would be expected to occur by change less than one time in 20. These results extend previous evaluations of the ATT to a clinically representative population and confirm that word-discrimination thresholds provide a useful complement to warble-tone and pure-tone audiometry.     

Clinical relevance of the i(12p) marker chromosome in germ cell tumors

BACKGROUND: Germ cell tumors in men are curable at all stages and are among the most sensitive of all cancers to chemotherapy. An isochromosome of the short arm of chromosome 12, i(12p), has been reported to be a frequent marker of these tumors and to have diagnostic and prognostic significance. PURPOSE: We evaluated the possible association between this cytogenetic marker and clinical outcome for men with germ cell tumors. METHODS: One hundred seventy-eight germ cell tumor samples from 150 men were studied using conventional and molecular cytogenetic techniques. Of these samples, 171 were evaluable. Patient characteristics, disease stage, treatment outcome, and disease status were correlated with the observed cytogenetic changes. In addition, 28 biopsy specimens obtained from 28 patients with tumors of uncertain histogenesis were evaluated to determine whether the presence of i(12p) could serve as a diagnostic marker of a germ cell origin for these tumors. RESULTS: Of the 171 evaluable tumor accessions, 101 (59%) yielded abnormal karyotypes. i(12p) was determined to be present in 79 of the 101 (79%) abnormal karyotypes, which were derived from all cell types and primary sites. An abnormal karyotype was more frequently obtained from nonseminomatous tumors (91/137 [81%]) than from seminomas (10/34 [30%] [P < .001]). Tumors resulting in a cytogenetic failure were more likely to respond completely to chemotherapy than tumors with an abnormal karyotype (P = .004). i(12)p copy number was not associated with response or survival. Fluorescence in situ hybridization using a chromosome 12 centromere-specific probe detected i(12p) in 47 of 47 tumors (100%) already shown to have i(12p) by cytogenetic analysis and in 13 of 49 tumors (27%) exhibiting either an abnormal karyotype or a cytogenetic failure. One or more copies of i(12p), excess 12p copy number, or a deletion on the long arm of chromosome 12 was found in seven of 28 (25%) midline tumors of uncertain histogenesis, thus establishing a diagnosis of a germ cell tumor in these patients. One partial and five complete responses were observed in these seven patients. Only two partial responses were seen in the 17 patients who had no detectable germ cell tumor-related cytogenetic marker (P = .009). CONCLUSIONS: i(12p) is a highly nonrandom chromosomal marker seen in about 80% of male germ cell tumors with evaluable cytogenetic abnormalities. The presence of this isochromosome has diagnostic and possibly prognostic importance for patients with these tumors. IMPLICATIONS: Cytogenetic studies of germ cell tumors in prospective clinical treatment trials are warranted to define more precisely the relationship between histologic subtype, serum tumor marker production, and prognosis.     

Genetic analysis of a kindred with X-linked mental handicap and retinitis pigmentosa

A kindred is described in which X-linked nonspecific mental handicap segregates together with retinitis pigmentosa. Carrier females are mentally normal but may show signs of the X-linked retinitis pigmentosa carrier state and become symptomatic in their later years. Analysis of polymorphic DNA markers at nine loci on the short arm of the X chromosome shows that no crossing-over occurs between the disease and Xp11 markers DXS255, TIMP, DXS426, MAOA, and DXS228. The 90% confidence limits show that the locus is in the Xp21-q21 region. Haplotype analysis is consistent with the causal gene being located proximal to the Xp21 loci DXS538 and 5'-dystrophin on the short arm of the X chromosome. The posterior probability of linkage to the RP2 region of the X chromosome short arm (Xp11.4-p11.23) is .727, suggesting the possibility of a contiguous-gene-deletion syndrome. No cytogenetic abnormality has been identified.     

Functional evaluation of invariant arginines situated in the mobile lid domain of phosphoribulokinase

Rhodobacter sphaeroides phosphoribulokinase contains four invariant arginines (R49, R168, R173, and R187). The high-resolution structure of this enzyme [Harrison, D. H. T., Runquist, J. A., Holub, A., and Miziorko, H. M. (1998) Biochemistry (submitted for publication)] reveals that it folds in a manner similar to that of adenylate kinase. Three invariant arginines (R168, R173, and R187) as well as arginine-186, which is conserved in prokaryotic phosphoribulokinases, have not been previously functionally evaluated. These arginine residues map within the mobile lid domain that is a distinctive feature of the adenylate kinase family of proteins. Precedent for the significant function of arginines in phosphotransferase reactions prompted substitution of glutamine for each of these three invariant arginines. Solution state characterization of the isolated mutant proteins indicated that they retained a high degree of structural integrity, as indicated by their stoichiometric binding of an alternative nucleotide substrate (trinitrophenyl-ATP) as well as the allosteric effector (NADH). Kinetic characterization indicated > 10(4)-fold diminution in V/KRu5P for R168Q, attributable to a > 300-fold decrease in catalytic efficiency and an increase (approximately 50-fold) in Km Ru5P. For R173Q, a 15-fold diminution in Vmax and a 100-fold increase in Km Ru5P were observed. These observations implicate new components of the ribulose 5-phosphate binding site. Additionally, they confirm assignment of the mobile lid domain as part of the phosphoribulokinase active site, even though this region is well separated from other active site elements in the structure of the open form of the protein. Characterization of R186Q and R187Q mutants suggests that they influence the cooperativity of substrate binding.     

George Lyman Duff Memorial Lecture. Arterial imaging and atherosclerosis reversal

This review explores evidence for the reversibility of atherosclerosis and augmentation of angiography with non-invasive arterial wall imaging. Meta-analysis from coronary angiographic trials demonstrates that regression and stabilization are 1.5 to 2 times more common in treated than placebo subjects, and progression is reduced by half in treated subjects. Odds ratios for clinical coronary events are significantly reduced with treatment. Lesion improvement occurs more readily in women than men and more so in women receiving concomitant estrogen replacement therapy. Lesions with > or = 50% diameter stenosis (%S) at baseline respond more readily to lipid lowering than those < 50% S, whereas reduction in coronary events is related to stabilization of lesions < 50% S. Lipoproteins have a differential effect on lesion progression according to lesion size, and triglyceride-rich lipoproteins play an important role in the progression of coronary artery lesions < 50% S. Improved therapeutic regimens to alter progression of atherosclerosis may require adjunctive therapy, such as with antioxidants or hormone replacement therapy, in concert with low-density lipoprotein cholesterol reduction to prevent new lesion formation or early lesion progression. Sequential coronary angiographic determination of progression evaluated by both quantitative coronary angiography and global change score, a visual assessment of overall lesion change, predicts clinical coronary events. Only inferences about the state of the arterial wall can be made from angiography, because it delineates only the lumen. Therapy testing and study of atherosclerosis progression can be improved with noninvasive B-mode ultrasonographic imaging of the distal common carotid artery far-wall intima-media thickness (IMT), a reliable measure of early preintrusive atherosclerosis. Measurement of common carotid IMT is useful for the study of coronary artery risk factors and can augment studies of coronary artery intrusive lesions, because it is associated with coronary artery disease. B-mode measurement of common carotid IMT has the potential of serving as a noninvasive surrogate end point for clinical coronary events. Screening for peripheral vessel changes indicative of high risk for coronary artery disease is possible and cost-effective with the noninvasive procedures now available.     

The radiological features of adult T-cell leukaemia/lymphoma

Adult T-cell leukaemia/lymphoma (ATLL) results from a monoclonal expansion of T-cells infected with the human T-cell lymphotropic virus type 1 (HTLV-1). Six cases of ATLL, three males and three females all from the Caribbean and with a mean age of 55 years, have been reviewed retrospectively, illustrating the wide spectrum of radiological changes. One patient had extensive mediastinal adenopathy, hitherto an unrecorded finding in ATLL.     

Tumor suppressor gene alteration in adult acute lymphoblastic leukemia (ALL). Analysis of retinoblastoma (Rb) and p53 gene expression in lymphoblasts of patients with de novo, relapsed, or refractory ALL treated in Southwest Oncology Group studies

To examine the impact of inactivation of tumor suppressor genes on outcome in adult ALL, we compared two groups of patients registered to SWOG treatment protocols for loss of the Rb gene product and p53 overexpression: (1) 89 patients with de novo ALL, and (2) 26 patients with relapsed/refractory ALL. The groups were comparable with respect to age, sex, and race. Cell lysates (> or = 80% blasts) were analyzed by immunoblotting which enabled detection of Rb or p53 proteins in as little as 1 microg of lysate. Loss of Rb expression (pRbneg) was found in 54/85 (64%) de novo and 11/19 (58%) relapsed patients (P = 0.79). Overexpression of p53 (p53abn), indicative of p53 point mutations, was found in 16/75 (21%) de novo and 8/19 (42%) relapsed patients (P = 0.08). Using a nonisotopic RNase cleavage assay, p53 point mutations in exons 5-9 were confirmed in 14/23 (61%) p53abn specimens. For the de novo ALL group, patients with normal Rb protein had higher WBC and higher peripheral blast and lymphocyte counts. Otherwise neither abnormal Rb or p53 expression correlated with any of a large panel of clinical and laboratory variables including FAB class, blast lineage, expression of myeloid antigens or CD34, and presence of the Ph1 chromosome or BCR-ABL. Analyses of treatment outcomes demonstrated no significant impact of Rb or p53 status alone on CR rates, relapse-free or overall survival. An identical percentage (11%) of both de novo and relapsed/refractory patients had concurrent abnormalities of both Rb and p53 expression (pRbneg/p53abn). The survival curve of these patients suggests an increased rate of early death, but the number of patients in this group was small. Summarizing, (1) loss of Rb expression is common in adult ALL; (2) overexpression of p53 may be more frequent in relapsed/refractory than de novo adult ALL; and (3) although Rb or p53 alterations alone are not strong independent predictors of outcome, their concurrent expression may predict a poor response to therapy.     

Paternally inherited chromosomal structural aberrations detected in mouse first-cleavage zygote metaphases by multicolour fluorescence in situ hybridization painting

We describe a fluorescence in situ hybridization (FISH) procedure for assessing zygotic risk of paternal exposure to endogenous or exogenous agents. The procedure employs multicolour FISH with chromosome-specific DNA painting probes plus DAPI staining for detecting both balanced and unbalanced chromosomal aberrations in mouse first-cleavage (1-Cl) zygote metaphases. Four composite probes specific for chromosomes 1, 2, 3 or X, each labelled with biotin, plus a composite probe specific for chromosome Y labelled with digoxigenin, were used. We applied this method to evaluate the effects of paternal exposure to acrylamide, a model germ cell clastogen. First-cleavage zygote metaphases, collected from untreated females mated to males whose sperm or late spermatids were treated with acrylamide, were scored for the induction of structural aberrations using both chromosome painting (PAINT analysis) and DAPI analysis. Structural chromosomal aberrations were observed in the sperm-derived, but not in the egg-derived, pronuclei. While 59.4% of the zygotes had structural aberrations by DAPI analysis, 94.1% of the same zygotes had structural aberrations by PAINT analysis (P < 0.001), illustrating the increased sensitivity for detecting translocations and insertions obtained by adding chromosome painting. These findings show that FISH painting of mouse 1-Cl zygotes when used in conjunction with DAPI analysis is a powerful model for investigating the cytogenetic defects transmitted from father to offspring.