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M Haas

Publications and source records attributed to M Haas.

At least 181 records · Page 10Linked to original sources
PubMed1993

Localization of Shaw-related K+ channel genes on mouse and human chromosomes.

Four related genes, Shaker, Shab, Shaw, and Shal, encode voltage-gated K+ channels in Drosophila. Multigene subfamilies corresponding to each of these Drosophila genes have been identified in rodents and primates; this suggests that the four genes are older than the common ancestor of present-day insects and mammals and that the expansion of each into a family occurred before the divergence of rodents and primates. In order to define these evolutionary relationships more precisely and to facilitate the search for mammalian candidate K+ channel gene mutations, we have mapped members of the Shaw-homologous gene family in humans and mice. Fluorescence in situ hybridization analysis of human metaphase chromosomes mapped KCNC2 (KShIIIA, KV3.2) and KCNC3 (KShIIID, KV3.3) to Chromosome (Chr) 19q13.3-q13.4. Inheritance patterns of DNA restriction fragment length variants in recombinant inbred strains of mice placed the homologous mouse genes on distal Chr 10 near Ms15-8 and Mdm-1. The mouse Kcnc1 (KShIIIB, NGK2-KV4, KV3.1) gene mapped to Chr7 near Tam-1. These results are consistent with the hypothesis that the generation of the mammalian KCNC gene family included both duplication events to generate family members in tandem arrays (KCNC2, KCNC3) and dispersion of family members to unlinked chromosomal sites (KCNC1). The KNCN2 and KCNC3 genes define a new synteny group between humans and mice.

Animals↗
PubMed1993

Dual mechanisms for Na-K-Cl cotransport regulation in airway epithelial cells.

To investigate cellular mechanisms involved in the regulation of basolateral Na-K-Cl cotransport in airway epithelia, we determined saturable basolateral [3H]bumetanide binding, a measure of functioning cotransporters, in primary cultures of canine tracheal and human nasal epithelial cells, including cells from patients with cystic fibrosis (CF). As we previously reported [M. Haas, L. G. Johnson, and R. C. Boucher. Am. J. Physiol. 259 (Cell Physiol. 28): C557-C569, 1990], isoproterenol and hypertonic cell shrinkage produce an equivalent stimulation of [3H]bumetanide binding to dog tracheal cells. We now find that apical ATP and UTP, which stimulate apical Cl channels and Cl secretion in normal and CF airway cells by an adenosine 3',5'-cyclic monophosphate (cAMP)-independent mechanism (S. J. Mason, A. M. Paradiso, and R. C. Boucher. Br. J. Pharmacol. 103: 1649-1656, 1991), increase basolateral [3H]bumetanide binding to dog tracheal cells to the same extent as do isoproterenol and hypertonic shrinkage. The stimulatory effects of ATP and UTP on binding are inhibited by apical addition of a Cl channel blocker, the indanyloxyacetic acid derivative IAA-94 (0.2 mM), or by raising basolateral K concentration ([K]b) from 3.3 to 40 mM, suggesting these effects are secondary to apical Cl efflux via channels. Apical IAA-94 and increased [K]b also inhibit stimulation of binding by isoproterenol by approximately 50%, suggesting that part (but not all) of the effect of the beta-agonist on basolateral cotransport is secondary to apical Cl efflux, with an additional component of direct stimulation of cotransport via cAMP. In support of this interpretation, we find that isoproterenol and a membrane-permeable cAMP analogue increase [3H]bumetanide binding to primary cultures of CF nasal epithelial cells, in which significant cAMP-mediated stimulation of apical Cl efflux does not occur. [3H]bumetanide binding to CF nasal cells is also stimulated by apical ATP, and levels of saturable [3H]bumetanide binding to CF cells are 1.3-1.5 times those in non-CF nasal cells under both basal and stimulated conditions. The results suggest that basolateral Na-K-Cl cotransport in airway cells may be upregulated in two distinct ways: 1) directly via a cAMP-dependent cascade, and 2) as a secondary response to apical Cl channel activation. Both of these mechanisms appear to be intact in CF.

Adenosine Triphosphate↗
PubMed1993

Transport defects of rabbit medullary thick ascending limb cells in obstructive nephropathy.

To characterize the sodium transport defect responsible for salt wasting in obstructive nephropathy, the major sodium transporters in the medullary thick ascending limb (mTAL), the apical Na-K-2Cl cotransporter and the basolateral Na-K-ATPase, were studied in fresh suspensions of mTAL cells and outer medulla plasma membranes prepared from obstructed and untreated kidneys. Oxygen consumption (QO2) studies in intact cells revealed marked reductions in the inhibitory effects of both furosemide and ouabain on QO2 in cells from obstructed, as compared with control animals, indicating a reduction in activities of both the Na-K-2Cl cotransporter and the Na-K-ATPase. Saturable [3H]bumetanide binding was reduced in membranes isolated from obstructed kidneys, but the Kd for [3H]bumetanide was unchanged, indicating a decrease in the number of functional luminal Na-K-2Cl cotransporters in obstructed mTAL. Ouabain sensitive Na-K-ATPase activity in plasma membranes was also reduced, and immunoblots using specific monoclonal antibodies directed against the alpha and beta subunits of rabbit Na-K-ATPase showed decreased amounts of both subunits in outer medullas of obstructed kidney. A significant decrease in [3H]bumetanide binding was detected after 4 h of ureteral obstruction, whereas Na-K-ATPase activity at this time was still not different from control. We conclude that ureteral obstruction reduces the amounts of both luminal Na-K-2Cl cotransporter and basolateral Na-K-ATPase in mTAL of obstructed kidney and that these reductions contribute to the salt wasting observed after release of obstruction.

Animals↗
PubMed1993

P53 mutation in acute T cell lymphoblastic leukemia is of somatic origin and is stable during establishment of T cell acute lymphoblastic leukemia cell lines.

Samples donated by patients with T cell acute lymphoblastic leukemia (T-ALL) were screened for mutations of the p53 tumor suppressor gene. Peripheral blood cells of T-ALL relapse patient H.A. were found to possess a heterozygous point mutation at codon 175 of the p53 gene. To determine whether this was an inherited mutation, a B cell line (HABL) was established. Leukemic T cell lines (HATL) were concurrently established by growing peripheral blood leukemic T cells at low oxygen tension in medium supplemented with IGF-I. Previously we had shown that > 60% of leukemic T cell lines possessed mutations in the p53 gene (Cheng, J., and M. Hass. 1990. Mol. Cell. Biol. 10:5502), mutations that might have originated with the donor's leukemic cells, or might have been induced during establishment of the cell lines. To answer whether establishment of the HATL lines was associated with the induction of p53 mutations, cDNAs of the HATL and HABL lines were sequenced. The HATL lines retained the same heterozygous p53 mutation that was present in the patient's leukemic cells. The HABL line lacked p53 mutations. Immunoprecipitation with specific anti-p53 antibodies showed that HATL cells produced p53 proteins of mutant and wild type immunophenotype, while the HABL line synthesized only wild-type p53 protein. The HATL cells had an abnormal karyotype, while the HABL cells possessed a normal diploid karyotype. These experiments suggest that (a) p53 mutation occurred in the leukemic cells of relapse T-ALL patient HA; (b) the mutation was of somatic rather than hereditary origin; (c) the mutation was leukemia associated; and (d) establishment of human leukemia cell lines needs not be associated with in vitro induction of p53 mutations. It may be significant that patient HA belonged to a category of relapse T-ALL patients in whom a second remission could not be induced.

Adult↗
PubMed1993

Functional analysis of phosphorylation sites in human lamin A controlling lamin disassembly, nuclear transport and assembly.

We have constructed point mutations in human lamin A cDNA at conserved serine and threonine residues, some of which were shown to be phosphorylated in vitro by cdc2-kinase and protein kinase C and in vivo. Using a functional in vivo assay system, we identified three categories of mutant phenotypes. (i) Dominant negative phenotypes in mitosis result from mutation of Thr-19 and Ser-22 within the amino-terminal cdc2-kinase motif of lamin A. An increase of aberrant mitotic phenotypes in the double mutants Thr-19/Ser-392 and Ser-22/Ser-392 suggests that concomitant phosphorylation of the three residues regulates mitotic lamin A disassembly. (ii) Mutation of both Ser-403/Ser-404 within a PKC motif flanking the nuclear localization signal inhibits transport of mutant lamin A to the nucleus in 64% of the cells. It is proposed that phosphorylation of the motif in vivo positively regulates nuclear localization together with the nuclear localization sequence. (iii) The assembly of lamin A into the perinuclear lamina is disturbed by mutation of the carboxy-terminal Ser-525, previously shown to be interphase-specifically phosphorylated (Eggert et al., Eur. J. Biochem. 213, 659-671 (1993)). The phenotype shows discontinuous and patch-like aggregates of the mutant protein in the nucleus. We suggest that phosphorylation of the site either regulates lamina assembly or lamina-chromatin interaction in interphase.

Amino Acid Sequence↗
PubMed1993

The interexaminer reliability of a galvanic skin response instrument.

OBJECTIVE: To determine the interexaminer reliability of a protocol of use of a galvanic skin resistance device for detection of low resistance areas along the spinal column, in relatively pain-free subjects. DESIGN: A blinded investigation of concordance of skin resistance examination findings over the spinal column using two clinicians experienced in the use of the instrument. SETTING: A private practice chiropractic outpatient clinic. PATIENTS: Sixty-four male and female chiropractic college students (mean age: 35 yr). The Visual Analog Pain Scale indicated a mean response of 7.6 mm on a 100-mm range. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Concordance of examiners evaluated with the kappa statistic. RESULTS: The results indicated modest levels of concordance for the first study sample (n = 46). The average kappa was 0.37. The second group assessed (n = 18) also demonstrated only modest levels of interexaminer concordance. The average kappa value for this sample was 0.36. CONCLUSION: The use of the Electrical Conductor Scanner instrument for evaluating putative spinal pathology through manifestations in skin resistance in relatively asymptomatic subjects is not supported by the results of this experiment. The unevenness of data generated from this experiment in certain spinal regions necessitates further investigation prior to making any strong conclusions regarding the usefulness of this instrument in the clinical setting.

Adult↗
PubMed1993

Reactivity of leg alignment to articular pressure testing: evaluation of a diagnostic test using a randomized crossover clinical trial approach.

OBJECTIVE: A study was undertaken to assess the reliability of detecting leg alignment changes (reactivity) and to determine if the observed leg alignment reactivity can be attributed to a rotatory articular pressure challenge. DESIGN: Prospective double-blind crossover trial of a diagnostic test. SETTING: Laboratory: Center for Technique Research. PARTICIPANTS: Forty-two chiropractic college students, faculty and staff. INTERVENTIONS: A standardized force of 2 or 3 kg was applied with a 1 cm2 rubber-tipped pressure algometer on the lateral aspect of the T3-T7 spinous processes and the posterior aspect of the lateral masses of C1. MAIN OUTCOME MEASURES: Leg alignment reactivity: an increase in leg alignment discrepancy (yes or no) following a diagnostic intervention. RESULTS: The reliability for detecting leg alignment reactivity was poor: on average, Kappa = 0.05 in the thoracics and 0.06 at C1. On average, the attributable risk of leg alignment reactivity (pressure test risk--sham test risk) was less than 4%. In many cases, the sham rate was greater than the pressure test rate. CONCLUSIONS: For the population investigated, leg alignment reactivity to rotatory pressure testing can, in the majority of cases, be attributable to background noise. This procedure was not found to be viable for identifying vertebrae for adjustment. Further research with different subject populations, regions of investigation, leg alignment measurement techniques and vertebral challenge techniques are indicated.

Adult↗
PubMed1993

Responsiveness of leg alignment changes associated with articular pressure testing to spinal manipulation: the use of a randomized clinical trial design to evaluate a diagnostic test with a dichotomous outcome.

OBJECTIVE: A study was undertaken to assess the stability of leg alignment reaction to a pressure challenge and its responsiveness to an adjustive intervention. DESIGN: Prospective, double-blind clinical trial of a diagnostic test. SETTING: Laboratory: Center for Technique Research. PARTICIPANTS: Forty-two chiropractic college students, faculty and staff. INTERVENTIONS: A high-velocity, low-amplitude, short lever adjustment of a single vertebra from among C1 and T3-T7; or a sham adjustment similar to a manual diagnostic pressure test at C1, T3-T7 or T9-T10. MAIN OUTCOME MEASURES: Leg alignment reactivity: An increase in leg alignment discrepancy (yes or no) following a metered pressure challenge to a vertebra. RESULTS: On average, stability was poor at T3-T7 (Kappa = 0.04), moderate at C1 (K = 0.47), and fair for sham pressure tests (K = 0.30). Responsiveness: The proportion of positive baseline leg alignment reactions that responded (became negative) to sham adjustment was 95% at T3-T7 and 55% at C1. Further analysis was untenable since too few vertebrae were implicated for an adjustment. CONCLUSIONS: For the population investigated, the majority of the responsiveness of the leg alignment diagnostic test to a rotatory adjustment appears to be a diagnostic illusion (i.e., background noise unrelated to a treatment intervention). Further research with different subject populations, regions of investigation, leg alignment measurement techniques and vertebral challenge techniques are indicated.

Adult↗
PubMed1992

The Na-K-Cl cotransport protein of shark rectal gland. I. Development of monoclonal antibodies, immunoaffinity purification, and partial biochemical characterization.

The Na-K-Cl cotransporter mediates the coupled transport of Na, K, and Cl across the plasma membrane of many animal cell membranes. It is inhibited by loop diuretics such as furosemide, bumetanide, and benzmetanide. We have developed a panel of monoclonal antibodies directed against the 195-kDa shark rectal gland Na-K-Cl cotransport protein. Four representative antibodies (J3, J4, J7, and J25), each of which recognizes a discrete structural domain, were selected for detailed characterization. When a radiolabeled loop diuretic is bound to the cotransporter prior to solubilization, each antibody immunoprecipitates the same diuretic-protein complex. Of the four antibodies, J4 favors the native protein over the denatured one and does not bind well to proteolytic fragments; in contrast, J7 recognizes the cotransporter only after it has been solubilized. J3, J7, and J25 each recognize a unique ensemble of proteolytic fragments of the 195-kDa protein; analysis of the patterns of recognition has yielded a tentative assignment of the approximate location of the epitopes within the peptide. When the cotransport protein is treated with N-glycanase to remove N-linked oligosaccharides, its apparent mass decreases to approximately 135 kDa. The deglycosylated form is recognized by each of the antibodies except J25; this suggests that the J25 epitope is within the oligosaccharide component or in a peptide domain whose folding is disturbed by carbohydrate removal. An immunoaffinity matrix constructed with the J4 antibody permits single-step purification of the 195-kDa protein; other proteins copurify with the large glycoprotein, but none of these appear to be subunits of a stoichiometric complex. The amino acid sequence of four fragments of the 195-kDa cotransport protein is reported. Immunofluorescence and immunoelectron microscopy demonstrates, in agreement with physiological evidence, that the 195-kDa protein is distributed along the basolateral membrane and excluded from the apical membrane of the rectal gland secretory cell.

Amino Acid Sequence↗
PubMed1992

Identification of a regulated Na/K/Cl cotransport system in a distal nephron cell line.

Lack of an adequate cell model has limited investigation of Na/K/Cl cotransporter regulation in the kidney. Using A6 cells, an amphibian distal renal cell line, we observed that 63% of rubidium uptake in confluent A6 monolayers was ouabain-insensitive. Ouabain-insensitive rubidium uptake was inhibited in a dose-dependent fashion by furosemide (IC50 6.6 microM) or bumetanide (IC50 1.7 microM). Kinetic studies confirmed that furosemide-sensitive rubidium uptake had features consistent with cotransporter activity in other cell lines. Furthermore, specific binding of [3H]bumetanide occurred with a capacity of 8.6 pmol/mg protein and a Kd of 1.6 microM bumetanide. Finally, furosemide-sensitive rubidium uptake was rapidly regulated by a calcium ionophore, the phorbol ester PDBu, forskolin, and adenosine. These data demonstrate an Na/K/Cl cotransport system in the A6 cell which will serve as a useful model for studying cotransporter regulation by endogenous signaling pathways.

Animals↗
PubMed1992

CD4 T cells in murine acquired immunodeficiency syndrome: polyclonal progression to anergy.

We have examined the kinetics of changes that occur in the helper T cell subset during murine acquired immunodeficiency syndrome, which occurs after infection with the mix of viruses known as BM5. We find that there is expansion of the CD4 T cells by 2 wk, 50% of the CD4 T cells become large as the disease progresses, and the CD4 T cell population is increasingly comprised of cells with a memory/activated phenotype. These effects are apparent by 2 wk postinfection, and the change is nearly complete by 6-8 wk. The phenotypic shift is paralleled by the loss of the ability of the CD4 T cells to proliferate or to produce interleukin 2 (IL-2), IL-3, IL-4, and interferon gamma in response to stimulation with mitogens, superantigen, or anti-CD3. There is no obvious expansion or deletion of CD4 T cells expressing particular V beta genes, as might be expected if a conventional superantigen were driving the changes. The results suggest, however, that the total CD4 population has been driven to anergy by some potent polyclonal stimulus directly associated with viral infection.

Animals↗
PubMed1992

Suppression of acute lymphoblastic leukemia by the human wild-type p53 gene.

Independent mutations in both alleles of the p53 tumor suppressor gene are a frequent finding in human T-cell acute lymphoblastic leukemia (T-ALL) cell lines and in the cells of some T-ALL patients in relapse. One major goal of studying the status of p53 (and other tumor suppressor genes) in human cancer is to facilitate the suppression of the tumorigenic phenotype through the restoration of the expression of the wild-type allele. While the efficient insertion of a suppressor into all cells of solid/metastatic human tumors may at present be impossible, insertion into leukemia cells may be feasible due to the accessibility of the leukemia cells in the body. To examine the feasibility of suppressing the tumorigenicity of human T-leukemia cells, the human T-ALL cell line Be-13, which lacks endogenous p53 protein, was infected with a recombinant retrovirus encoding the wild-type allele of human p53 (hwtp53). Expression of p53 reduced the growth rate of infected Be-13 cells in vitro, suppressed colony formation in methylcellulose cultures, and abrogated their tumorigenic phenotype in nude mice in vivo. These results suggest that suppression of the leukemic phenotype of relapse T-ALL-derived Be-13 cells is feasible. Acute leukemia cell suppression via high-efficiency infection with retroviruses encoding wtp53 may be feasible and beneficial in T-ALL cases as part of a bone marrow transplantation regimen in an effort to reduce the frequency of posttransplantation relapse.

Animals↗
PubMed1992

Association of induction of a fully tumorigenic phenotype in murine radiation-induced T-lymphoma cells with loss of differentiation antigens, gain of CD44, and alterations in p53 protein levels.

We investigated the mechanism of radiation induction of murine thymic lymphomas by studying the characteristics of primary x-ray-induced thymic lymphoma (PXTL) cell lines and of their oncogene-induced, progressed progeny. It is widely thought that proto-oncogene alterations are associated with the induction of murine lymphomas; however, few, if any primary murine radiation-induced lymphomas possess (proto-)oncogene alterations. Independently derived cell lines grown directly (i.e., without in vivo transplantation) from thymic lymphomas of irradiated C57BL/6 mice possess the properties of immortalized pre-T cells and lack many of the characteristics of "tumor cells". PXTL cells are poorly tumorigenic upon transplantation, do not clone in methylcellulose cultures, are growth factor dependent and autocrine, and lack consistent chromosome and oncogene abnormalities. However, the thymic lymphomas are malignant and cause the death of each afflicted mouse. PXTL cells expressed two immunologically distinct forms of the tumor suppressor protein p53 that have moderately increased stability (t1/2 = 1 h) when compared with p53 of normal splenic T lymphocytes. Early PXTL cells could progress in vitro to a fully tumorigenic phenotype after infection with retroviruses encoding the c-myc and v-ras oncogenes. Progressed T-lymphoma cells acquired growth factor independence, a highly transplantable and tumorigenic phenotype, and the ability to quantitatively clone in methylcellulose cultures. Progressed lymphoma cells coordinately downregulated the expression of five T-cell differentiation markers, upregulated the expression of CD44 (Pgp-1), and harbored vastly elevated levels of two immunologically distinct forms of p53. Our results suggest that the early thymic lymphomas consist of differentiation-inhibited, immortal pre-T cells that are precursors to progressed, fully tumorigenic T-lymphoma cells.

Animals↗
PubMed1992

Interexaminer reliability and discriminant validity of inclinometric measurement of lumbar rotation in chronic low-back pain patients and subjects without low-back pain.

The interexaminer reliability of an inclinometer procedure to measure lumbar rotation was evaluated by two chiropractic clinicians who examined 25 chronic (greater than 6 months) low-back pain patients and 25 subjects without low-back pain. These groups were compared for differences in mean left, right, and total rotation. Patients who had lumbar spinal surgery were excluded. Twenty-eight men and 22 women, ranging in age from 28-38 years, were evaluated. Reliability between examiners was evaluated by Pearson's correlation coefficient and the intraclass correlation coefficient. All coefficients were significant (P less than 0.01). Errors in prediction and examiner disagreement were evaluated by the standard error of estimate and the interexaminer measurement error. The standard errors of estimate (range: 1.4-4.4) and the interexaminer measurement errors (range: 3.8-10.4) were large compared to the scale of measurement. An analysis of variance of differences between the chronic low-back pain patients and asymptomatics revealed significantly more left rotation in the asymptomatic subjects (F = 8.4; df = 1; P less than 0.006). Also, there was significantly more total rotation in the asymptomatic subjects (F = 4.143; df = 1; P less than 0.048). However, because of the large error attributed to this procedure, it is not possible to say whether the difference between the two groups is a result of the large error or some "real" difference. Therefore, the procedure described in this study should not be used as a clinical outcome measure.

Adult↗