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Biomedical subjects

M Haas

Publications and source records attributed to M Haas.

At least 163 records · Page 9Linked to original sources
PubMed1995

Assignment of the human heart tetrodotoxin-resistant voltage-gated Na+ channel alpha-subunit gene (SCN5A) to band 3p21.

The chromosomal location of SCN5A, the gene encoding the principal voltage-gated Na+ channel expressed in human heart, has been determined by three independent methodologies: somatic cell hybrid mapping, chromosomal microdissection-polymerase chain reaction, and fluorescence in situ hybridization. The SCN5A gene was assigned to the short arm of chromosome 3 (band 3p21) by all three approaches. These data are further evidence that striated muscle Na+ channel genes are dispersed in the genome.

Animals↗
PubMed1995

Glomerular permselectivity in proteinuric patients after kidney transplantation.

To characterize the defect in glomerular permselectivity responsible for proteinuria after renal transplantation, we studied 10 patients with moderate proteinuria (median 0.37 g/d, range 0.20-0.79), 16 patients with the nephrotic syndrome (6.73 g/d, 3.9-14.6), 8 living related donor transplant recipients without any history of rejection (median proteinuria 0.26 g/d, 0.06-0.58), and 12 healthy volunteers. The fractional clearance of neutral dextrans > 54 A was significantly higher in nephrotic patients, demonstrating a defect in glomerular size selectivity. Using a log-normal model of glomerular pore size distribution, r*(5%) and r*(1%), indices for the presence of large pores, were increased in the nephrotic patients. The fractional clearance of negatively charged dextran sulfate was significantly higher in all patient groups, indicating a loss of glomerular charge selectivity. Biopsy findings showed more prominent glomerular lesions in the nephrotic group compared with the moderately proteinuric group. We conclude that mild proteinuria late after renal transplantation is associated with a defect in glomerular charge selectivity. The development of nephrotic range proteinuria is associated also with a defect of glomerular size selectivity, which correlates with prominent glomerular pathology.

Adult↗
PubMed1995

How to evaluate intraexaminer reliability using an interexaminer reliability study design.

Examiner reliability is often investigated to make generalizations about a profession's performance with various diagnostic tests. Although the evaluation of interexaminer reliability is straightforward, assessment of intraexaminer reliability can be problematic. Estimations of intrarater reliability can be inflated due to correlated error and the difficulty in blinding the examiners. A statistical method is presented that permits the investigator to compute intraexaminer as well as interexaminer reliability and precision from the findings of an interexaminer reliability investigation. In this approach, intraclass correlation coefficients are constructed from variance components estimated from a simple repeated-measures design: each of two or more raters evaluates each subject one time. The method can be applied to nominal and ordinal as well as interval data. Not only can spurious results be avoided, but time and funds can be saved by reducing the required number of subject ratings.

Analysis of Variance↗
PubMed1995

Delegation and supervision in school settings. Standards, issues and guidelines for practice (Part 1)

Part one of this article reviews nursing standards and practice issues related to the delegation of nursing activities to, and the supervision of, unlicensed assistive personnel. Part two, which will be published in a future issue, provides specific strategies, practices and resources to assist school nurses to apply these standards and concepts in their everyday school nursing practice.

Humans↗
PubMed1995

Low back pain outcome measurement assessment in chiropractic teaching clinics: responsiveness and applicability of two functional disability questionnaires.

OBJECTIVE: The major aims were to evaluate responsiveness and clinical/research applicability of the Revised Oswestry Disability Questionnaire (ODQ) and the Dallas Pain Questionnaire (DPQ). Construct and content validity were assessed. Patient characteristics and outcomes were also documented. DESIGN: Longitudinal observational study. SETTING: College outpatient clinics. PARTICIPANTS: Six hundred sixty-three consecutive new patients accepted for treatment of low back pain (LBP) at the clinics over a 1-yr period, age 18 or older. INTERVENTIONS: Treatment of low back pain by senior interns under the supervision of staff clinicians. MAIN OUTCOME MEASURES: ODQ and DPQ administered at baseline, 2 wk, 1 month, and monthly up to 6 months. Responsiveness: mean standardized change score (delta' = mudif/sigma dif), relative efficiency ([RE = delta' ODQ/delta DPQ']2), and improvement rates (IR). Applicability: instrument completion rates. Construct validity: correlation with VAS for pain intensity. RESULTS: ODQ responsiveness was generally consistent over time (delta' = .70-.83) and negligibly better than the DPQ activities of daily living scale (RE = 1.00-1.35); most patients self-reporting improvement showed positive outcomes (IR = 97%). For large samples (n > 100): delta' = .47-.63 and IR = 81% for the DPQ work/leisure scale; delta' = .17-.40 and IR = 54% for the DPQ anxiety/depression and social dimensions. Completion rates: 65%-78% of all instruments; 81%-100% of individual scales. Construct validity: r = .44-.68 for the ODQ, DPQ activities of daily living, and DPQ work/leisure scales; r = .20-.40 for the anxiety/depression scale. CONCLUSIONS: The ODQ and the activities of daily living and work/leisure scales from the DPQ appear appropriate for monitoring LBP patients returning for care to chiropractic teaching clinics. The social and anxiety/depression dimensions of the DPQ do not appear to be responsive in this population. The latter scale may be unsuitable on the grounds of misinterpretations.

Activities of Daily Living↗
PubMed1994

Nonhereditary p53 mutations in T-cell acute lymphoblastic leukemia are associated with the relapse phase.

We have previously reported that greater than 60% of human leukemic T-cell lines possess mutations in the p53 tumor suppressor gene. To determine whether T-cell acute lymphoblastic leukemia (T-ALL) patient samples possess p53 mutations, we screened peripheral blood-and bone marrow-derived leukemia samples, taken at diagnosis and at relapse, for p53 mutations. Exons 4 through 9 and selected intron regions of the p53 gene were analyzed using polymerase chain reaction-single-strand conformation polymorphism and direct sequencing. p53 mutations were found in 0 of 15 T-ALL diagnosis samples, as compared with 10 of 36 (28%) T-ALL relapse samples. To determine whether p53 mutations play a role in the recurrence (relapse) of T-ALL, two special groups of T-ALL patients were studied: (1) a group of 8 relapse patients whose disease was refractory to chemotherapeutic treatment, and (2) a group of 6 "paired" T-ALL cell samples from patients for whom we possess both diagnosis and relapse samples. Three of 8 relapsed patients (37.5%) whose disease was refractory to the reinduction of remission by chemotherapy possessed missense mutations of the p53 gene. All 3 cases had mutations in exon 5. Among the paired samples, 3 of 6 patients harbored p53 mutations at disease recurrence, but possessed only wild-type p53 alleles at diagnosis. One case had mutation on exon 4, 1 case in exon 5, and 1 case in exon 8 with loss of heterozygosity. These data clearly indicate that recurrence of T-ALL is associated with missense mutations in p53. Our results indicate that (1) mutations of p53 do occur in T-ALL in vivo, and such mutations are associated with the relapse phase of the disease; and (2) p53 mutation is involved in the progression of T-ALL. This conclusion is supported by our observation that the introduction of T-ALL-derived mutant p53 expression constructs into T-ALL cell lines further increases their growth rate in culture, enhances cell cloning in methylcellulose, and increases tumor formation in nude mice.

Base Sequence↗
PubMed1994

Crystallization and preliminary crystallographic studies of the precursor and mature forms of a neutral lipase from the fungus Rhizopus delemar.

A neutral lipase from the filamentous fungus Rhizopus delemar has been crystallized in both its proenzyme and mature forms. Although the latter crystallizes readily and produces a variety of crystal forms, only one was found to be suitable for X-ray studies. It is monoclinic (C2, a = 92.8 A, b = 128.9 A, c = 78.3 A, beta = 135.8) with two molecules in the asymmetric unit related by a noncrystallographic diad. The prolipase crystals are orthorhombic (P2(1)2(1)2(1), with a = 79.8 A, b = 115.2 A, c = 73.0 A) and also contain a pair of molecules in the asymmetric unit. Initial results of molecular replacement calculations using the refined coordinates of the related lipase from Rhizomucor miehei identified the correct orientations and positions of the protein molecules in the unit cells of crystals of both proenzyme and the mature form.

Crystallization↗
PubMed1994

IRE-bubble PCR: a rapid method for efficient and representative amplification of human genomic DNA sequences from complex sources.

A significant issue in the analysis of any genomic DNA segment is the generation of a unique set of short single-copy sequences that are representative of that region. In this report we describe a novel technique, IRE-bubble PCR, which was designed to amplify the human DNA content of somatic cell hybrids, YACs, cosmids, and lambda phage and result in greater complexity and representation than standard inter-IRE PCR. Here we demonstrate that IRE-bubble PCR is species specific and that it results in the generation of a product that is at least 10-fold more complex and representative than that produced by standard inter-IRE PCR. In addition, we have addressed the factors that contribute to the representation of the IRE-bubble PCR product and show how they may be used to further increase the complexity of this reaction. Finally, we have illustrated how the complexity and distribution of products generated by IRE-bubble PCR can be exploited and applied to FISH mapping and "chromosome painting" as well as to the generation of STSs targeted to specific chromosomal or subchromosomal regions.

Animals↗
PubMed1994

Analysis of the entire nucleotide sequence of the cryptic plasmid QpH1 from Coxiella burnetti.

The complete plasmid QpH1 from Coxiella burnetti, isolate 'Nine Mile', phase I, was cloned as NotI fragment with a size of 37329 bp. The entire plasmid was sequenced by the chain termination method after EcoRI subcloning. 37 open reading frames coding for polypeptides larger than 100 amino acid residues were determined. The predicted polypeptide products of the open reading frames were compared by computer analysis with reported protein sequences. Homologies of predicted polypeptide products to analogous proteins are described.

Amino Acids↗
PubMed1994

IgG subclass deposits in glomeruli of lupus and nonlupus membranous nephropathies.

Membranous nephropathy (MN) accounts for approximately 10% of all renal lesions of systemic lupus erythematosus (SLE). These membranous lesions often have a clinical presentation similar to that of idiopathic MN and may present before SLE is apparent clinically. However, unlike proliferative lesions of lupus nephritis (LN), membranous LN often does not show a "full-house" pattern of glomerular immunoglobulin and complement (C) deposits by immunofluorescence (IF); only nine of 14 such lesions that we examined stained for all of the following: IgG, IgA, IgM, C3, and C1q. Iskandar et al reported in 1992 that most cases of diffuse proliferative LN showed IgG3 as the major IgG subclass present in glomerular deposits; by contrast, IgG4 predominated in six of seven cases of MN of unspecified etiology. If IgG subclass deposition is similar in membranous and proliferative lesions of LN, then IF staining for IgG3 and IgG4 may be helpful in distinguishing lupus from nonlupus lesions in patients with MN who are lacking a firm diagnosis of SLE. We therefore stained cryostat sections of renal biopsies from 14 patients with SLE and MN (without a proliferative component; World Health Organization [WHO] classes Va and Vb) and 28 non-SLE patients with MN for IgG subclasses by direct IF; the observer was blind to the diagnosis for each case until all were read. The intensity of glomerular staining was graded on a 0 to 4+ scale with increments of 0.5+.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1994

Characterization of the endogenous Na(+)-K(+)-2Cl- cotransporter in Xenopus oocytes.

Over time, Xenopus laevis changed from producing stage V and VI oocytes with little native Na(+)-K(+)-2Cl- cotransport activity to those with substantial activity. In oocytes with high endogenous activity, K+ uptake, using the tracer 86Rb+ was approximately 20 pmol.min-1.oocyte-1 in the presence of blockers of Na(+)-K(+)-ATPase and conductive K+ transport. Bumetanide (10 microM) inhibited > 90% of this uptake, suggesting involvement of Na(+)-K(+)-2Cl- cotransport. This was confirmed by two observations that are found in this cotransporter in other tissues: 1) The related diuretics, thiobenzmetanide [50% inhibitory concentration (IC50), 2 x 10(-11) M] > bumetanide (IC50, 7 x 10(-8) M) > furosemide (IC50, 2.5 x 10(-6) M) inhibited the cotransporter in a dose-dependent manner. 2) There was little uptake of K+ in the absence of extracellular Na+ or Cl-. Halving medium osmolarity to 92 mosM decreased bumetanide-sensitive K+ uptake by approximately 75%, whereas a doubling of medium osmolarity increased it by approximately 50%. The cotransport activity was increased fourfold by the phosphatase inhibitor calyculin A (200 nM) but was unaffected by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, 8-bromoguanosine 3',5'-cyclic monophosphate, ATP, ionomycin, or okadaic acid. Both the photoaffinity bumetanide analogue, 4-[3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid, and an antiserum raised against Ehrlich ascites cell cotransporter specifically labeled an approximately 140-kDa oocyte membrane protein. These results demonstrated that, in addition to the Na+ pump and K+ channels, K+ uptake in Xenopus oocytes occurs via a loop-diuretic-sensitive Na(+)-K(+)-2Cl- cotransporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Na-K-Cl cotransport in nystatin-treated tracheal cells: regulation by isoproterenol, apical UTP, and [Cl]i.

Chloride secretion in mammalian airway epithelia is stimulated by beta-adrenergic agonists via an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent mechanism and by apical triphosphate nucleotides (ATP, UTP) via a cAMP-independent mechanism. Both types of secretagogues are known to stimulate apical Cl channels in airway cells; however, to maintain a stimulated rate of secretion, basolateral Cl influx via Na-K-Cl cotransport must be upregulated in parallel with apical Cl efflux. To examine the regulation of basolateral cotransport activity and its relationship to apical Cl efflux, we examined Cl transport in confluent primary cultures of dog tracheal epithelial cells treated with nystatin, an antibiotic that increases the permeability of plasma membranes to small monovalent ions, including Cl. By applying nystatin to the apical membrane of these cultures, apical Cl permeability could be increased to the point where transepithelial Cl transport is limited by transport across the basolateral membrane, which reflects primarily the activity of the cotransporter. In cultures of tracheal cells not treated with nystatin, transepithelial (basolateral-to-apical) 36Cl flux was increased two- to threefold by exposure to isoproterenol (5 microM, basolateral) or apical UTP (10 microM). Apical application of nystatin (400 units/ml) increased the basal level of transepithelial 36Cl flux approximately 1.5-fold and eliminated UTP stimulation of this flux, although an approximately twofold stimulation by isoproterenol persisted. Nystatin treatment also abolished UTP stimulation of saturable, basolateral [3H]bumetanide binding, a measure of functioning Na-K-Cl cotransporters in these cells; isoproterenol stimulation of binding was only mildly inhibited by nystatin treatment. Lowering intracellular Cl concentration ([Cl]i) by incubating cultures with apical media containing nystatin and reduced [Cl] (NO3 replacement) increased both basolateral-to-apical 36Cl flux and [3H]bumetanide binding in the absence of secretagogues or cell shrinkage. The results support our previous suggestion, based entirely on [3H]bumetanide binding [M. Haas, D. G. McBrayer, and J. R. Yankaskas. Am. J. Physiol. 264 (Cell. Physiol. 32): C189-C200, 1993], that UTP stimulation of basolateral Na-K-Cl cotransport in airway epithelial cells is entirely secondary to, and requires, an increase in apical Cl efflux, and further suggest that a decrease in [Cl]i may be a signal for cotransport activation in response to UTP. In addition, a cAMP-dependent cascade initiated by isoproterenol appears to directly stimulate the cotransporter.

Animals↗
PubMed1994

Characterization of the proteins of the intestinal Na(+)-K(+)-2Cl- cotransporter.

Absorptive intestinal epithelia, such as that of the winter flounder, absorb salt via a bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport mechanism on the brush-border membrane (BBM). The present study demonstrates the first molecular characterization of the intestinal Na(+)-K(+)-2Cl- cotransporter and its unique regulation. The photoaffinity bumetanide analogue, 4-[3H]benzoyl-5-sulfamoyl-3- (3-thenyloxy)benzoic acid, specifically labeled three groups of proteins in flounder intestinal microsomal membranes (MM): a approximately 180-kDa peptide, prominently labeled, and diffuse bands at approximately 110-70 and 50 kDa, less intensely labeled. Subcellular fractionation revealed a single prominently labeled protein of approximately 170 kDa in BBM but not in basolateral membranes (BLM) and little or no labeling of proteins of approximately 110-70 or 50 kDa. Polyclonal antiserum raised against the Ehrlich ascites cell cotransporter identified a 180-kDa peptide in MM and a 175-kDa peptide (pI approximately 5.4) in BBM but none in BLM or in the cytosol of flounder intestine. As predicted from the regulation of cotransport in this tissue, phosphorylation of this protein is increased by guanosine 3',5'-cyclic monophosphate (cGMP)-dependent but not by adenosine 3',5'-cyclic monophosphate-dependent protein kinase. In addition, phosphorylation of the protein is not increased by protein kinase C or Ca2+/calmodulin-dependent protein kinase but is increased by the phosphatase inhibitor calyculin A. Finally, calyculin A preserves the inhibitory effect of cGMP on ion transport, even in the absence of the nucleotide, suggesting that phosphorylation-dephosphorylation mechanisms are crucial in cotransporter regulation. Thus the flounder intestinal cotransporter is a approximately 175-kDa BBM protein that can be regulated by phosphorylation.

Affinity Labels↗
PubMed1994

The Na-K-Cl cotransporters.

The Na-K-Cl cotransporters are a class of membrane proteins that transport Na, K, and Cl ions into and out of cells in an electrically neutral manner, in most cases with a stoichiometry of 1Na:1K:2Cl. Na-K-Cl cotransporters are present in a wide variety of cells and tissues, including reabsorptive and secretory epithelia, nerve and muscle cells, endothelial cells, fibroblasts, and blood cells. Na-K-Cl cotransport plays a vital role in renal salt reabsorption and in salt secretion by intestinal, airway, salivary gland, and other secretory epithelia. Cotransport function also appears to be important in the maintenance and regulation of cell volume and of ion gradients by both epithelial and nonepithelial cells. Na-K-Cl cotransport activity is inhibited by "loop" diuretics, including the clinically efficacious agents bumetanide and furosemide. The regulation of Na-K-Cl cotransport is mediated, at least in some cases, through direct phosphorylation of the cotransport protein. Cotransporter regulation is highly tissue specific, perhaps in part related to the presence of different Na-K-Cl cotransporter isoforms. In epithelia, both absorptive (kidney-specific) and secretory isoforms have been identified by cDNA cloning and sequencing and Northern blot analysis; alternatively spliced variants of the kidney-specific isoform have also been identified. The absorptive and secretory isoforms exhibit approximately 60% identity at the amino acid sequence level; these sequences in turn show approximately 45% overall homology with those of thiazide-sensitive, bumetanide-insensitive, Na-Cl cotransport proteins of winter flounder urinary bladder and mammalian kidney. This review focuses on recent developments in the identification of Na-K-Cl cotransport proteins in epithelial and on the regulation of epithelial Na-K-Cl cotransporter function at cellular and molecular levels.

Animals↗
PubMed1994

Gain-of-function mutations of the p53 gene induce lymphohematopoietic metastatic potential and tissue invasiveness.

Leukemia cell infiltration and the induction of lethal hematopoietic disease in immune-deficient SCID mice transplanted with human T cell acute lymphoblastic T leukemia (T-ALL) cells occurred only when the cells possessed mutant p53 genes and lacked a wild-type allele or when T-ALL cells lacking p53 protein were infected with specific mutant p53 genes. A series of six mutant p53 genes were cloned from relapse T-ALL-derived cell lines and were constructed into defective retroviral expression vectors. Viruses encoding mutant p53 proteins were used to infect relapse T-ALL cells in a study designed to compare their pathogenic potency. The mutant p53 genes possessed a distinct hierarchy in vivo and in vitro: mutants inducing the greatest increase in proliferation of different T-ALL lines in vitro and colony formation in methylcellulose cultures also induced tissue invasiveness of infected T-ALL cells in vivo. Mutant p53 gene transfer to a cell line lacking p53 protein showed that the more potent p53 mutants possessed a distinctive dominant oncogenic activity in vitro and in vivo. The dominant oncogenic activity of these mutant p53 proteins was not dependent on the presence of and on complex formation with wild-type p53 protein. These "hot" p53 mutations thus represent bona fide gain-of-function mutations. Infection of p53-negative T-ALL cells with viruses encoding gain-of-function mutant p53 genes resulted in the acquisition of metastatic potential and tissue invasiveness. Taken together, our results suggest that specific mutant p53 genes play a role in the generation of lymphohematopoietic metastatic potential and tissue invasiveness as assayed in SCID mice, whereas the expression of wild-type p53 is capable of keeping this metastatic potential in check.

Animals↗
PubMed1993

Isolation and FISH mapping of 80 cosmid clones on the short arm of human chromosome 3.

We have isolated and mapped by fluorescence in situ hybridization 80 new cosmids on the short arm of chromosome 3. These markers were isolated from a radiation-reduced hybrid, DM1, made from a cell line that was monochromosomal for human chromosome 3. Selected cosmids were used in double-label cohybridization experiments in which polymerase chain reaction products, generated by an Alu oligonucleotide primer from genomic DNA, were used for chromosome banding. Fifty-six of the cosmid probes map between 3p14.3 and 3p22 while 24 other probes cluster around bands 3p23-3p25. Three probes that appeared to map close to the chromosome 3 region bearing a t(3,8)p14.2; q24.1 translocation associated with renal cell carcinoma were analyzed by interphase mapping techniques and hybridized to metaphase spreads from the translocation cell line. These 80 probes will be useful in the elucidation of genetic alterations associated with diseases such as small cell lung carcinoma, renal cell carcinoma, and von Hippel-Lindau disease.

Animals↗