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Biomedical subjects

M Haas

Publications and source records attributed to M Haas.

At least 19 recordsLinked to original sources
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↗
PubMed1992

The rationing of health care: should Oregon be transported to Australia?

The Oregon Plan is an ambitious attempt to address the widespread problem in the United States of a growing number of individuals who are without private health insurance and are not eligible for federal assistance programs. Its aim is to provide universal access for all Oregonians, without increasing total health care expenditure, by restricting the cover of some treatments. It has aroused interest in Australia and elsewhere. The appeal of the Oregon Plan lies in its explicit approach to rationing, in community participation in setting priorities, and the use of a cost-effectiveness framework. This paper describes the beginnings and the development of the Oregon Plan, and compares the actual development of the Plan with the rhetoric. There is a gap between the rhetoric of the Plan and its reality. The Oregon plan should be considered in the context of the United States health care system. We compare the American problems with those facing the Australian health care system and conclude that the answer to the question of whether Oregon should be transported to Australia is no. Nevertheless there are elements of the rhetoric of the Plan which could be applied in rationing health care in Australia.

Health Care Rationing↗
PubMed1992

Na-K-Cl cotransport in the shark rectal gland. I. Regulation in the intact perfused gland.

To investigate regulation of the Na-K-Cl cotransport system in the rectal gland of the dogfish shark Squalus acanthias, we examined binding of the loop diuretic [3H]benzmetanide to the intact gland. Glands were perfused with a shark Ringer solution, either in a basal state or stimulated with vasoactive intestinal peptide (VIP). [3H]benzmetanide was added to the perfusion solution for the last 25 min of perfusion, after which the gland was homogenized and the amount of bound [3H]benzmetanide was determined in the membrane fraction. Most of the membrane-associated [3H]-benzmetanide appeared to be associated with the Na-K-Cl cotransporter as judged by the dissociation rates at 0 degree C and 20 degrees C, by labeling with a photosensitive analogue, and by continued association of [3H]benzmetanide with membrane protein on solubilization. With the use of [3H]4-benzoyl-5-sulfamoyl-3-(3- thenyloxy)benzoic acid, a photosensitive analogue of benzmetanide, a 200-kDa protein was selectively labeled on exposure to ultraviolet light. It was also possible to detect [3H]-benzmetanide binding during the perfusion period as an arterial-venous difference, thereby providing a time course of the binding process. In comparing two groups of five glands each, VIP stimulated NaCl secretion 20-fold and [3H]benzmetanide binding 16-fold, providing strong evidence that the Na-K-Cl cotransport system is activated as part of the process of stimulation of secretion. The VIP-stimulated increase in [3H]benzmetanide binding was completely inhibited when Ba was added to the perfusate to block K channel-mediated K exit across the basolateral membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Deposition and removal of cutaneous beta 2-microglobulin.

These studies were designed to track the cutaneous deposition of beta 2-microglobulin (beta 2M) in patients on chronic hemodialysis, patients with chronic renal insufficiency and patients with successful renal transplants. Immunoperoxidase staining of skin biopsies from dialysis patients demonstrated significantly increased amounts of beta 2M compared to controls (p < 0.01). There was a strong positive correlation between the skin beta 2M content and the years of dialysis treatment. Renal transplant recipients had decreased skin content of beta 2M compared to hemodialysis patients. There was no difference in the skin beta 2M content in patients with chronic renal insufficiency not on hemodialysis and controls. No dialysis patient had amyloid in the skin by Congo red stain. We conclude that beta 2M accumulates in the skin of patients on chronic hemodialysis. This beta 2M is not in the form of amyloid. Successful renal transplantation allows for the removal of beta 2M from the skin indicating that beta 2M not in the form of amyloid can be mobilized from tissue sites.

Adult↗
PubMed1992

Lumbar motion trends and correlation with low back pain. Part II. A roentgenological evaluation of quantitative segmental motion in lateral bending.

OBJECTIVE: A radiographic study was undertaken to describe the relationship between the magnitude of coupled lumbar motion in lateral bending and the presence of low back pain: correlation between pain and motion, relationship between motion category and motion and symmetry of lumbar motion. DESIGN: Survey. SETTING: Chiropractic college student health center and private chiropractic clinic. PARTICIPANTS: 249 subjects: 114 with low back pain, 29 asymptomatic with no history and 106 asymptomatic with history. Of these, 194 were freshman volunteers and 55 were new private clinic low back pain patients. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Net lumbar segmental tilt and rotation in lateral bending: corrected and uncorrected for segmental malposition with the patient standing in the upright neutral position. RESULTS: Statistical analysis demonstrated no significant relationship between coupled lumbar motion and low back pain (p greater than .01). The presence of type II motion could account for, on average, less than 5% loss of segmental tilt in the lumbar spine. Asymmetries between left and right side motion averaged 45 to 100% of unilateral range of motion. CONCLUSIONS: This study suggests that back pain is not an indication for the routine use of lateral bending films for the identification of alterations in the magnitude of lumbar segmental motion in lateral bending. It further indicates that type II motion cannot be ruled out as a normal variant. The paucity of symmetrical lumbar motion suggests that segmental tilt or coupled rotation asymmetry, in and of itself, should not be considered an indication for spinal manipulation.

Adolescent↗
PubMed1992

Diagnostic utility of the McGill Pain Questionnaire and the Oswestry Disability Questionnaire for classification of low back pain syndromes.

Verbal pain description and assessment of functional limitations are key components in the clinical evaluation of patients with low back pain syndromes. Using the McGill Pain Questionnaire (MPQ) to quantify the pain experience and the Oswestry Disability Questionnaire (ODQ) to quantify functional disability, a study was undertaken to determine the efficiency with which the MPQ and ODQ were capable of enhancing the differential diagnosis of three broad categories of low back syndromes. Three discriminative models were employed. The combined discriminant model (MPQ/ODQ) yielded the highest accuracy, 0.90, and it was the only model with acceptable predictive power. The greatest utility of the discriminant models was found to be ruling out nonspecific low back pain and ruling in radiculopathy, with and without neurological deficits. Subjective pain and disability appear to have the potential for successfully differentiating broad categories of low back pain. Further studies need to be performed to assess the discriminant power of the MPQ and ODQ for specific diagnostic entities.

Activities of Daily Living↗
PubMed1992

Role of the p53 tumor suppressor gene in the pathogenesis and in the suppression of acute lymphoblastic T-cell leukemia.

The tumor suppressor gene p53 has been shown to be mutated in 50% of acute lymphoblastic T-cell-leukemia (T-ALL) cell lines, all of which were established from T-ALL cases in relapse. In these lines both alleles of the p53 gene were independently affected by point mutation. In contrast, in human solid tumors possessing a mutated p53 allele, the second wild-type p53 suppressor allele is often lost by deletion rather than altered by mutation. This suggests that in T-ALL cell lines, the product encoded by the second mutated allele provides the cells with an additional oncogenic stimulus, beyond the loss of suppressive activity. While different p53 mutations have been shown to possess differential oncogenic potential in the p53 plus ras cotransformation assay, in T-ALL cells different mutations may in addition possess distinct functions, further contributing to the tumorigenic phenotype.

Animals↗
PubMed1992

[Clinical results and material behavior of composite, ceramic and gold inlays].

Laboratory-manufactured gold, ceramic and composite inlays were compared with the CAD/CAM produced CEREC inlay experimentally and clinically. The following systems were examined: Dicor, Optec, Hi-Ceram, Du-Ceram, Cerec, Kulzer, Coltene, SR-Isosit composite inlays, and gold inlays (Degulor C) with adhesive fixation (group I) and with zinc oxide phosphate cement (group II). In the clinical trial inlays in 270 teeth were re-evaluated in a total of 73 patients after 2.3 to 5 years. After 3 years the results obtained with Optec, Hi-Ceram and the Coltene composite inlay were less favorable than those of other systems--statistically however, this difference was of rather low significance (p less than 0.05). Fractures of ceramic inlays occurred in 6 cases within 8 months. 10 failures of ceramic and composite inlays were due to secondary caries.

Composite Resins↗
PubMed1991

[3H]bumetanide binding to mouse kidney membranes: identification of corresponding membrane proteins.

Crude plasma membranes from whole mouse kidneys have two classes of [3H]bumetanide binding sites. High-affinity sites (K1/2 approximately equal to 0.04 microM; Bmax = 1-2 pmol/mg protein) are similar to those identified on dog kidney membranes (B. Forbush and H.C. Palfrey. J. Biol. Chem. 258: 11787-11792, 1983) both with respect to affinity and in that Na, K, and Cl are required for [3H]bumetanide binding. Low-affinity sites (K1/2 approximately equal to 1 microM; Bmax = 7-14 pmol/mg) are unaffected by removal of these ions; such sites are not seen with dog kidney. When mouse kidney membranes are photolabeled with 4-[3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid [( 3H]BSTBA), a photoreactive bumetanide analogue, specific incorporation of the label is seen in two regions. As with dog kidney [M. Haas and B. Forbush. Am. J. Physiol. 253 (Cell Physiol. 22): C243-C252, 1987], an approximately 150-kDa protein is labeled with high affinity (K1/2 approximately equal to 0.05 microM). This labeling also requires Na, K, and Cl and appears to correspond to the high-affinity [3H]bumetanide binding sites and to the Na-K-Cl cotransport system. A second peak of [3H]BSTBA photolabeling, centered at approximately 75 kDa, incorporates the label with lower affinity (K1/2 = 2-3 microM). The photolabeling at approximately 75 kDa is unaffected by Na, K, and Cl concentrations and thus may correspond, at least in part, to the low-affinity [3H]bumetanide binding sites. Western blot analysis of [3H]BSTBA-labeled mouse kidney membranes was performed using an antiserum raised to proteins of approximately 82 and approximately 39 kDa isolated from mouse Ehrlich ascites tumor cells using a bumetanide affinity gel (P. B. Dunham, F. Jessen, and E. K. Hoffmann. Proc. Natl. Acad. Sci. USA 87: 6828-6832, 1990). This antiserum cross-reacts with a approximately 150-kDa mouse kidney protein, the staining profile of which on Western blot corresponds very closely to the peak of specific [3H]BSTBA incorporation in this region. The antiserum also reacts with proteins in the range of 65-85 kDa, overlapping the low-affinity peak of [3H]BSTBA incorporation.

Affinity Labels↗
PubMed1991

Intrabacterial sodium-to-potassium ratios and ATP contents of Mycobacterium leprae from ofloxacin-treated patients.

In a clinical trial including 17 multibacillary leprosy patients the in vivo effectiveness of ofloxacin on Mycobacterium leprae was tested via mass spectrometric determination of intrabacterial ratios of the concentrations of the sodium and potassium ions of individual organisms and of the ATP content per 10(6) bacteria isolated from skin biopsies. After 3 months of treatment, the in vivo drug effect could be determined with at least one of the two methods in 14 cases. Both methods revealed that in two cases the bacteria definitely did not respond to a 3-month ofloxacin monotherapy (200 mg twice daily). In three further cases a nonresponse of the M. leprae organisms was suspected from the mass spectrometric measurements. In the responder cases, the M. leprae were severely impaired. From the intrabacterial cation ratios the percentage of viable organisms averaged over all untreated biopsies was determined to be 58% and the percentage-killing during the first 3 months of treatment was 72%.

Adenosine Triphosphate↗
PubMed1991

[Caries status and periodontal health in 6- to 10-year old elementary school children in Graz compared to Swiss, Swedish and WHO principles].

In a complete survey, 7500 primary pupils in Graz aged between six and ten years were examined for caries and periodontal health. Comparison with the DMF-T values of primary school pupils in Switzerland and Sweden indicated that considerable efforts are required in Austria if preventive dental health education is to achieve the standard status it has enjoyed for years in the other countries mentioned.

Austria↗