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W Haase

Publications and source records attributed to W Haase.

At least 91 records · Page 5Linked to original sources

Monoclonal antibodies against luminal membranes of renal proximal tubules which are kidney-specific.

After immunization with porcine brush-border membrane proteins, 11 monoclonal antibodies were generated which react with proximal tubules. Their antigenic polypeptides were characterized with respect to apparent molecular weight, histochemical localization in porcine and human kidney, and tissue distribution in pig. In porcine kidney, six antibodies bind selectively to the proximal tubule whereas the others also react with other nephron segments. With the exception of one antibody which reacts with the luminal and the basolateral membrane of the porcine proximal tubule, the other antibodies specific for the proximal tubule only stain the brush-border membrane. Four of them react along the entire length of the porcine proximal tubule, whereas one (R1A2) binds to the S3-segment in pig and to the entire length of the proximal tubule in man. This indicates that segment-specific expression may be species-dependent. Testing the antibodies in 21 different extrarenal tissues it was found that three of the antibodies, specific for the brush-border membrane in renal proximal tubules, only react in kidney. Two of these are specific for pig kidney whereas one also reacts with human kidney. This antibody (N4A4) is directed against a polypeptide with an apparent molecular weight of 400,000. Electron microscopic immunohistochemistry showed that N4A4 binds to the intervillus region of the brush-border membrane and to subapical vesicles.

Animals↗

Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+. I. Microscopic and general electrophysiological observations.

Following the publications by Duffey et al. [Nature 294:451 (1981)] and Palant et al. [Am J Physiol 245: C203 (1983)] it is generally accepted that tight-junction tightness of Necturus gall bladder epithelium is up-regulated by cAMP-mediated and Ca(2+)-mediated stimulation. This conclusion was mainly based on observed increases in transepithelial resistance (Rt). However, since in leaky epithelia Rt cannot be simply equated with the tight junction resistance (Rj), but may include large contributions from the lateral space resistance (Rlis), we asked whether the observed increases in Rt resulted indeed from Rj or whether Rlis also increased. The experiments were performed on Necturus gall bladders using forskolin or the Ca2+ ionophore A23187 as stimulants. Forskolin (2 mumol/l) had a biphasic effect. In the first 5 min Rt decreased from 128 +/- 13 to 119 +/- 14 omega cm2 (P < 0.05, n = 10) which probably reflects stimulation of an apical cell membrane Cl- conductance (see accompanying paper). Subsequently Rt increased in approximately 30 min to 184 +/- 20 omega cm2 and then remained fairly constant. Simultaneously the lateral spaces collapsed. If the spaces were now transiently opened by passing mucosa-positive direct current across the epithelium, Rt fell transiently to 111 +/- 7 omega cm2, but returned gradually to its elevated level when the spaces collapsed again. When the spaces were constantly dilated by a serosa-positive hydrostatic pressure of 1 cm H2O, forskolin neither affected the space width nor increased Rt, and current passage was virtually ineffective, although the cells depolarized in response to forskolin as usual.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Elevated cAMP levels induce multilayering of MDCK cells without disrupting cell surface polarity.

The effect of hormones on the morphology and cell surface polarity of the epithelial cell line MDCK was examined. When MDCK cells were seeded in high densities in media containing FCS a regular monolayer was formed. However, in serum-free medium supplemented with insulin, transferrin, prostaglandin E1, hydrocortisone and triiodothyronine, the development of a multilayer with intercellular lumina was observed. In hormone-depletion studies we identified PGE1 as the inducer of these multilayers. Since dibutyryl cyclic AMP and the phosphodiesterase inhibitor isobutyl methylxanthine could substitute for PGE1, we conclude that an elevated intracellular cAMP level resulted in formation of the multilayer. Further analysis by electron microscopy and immunocytochemistry revealed a polarized organization of the multilayered cells. Junctional complexes, enclosing microvilli-rich membrane domains, were found at the apices of adjacent cells facing the medium and those surrounding the intercellular lumina. Surprisingly, cells participating in the formation of both the free surface and the surface of the intercellular lumen, exhibited two distinct membranes with microvilli, each separated by junctional complexes. Immunolocalization of membrane marker proteins demonstrated that an apical 114 kDa membrane protein was localized to the free cell surfaces, the same membrane domains where extensive microvilli were also observed. The distribution of a basolateral 58 kDa membrane protein was restricted to sites of cell contact. These results provided evidence that nontransformed epithelial MDCK cells form multilayers in response to elevated cAMP levels; however, they retain the potential of developing cell surface polarity.

Alprostadil↗

Quality control review for radiotherapy of small breast cancer: analysis of 708 patients in the GBSG I trial. German Breast Study Group (GBSG).

Within six years between November 1983 and December 1989 the German Breast Cancer Study Group (GBSG) conducted a prospective multicenter trial on the treatment of pT1 pN0 M0 breast carcinoma. Out of 1036 eligible patients from 69 hospitals, a total of 733 underwent breast preservation surgery and postoperative radiotherapy. A detailed quality control (QC) screening of 708 available radiotherapy records was performed by the radiotherapy reference center. The following quality control scoring system was used for all radiotherapy variables: Radiotherapy treatment "completely according to study protocol" (QC score = 0); "acceptable protocol deviations" (QC score = 1); "unacceptable protocol violations" (QC score = 2); the "overall QC score" was the worst judgement for any single quality control criterion, while the "total QC score" was the summation of all single quality control scores (range: 0 to 14). 292 (41.2%) patients were treated per protocol; 290 (41.0%) had acceptable protocol deviations; and 126 (17.8%) had unacceptable protocol violations. 107 (84.9%) single violations, 15 (11.9%) combinations of two, three (2.4%) combinations of three, and one (0.8%) combination of four unacceptable violations were observed. 564 (79.4%) patients achieved a total QC score of < or = 2, 124 (17.5%) of 3 to 5, and 20 (2.8%) of > or = 6. The criterion "radiotherapy treatment duration" had the highest rate of protocol violations, 67/708 (9.5%); other quality control criteria with protocol violations were "radiotherapy reference dose" (27 = 3.8%), "radiotherapy treatment initiation" (15 = 2.1%) and "other protocol violations" (33 = 4.7%). No protocol violations were observed for the criterion "target volume coverage". The protocol compliance improved slightly during the conduct of the study. Institutional differences in protocol compliance were observed depending upon the accrual rate, the institutional treatment preference and between academic and non-academic hospitals. After a short median follow-up of 48 months, differences in protocol compliance, i.e. protocol violations, have not resulted in a reduced disease-free survival; however, the final prognostic evaluation of the quality control review may require longer follow-up and eventually different treatment endpoints to be analyzed.

Breast Neoplasms↗

[Effective vision screening can decrease the rate of amblyopia].

The quality of screening for testing visual disorders is unsatisfactory. Diagnosis and therapy are generally too late to exploit the advantage therapy can give during the sensitive period of visual acuity. About 10% of the adult population show decreased visual acuity (below 0.8). These persons have a more or less distinct amblyopia. Commonly used visual acuity tests do not allow the crowding phenomenon to be measured, although it is an easily measurable amblyopia characteristic. The most frequently used visual acuity test by German health authorities (R 5/R 11 test with plate No. 120) shows 48% false-negative results. We introduced the H test for preschool children. For school children and adults we developed the C test. To detect amblyopia precisely we designed both tests so that the crowding phenomenon could be measured. Both tests were validated in more than 700 subjects. All persons received an extensive ophthalmological checkup, which was used as the reference for normality. The H test is based on the matching principle and works with musical feedback. The test can be used from the age of 3 years onward. Therefore, the advantage of early therapy can be fully exploited. The H test has a 3% percentage false-negative rate. The C test is based on the Landolt ring. The visual acuity charts are presented as a book (40 cm distance) for measuring near visual acuity and (5 m) for measuring distance visual acuity. The C test has less than 5% false-negative results. Both tests can help in different age groups to diagnose amblyopic cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

[Effectiveness and tolerance of amitriptyline oxide in chronic tension headache--a multicenter double-blind study versus amitriptyline versus placebo].

Tricyclic antidepressants, especially amitriptyline, are the medication of first choice in the treatment of chronic tension headache. Few previous studies meet modern standards of study design and statistical analysis. Tolerability and efficacy of 60-90 mg amitriptyline oxide (AO) as a single dose in the evening were compared with 50-75 mg amitriptyline (AM) and placebo (PL) in a double-blind, parallel-group trial consisting of a 4-week baseline phase and 12 weeks of treatment. The 3-armed study was conducted in 7 centers. The inclusion criterion was tension-type headache on at least 15 days monthly with a duration of at least 6 months. Exclusion criteria were a migraine history, previous participation in another clinical trial within the last 3 months, drug abuse, medication with other antidepressants or tranquilizers, current use of other acknowledged prophylactic headache medication, lack of compliance, major psychiatric disorder according to DSM-III and medical contra-indications against tricyclic antidepressants. The primary study endpoint was a reduction at least 50% of the product of headache duration and frequency and a reduction at least 50% in headache intensity. Statistics used were Fisher's Exact Test and an analysis of variance. A total of 211 patients were included in this trial. One hundred ninety-seven cases, 87 males and 110 females, with a mean age of 38 +/- 13 (18-68) years, could be analysed completely (66 AO, 67 AM, 64 PL). With regard to the strictly defined primary study endpoint, no significant difference emerged between AO, AM and PL: treatment responders were 30.3% with AO, 22.4% with AM and 21.9% with PL (PAO-PL = 0.3210, PAM-PL = 1.000, PAO-AM = 0.3299 respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

Sequence analysis of the catalytic subunit of H(+)-ATPase from porcine renal brush-border membranes.

The catalytic subunit of the H(+)-ATPase from brush-border membranes of porcine renal proximal tubules was labeled with the hydrophobic SH-group reagent 10-N-(bromoacetyl)amino-1-decyl-beta-glucopyranoside (BADG) which irreversibly inhibits proton pump activity in the absence but not in the presence of ATP. The labeled protein was purified and digested with proteinases. After isolation and sequencing of proteolytic peptides two BADG-labeled cysteines were identified. The amino acid sequences of the obtained proteolytic peptides were homologous to the catalytic subunit of V-ATPases. From mRNA of porcine kidney cortex a catalytic H(+)-ATPase subunit was cloned. 181 of the 183 amino acids which overlap in the sequence derived from the cDNA and the proteolytic peptides were identical, and the two deviations are due to single base exchanges. A comparison of the amino acid sequence derived from the cloned cDNA with sequences of catalytic H(+)-ATPase subunits communicated by other laboratories revealed 98%, 96% and 94% identity with sequences from bovine adrenal medulla, from bovine kidney medulla and from clathrin-coated vesicles of bovine brain. Between 64% and 69% identity was obtained with sequences from fungi and plants. The data show that the catalytic subunit of V-ATPases is highly conserved during evolution. They indicate organ and species specificity in mammalians.

Acetylglucosamine↗

A monoclonal antibody against a Na(+)-L-glutamate cotransporter from rat brain.

A monoclonal mouse IgM antibody (Z8E9) was raised against the Na(+)-L-glutamate cotransporter from rat brain. In a preparation of brain plasma membrane vesicles, Z8E9 binds specifically to a polypeptide with an apparent molecular weight of 70,000 and inhibits Na+ gradient-dependent L-glutamate cotransport (up to 50%) in brain membrane vesicles. In the membrane vesicles, the antibody does not alter the membrane permeability for Na+ and K+ nor the Na+ gradient-dependent uptake of gamma-aminobutyric acid. Kinetic experiments showed that Z8E9 does not alter the K0.5 values for L-glutamate and Na+ activation of L-glutamate transport. However, an apparent cooperativity observed for L-glutamate activation was increased, and the Vmax of L-glutamate transport was decreased. Immunostaining of rat cerebellum identified antigenic sites of Z8E9 in Golgi epithelial cells and astrocytes (by light and electron microscopy), whereas no labeling at nerve terminals was detected. The data suggest that a component of a Na(+)-L-glutamate cotransporter subtype has been identified that is specific for glia cells in brain.

Amino Acid Transport System X-AG↗

Intravesicular acidification correlates with binding of ADP-ribosylation factor to microsomal membranes.

The ADP-ribosylation factor (ARF), a highly conserved low molecular weight GTP-binding protein, has been implicated to function in intracellular protein transport to and within the Golgi complex. In pancreatic acinar cells the ARF is confined to the cytoplasmic faces of trans-Golgi stack membranes, a compartment known to maintain a low intravesicular pH, which is established by a chloride-dependent MgATP-driven proton pump. The present study shows that MgATP (2mM), but neither adenosine 5'-[gamma-thio]triphosphate in the presence of Mg2+ nor ATP in the absence of Mg2+, increases transfer of ARF from the surrounding medium into the vesicle membranes. The specific vacuolar-type proton pump inhibitor bafilomycin B1 (10 nM), the protonophore carbonylcyanide m-chlorophenylhydrazone (10 microM), and replacement of chloride in the incubation buffer by acetate or nitrate resulted in an almost complete inhibition of the MgATP-dependent association of ARF to the vesicle membranes. The results demonstrate that redistribution of ARF to the vesicle membrane correlates with the intravesicular pH established by a vacuolar-type H(+)-ATPase. The intravesicular pH appears to be one mechanism by which certain low molecular weight GTP-binding proteins become relocated from the cytosol to their specific membrane vesicles.

ADP-Ribosylation Factors↗

Antibodies against synthetic peptides used to determine the topology and site of glycosylation of the cGMP-gated channel from bovine rod photoreceptors.

Peptides corresponding to amino acids 321-339 (peptide GS21) and 416-431 (peptide GS31) of the cGMP-gated channel from bovine rod photoreceptors were synthesized and used as antigens for the preparation of polyclonal antibodies. After affinity purification, both antipeptide antibodies were found to bind specifically to the channel protein after Western blotting, but only the antibody against GS21 gave satisfactory results on enzyme-linked immunosorbent assay and electron microscopy. Using immunocytochemistry, we were able to localize amino acids 321-339 to the extracellular side of the rod photoreceptor plasma membrane. By synthesizing heptapeptides corresponding to amino acids 324-330 (peptide GS2s) and 420-426 (peptide GS3s), we were able to affinity purify antibodies specific for two N-glycosylation consensus sites in the channel protein. As assessed by Western blotting, antibodies against GS3s were found to bind to both the glycosylated and deglycosylated channel proteins, whereas antibodies against GS2s only bound to the channel protein after enzymatic deglycosylation. Together, these results allow the refinement of folding models for the cGMP-gated channel and implicate Asn-327 as being the sole site of N-glycosylation.

Amino Acid Sequence↗

Large-scale purification of calf pancreatic zymogen granule membranes.

A protocol for isolating milligram quantities of highly purified zymogen granule membranes from calf pancreas was developed. The method provides a fivefold enriched zymogen granule fraction that is virtually free from major isodense contaminants, such as mitochondria and erythrocytes. Isolated granules are osmotically stable in isosmotic KCl buffers with half-lives between 90 and 120 min. They display specific ion permeabilities that can be demonstrated using ionophore probes to override intrinsic control mechanisms. A Cl- conductance, a Cl-/anion exchanger, and a K+ conductance are found in the zymogen granule membrane, as previously reported for rat pancreatic, rat parotid zymogen granules, and rabbit pepsinogen granules. Lysis of calf pancreatic secretory granules in hypotonic buffers and subsequent isolation of pure zymogen granule membranes yield about 5-10 mg membrane protein from approximately 1000 ml pancreas homogenate. The purified zymogen granule membranes are a putative candidate for the rapid identification and purification of epithelial Cl- channels and regulatory proteins, since they contain fewer proteins than plasma membranes.

Animals↗

Generation of anti-idiotypic monoclonal antibodies recognizing vasopressin receptors in cultured cells and kidney sections.

To produce anti-idiotypic antibodies against receptors for the neurohypophyseal hormone vasopressin, an anti-vasopressin monoclonal antibody with a ligand specificity similar to that of vasopressin receptors was employed for immunization. Three anti-idiotypic monoclonal antibodies were obtained which induced, like vasopressin, plasminogen activator production in the renal epithelial cell line LLC-PK1 (expressing V2-receptors). Induction of plasminogen activator synthesis by the anti-idiotypic antibodies could be inhibited by coincubation with a vasopressin antagonist. In a fashion similar to that of vasopressin itself, the anti-idiotypic antibodies induced receptor down-regulation. The anti-idiotypic antibodies were employed to visualize vasopressin receptors on LLC-PK1 and A7r5 (V1-receptor-expressing) smooth muscle cells by immunofluorescence. Antibody-mediated fluorescence was not observed in receptor-deficient mutant cell lines or vasopressin-receptor-down-regulated cells. Furthermore, these antibodies were used for immunohistochemical localization of vasopressin receptors in rat and bovine kidney preparations. In accordance with earlier physiological and biochemical observations, vasopressin receptors were detected predominantly in collecting ducts in cortex and medulla. On the cellular level, a differential staining pattern was observed.

Animals↗

Effects of agonists on p21ras and ras-related proteins in rat pancreatic acinar cells.

This study shows the presence of seven different low-molecular-weight GTP binding proteins (smg proteins) with molecular masses between 18 and 27 kDa in subfractions of rat pancreatic acinar cells. After stimulation of isolated intact and permeabilized pancreatic acinar cells with cholecystokinin octapeptide (CCK-OP), the diacylglycerol (DG) analogue 12-O-tetradecanoylphorbol 13-acetate (TPA), vasoactive intestinal peptide (VIP), adenosine 3',5'-cyclic monophosphate (cAMP), or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), [alpha-32P]GTP binding to 21- to 22-kDa smg protein(s) in microsomal membranes (MM) was reduced, whereas the [alpha-32P]GTP binding to 23-kDa protein(s) was enhanced. In addition, prestimulation of permeabilized cells with GTP gamma S caused enhancement of [alpha-32P]GTP binding to a 19-kDa protein in MM [immunologically identified as the ADP-ribosylation factor (arf)]. In the presence of cytosol, direct addition of GTP gamma S to isolated MM resulted in an apparent translocation of the 19-kDa protein (arf) from the cytosol to membranes. This indicates increased association of arf with the membrane in its GTP-bound state. In CCK-OP-prestimulated acinar cells, [alpha-32P]GTP binding to plasma membrane-located 21- to 22-kDa proteins (immunologically identified as p21ras proteins) was enhanced, suggesting that there is an interrelationship between p21ras proteins and CCK receptors. Our results give evidence for a role of 19-kDa, 21- to 22-kDa, and 23-kDa smg proteins in cAMP-protein kinase A- and DG-protein kinase C-mediated stimulation of intracellular pathways in pancreatic acinar cells.

Animals↗

[Therapy of stomach ulcer--a comparison between the low dosage antacid hydrotalcite and ranitidine--results of a randomized multicenter double-blind study. Talcivent Study Group].

In a 8 week double-blind randomized multicenter trial in 159 patients with benign gastric ulcer the efficacy of hydrotalcite vs. ranitidine in expediting ulcer healing and in achieving pain relief was determined. 79 patients received hydrotalcite 1000 mg q.i.d. as tablets equalling a total neutralizing capacity of 111.2 mval and 80 patients received ranitidine (300 mg at night). Endoscopically controlled healing rates after 4 weeks of therapy amounted to 41.8% with hydrotalcite and 53.8% with ranitidine. After 8 weeks both regimen showed significant equivalent healing rates (hydrotalcite: 81.0%, ranitidine: 78.8%, p < 0.003). Ulcer pain decreased parallel in both groups. By the end of therapy 92.4% of the patients treated with hydrotalcite and 86.3% of those receiving ranitidine were free of pain. Incidence of helicobacter pylori in antral mucosal biopsies was not influenced by both treatments. We conclude that an 8-week treatment with low dose hydrotalcite therapy is as effective as ranitidine in healing benign gastric ulcers and achieving pain relief.

Adult↗

Problems in performing a double-blind multicenter study using a hypnotic in private practice.

1. The procedure described--the immediate data transfer and entry system (ITES)--is suitable to improve the quality of data collected in multicentre studies in private practice. 2. The results of the study show that regarding influence on sleep quality and daytime well-being the non-benzodiazepine Z is significantly superior to placebo and slightly superior to the benzodiazepines F and T. 3. The advantage of Z treatment is the better daytime well-being after taking the hypnotic for sleep induction the night before. 4. This study shows that a quality standard equal to that in clinical practice may be achieved in private practice.

Adolescent↗

Molecular cloning of gp 80, a glycoprotein complex secreted by kidney cells in vitro and in vivo. A link to the reproductive system and to the complement cascade.

cDNA clones coding for the gp 80 heterodimeric glycoprotein complex secreted constitutively at the apical surface of Madin-Darby canine kidney (MDCK) cells have been isolated from MDCK cDNA libraries in lambda gt11 and lambda gt10. The cloned sequences encode a polypeptide chain of 445 amino acids. The deduced amino acid sequence of the gp 80 protein reveals 80% homology to rat SGP-2, a major secretory protein of the testes epithelium and 83% homology to SP-40,40, a human complement-associated protein. SGP-2 and SP-40,40 have been proposed to be serum and seminal forms of the same protein. The sequence homology as well as the results of Southern and Northern blot analyses and immunological studies suggest that gp 80 is the canine homolog of the rat SGP-2 and the human SP-40,40. The protein is expressed in the embryonic kidney already early during organogenesis. In the adult kidney the protein has been localized along the luminal surfaces of the proximal and distal tubule and the collecting duct cells.

Amino Acid Sequence↗

Biosynthesis and secretion of an osteopontin-related 20-kDa polypeptide in the Madin-Darby canine kidney cell line.

We describe a 20-kDa phosphorylated polypeptide, which is secreted constitutively at the apical surface of the kidney-derived Madin-Darby canine kidney cell line. Using polyclonal antibodies raised against this protein, we show that it is generated from a 60-kDa O-glycosylated, sulfated, and phosphorylated precursor protein by an intracellular proteolytic maturation step, which is pH-sensitive. Amino acid sequence analysis of the 20-kDa secreted polypeptide demonstrated that it displays 70% identity with the carboxyl-terminal amino acids of human osteopontin. The amino-terminal amino acid of the 20-kDa polypeptide corresponds to amino acid 213 of human osteopontin. Thrombin has been shown to cleave rat osteopontin in vivo and in vitro at amino acid 153, yielding two fragments of 28 and 26 kDa. A similar cleavage product can be detected by thrombin treatment of the 60-kDa precursor, suggesting that the precursor is identical or closely related to osteopontin. In the rat nephron, the protein has been localized along the luminal surfaces of the proximal and distal tubule and the collecting duct cells. These results show that in the kidney-derived cell line Madin-Darby canine kidney osteopontin or a closely related protein is proteolytically processed to a 20-kDa polypeptide, raising the possibility that diverse functions of osteopontin in various tissues might be attributed to specific processing to distinct polypeptides.

Amino Acid Sequence↗

Immunolocalization of 15-kDa membrane proteins in the kidneys of normal and acidotic rats.

Proteins with apparent molecular masses between 15 kDa and 17 kDa were enriched from rat renal brush-border membranes by preparative gel electrophoresis and used for immunization of rabbits. The serum of one of the rabbits reacted in Western blots of separated renal brush-border proteins with a single 15-kDa band. A comparably strong reaction is seen with a 15-kDa band of renal endosomal proteins. Basolateral membranes show a much weaker reaction. In light- and electron-microscopic studies the serum stains brush-border membranes and endosomes in rat proximal tubule cells, but not mitochondria and basolateral membranes. In cortical collecting ducts, principal cells are not stained with the antiserum. alpha-type (H(+)-secreting) intercalated cells bind the antibodies at apical tubulovesicles. The luminal membrane is scarcely labelled. Conversely, beta-type (HCO3(-)-secreting) intercalated cells exhibit antibody binding to their basolateral membrane. Thus, the antiserum detects 15-kDa proteins differently sorted in alpha- and beta-intercalated cells. After induction of an acute (6 h) metabolic acidosis, the antibody-binding pattern changes only in intercalated cells, type alpha, and occurs at the markedly enlarged luminal plasma membrane. The amount of alpha-type intercalated cells with enlarged luminal membrane ("secreting cell") increases at the expense of alpha cells with apical tubulovesicles ("resting cell"). Taken together, the antiserum detects 15-kDa proteins, the localization and adaptive changes to metabolic acidosis of which are similar to H(+)-ATPases. The functional role of the 15-kDa proteins needs to be established in further studies.

Acidosis↗