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Biomedical subjects

K Bauer

Publications and source records attributed to K Bauer.

At least 55 records · Page 3Linked to original sources
PubMed2000

Analysis of the thyrotropin-releasing hormone-degrading ectoenzyme by site-directed mutagenesis of cysteine residues. Cys68 is involved in disulfide-linked dimerization.

Thyrotropin-releasing hormone-degrading ectoenzyme is a member of the M1 family of Zn-dependent aminopeptidases and catalyzes the degradation of thyrotropin-releasing hormone (TRH; Glp-His-Pro-NH2). Cloning of the cDNA of this enzyme and biochemical studies revealed that the large extracellular domain of the enzyme with the catalytically active site contains nine cysteine residues that are highly conserved among species. To investigate the functional role of these cysteines in TRH-DE we used a site-directed mutagenesis approach and replaced individually each cysteine by a serine residue. The results revealed that the proteolytically truncated and enzymatically fully active enzyme consists of two identical subunits that are associated noncovalently by protein-protein interactions but not via interchain S-S bridges. The eight cysteines contained within this region are all important for the structure of the individual subunit and the enzymatic activity, which is dramatically reduced in all mutant enzymes. This is even true for the four cysteines that are clustered within the C-terminal domain remote from the Zn-binding consensus sequence HEICH. In contrast, Cys68, which resides within the stalk region seven residues from the end of the hydrophobic membrane-spanning domain, can be replaced by serine without a significant change in the enzymatic activity. Interestingly, this residue is involved in the formation of an interchain disulfide bridge. Covalent dimerization of the subunits, however, does not seem to be essential for efficient biosynthesis, enzymatic activity and trafficking to the cell surface.

Aminopeptidases↗
PubMed2000

Molecular characterization of a puromycin-insensitive leucyl-specific aminopeptidase, PILS-AP.

The family M1 of Zn-dependent aminopeptidases comprises members of closely related enzymes which are known to be involved in a variety of physiologically important processes. On the basis of two highly conserved peptide motifs, we have identified a new member of this family by PCR amplification and cDNA-library screening. The longest ORF encodes a protein of 930 residues. It contains the HEXXH(X)18E Zn-binding motif and displays high homology to the other M1 family members except for its N-terminus for which a signal sequence of 20 residues can be predicted. This interpretation was supported by expressing fusion proteins formed with green fluorescent protein which localized to intracellular vesicles in COS-7 and BHK cells. Northern-blot analysis revealed ubiquitous expression of a major 3. 1-kb transcript. For enzymatic studies, the complete protein was expressed in Sf 9 insect cells. When aminoacyl beta-naphthylamides were used as substrates, efficient hydrolysis was only observed for Leu (and to a lesser extent Met). The activity was inhibited by chelators of bivalent cations and by other known aminopeptidase inhibitors, but surprisingly puromycin was without effect. This newly identified puromycin-insensitive leucyl-specific aminopeptidase is a signal-sequence-bearing member of family M1 and may be another example of the small subset of substrate-specific peptidases.

Amino Acid Motifs↗
PubMed2000

Elevation of plasma prolactin in patients undergoing autologous blood stem-cell transplantation for breast cancer: is its modulation a step toward posttransplant immunotherapy?

Prolactin is a suspected promotor of breast cancer cell growth, and it shares pleiotropic immunoregulatory properties. We studied plasma prolactin and its drug-induced modulation in 20 women with breast cancer undergoing high-dose chemotherapy and autologous blood stem-cell transplantation. Plasma prolactin levels were serially assayed before and during conditioning and within and beyond 30 days after transplant. Before transplant, prolactin plasma levels were in the age-adjusted range of normal women. During conditioning and within 30 days after transplant, prolactin levels increased in all patients (p < 0.0001), but remained in the normal range. Antiemetic drugs such as metoclopramide and phenothiazines, known to enhance pituitary prolactin secretion, further elevated prolactin plasma levels (p < 0.00001). Patients remaining in continuous complete remission after transplant (median follow-up, 3 years) disclosed higher prolactin levels compared with those obtaining only partial remission or ensuing early relapse. Prolactin levels are regularly elevated during conditioning and within 30 days after autologous transplantation for breast cancer. Further elevations of prolactin plasma levels are induced by metoclopramide and other antiemetic drugs. Elevated plasma prolactin had no adverse effect on disease-free survival after transplant. We propose to investigate further the upregulation of prolactin after transplant aiming to induce a posttransplant consolidative immune reaction.

Adult↗
PubMed2000

Postnatal changes of extracellular volume, atrial natriuretic factor, and diuresis in a randomized controlled trial of high-frequency oscillatory ventilation versus intermittent positive-pressure ventilation in premature infants <30 weeks gestation.

OBJECTIVES: High-frequency oscillatory ventilation (HFOV) with a high lung volume strategy is an experimental mode of ventilating preterm infants aimed at achieving maximal alveolar recruitment Higher mean airway pressures are used during HFOV than during intermittent positive-pressure ventilation (IPPV), and the intrathoracic volume increase is relatively constant. Both factors increase the risk to depress organ blood flow and diuresis. Our objective was to test the hypothesis that high lung volume HFOV attenuates the postnatal reduction of extracellular volume in preterm infants by reducing plasma atrial natriuretic factor and diuresis. DESIGN: Prospective, randomized, controlled clinical trial. SETTING: University hospital, Level III neonatal intensive care unit. PATIENTS: Premature infants <30 wks gestation requiring intubation for respiratory distress syndrome within the first 6 hrs of life; 15 infants (gestational age, 26 [24-29] wks, birth weight 814 [452-1340] g) were randomized to HFOV, 19 infants (gestational age 27 [24-39] wks, birth weight 930 [644-1490] g) to IPPV. INTERVENTIONS: The randomized mode of ventilation was assigned within 1 hr after intubation. During HFOV mean airway pressure was increased as long as oxygenation improved and no lung overinflation was seen on chest radiograph. IPPV rates were > or =60/min. MEASUREMENTS AND MAIN RESULTS: We measured extracellular volume (sucrose dilution) and atrial natriuretic factor on Day 1 and Day 3. Mean airway pressure, body weight, diuresis, and fluid intake were measured daily. During HFOV mean airway pressure was higher at 12 hrs (median 7 cm H2O vs. 4 cm H2O; p = .001) and 24 hrs (median 6 cm H2O vs. 3 cm H2O; p = .01). In both groups, extracellular volume decreased between Day 1 and Day 3 (HFOV from 428 +/- 126 mL to 344 +/- 145 mL [p = .003], IPPV from 466 +/- 108 mL to 414 +/- 124 mL [p = .01]) and diuresis increased (HFOV, from 2.5 +/- 1.7 to 4.6 +/- 0.9 mL/kg/hr [p = .001]; IPPV, from 2.8 +/- 1.6 to 4.2 +/- 1.0 mL/kg/hr [p = .01]). Plasma atrial natriuretic factor was not decreased in the HFOV group. CONCLUSIONS: High lung volume HFOV as primary mode of ventilation in preterm infants <30 wks gestation did not result in unwanted fluid retention and a decrease in diuresis in the first days of life.

Atrial Natriuretic Factor↗
PubMed2000

Prolactin-releasing peptides do not stimulate prolactin release in vivo.

The prolactin (PRL)-releasing activity of the novel prolactin-releasing peptides (PrRPs) was studied in vivo using male and lactating female rats. Whereas thyrotropin-releasing hormone effectively stimulated PRL and thyrotropin release as expected, PrRP in both animal models neither stimulated PRL secretion nor affected the release of other pituitary hormones. At the anterior pituitary level, in situ hybridization (ISH) histochemistry and Northern blot analysis revealed significantly higher expression levels of PrRP receptor (UHR-1) transcripts in female compared to male rats but not between lactating and nonlactating animals. By ISH, expression of UHR-1 mRNA was also detected in the intermediate lobe but not in the posterior pituitary. UHR-1 transcripts were also readily detectable in various hypothalamic brain areas whereas expression of PrRP mRNA was restricted to the ventral part of the dorsomedial hypothalamic nucleus but was not detected in neuroendocrine hypothalamic nuclei (e.g. PVN, SON). We thus assume that in the central nervous system, PrRP may likely have functions as a neuromodulator. However, together with the detailed cytochemical studies of various investigators that failed to detect PrRP-immunopositive nerve endings in the median eminence, our results strongly suggest that the hypothalamic PrRPs cannot be classified as hypophysiotrophic factors.

Animals↗
PubMed2000

Leukocyte and endothelial activation in a laboratory model of extracorporeal membrane oxygenation (ECMO).

An inflammatory response and a capillary leak syndrome frequently develop during the treatment of neonatal respiratory failure by extracorporeal membrane oxygenation (ECMO). The present study was performed to investigate leukocyte activation and endothelial cell dysfunction that are associated with prolonged contact of blood components with synthetic surfaces. Laboratory ECMO was performed with fresh human blood at 37 degrees C for 8 h (n = 6). Leukocyte activation was measured by L-selectin (CD62L) and CD18 integrin surface expression and by neutrophil-derived elastase release. To monitor endothelial activation, endothelial cell ICAM-1 (CD54) expression was measured in cultured endothelial cells from human umbilical veins (HUVEC) after incubation with plasma from the ECMO experiments. CD18 integrin expression was found significantly up-regulated on polymorphonuclear neutrophils and monocytes after 2-4 h of laboratory ECMO. L-selectin was reduced on both cell types during the total duration of the experiments. Soluble L-selectin (sCD62L) and total and differential leukocyte counts remained unchanged during the experiment. Neutrophil-derived elastase content was maximal after 8 h of ECMO. Plasma from the ECMO experiments did not induce ICAM-1 expression of cultured HUVEC. We conclude that prolonged contact with synthetic surfaces during ECMO activates phagocytes, which may contribute to the inflammatory response seen in ECMO-treated patients. Activated phagocytes do not accumulate in the extracorporeal system nor release humoral factors inducing ICAM-1 expression on endothelial cells.

CD18 Antigens↗
PubMed2000

Analysis of thyrotropin-releasing hormone-signaling components in pituitary adenomas of patients with acromegaly.

In many acromegalic patients the paradoxical release of GH in response to TRH has been well documented, but the mechanisms underlying this phenomenon are not understood. It has been suggested that aberrant GH secretion may result from TRH endogenously synthesized by the adenoma. In 32 adenomas from acromegalic patients, TRH-like immunoreactivity (TRH-LI) was measured using 2 well characterized antisera. TRH-LI was not detectable in 10 samples, and in 19 samples, TRH-LI was measured only by the less specific antibody. With the TRH-specific antibody, TRH-LI was identified only in 3 samples, 2 of which contained exceedingly high concentrations (40 and 96 pg/mg tissue). In the latter 2 samples, prepro-TRH messenger ribonucleic acid was identified by Northern blot analysis, but not in the control tissue sample of a patient without pituitary disease and also not in the other adenomas analyzed by this technique. Transcripts of the TRH receptor were almost undetectable in all adenomas analyzed. For the TRH-degrading ectoenzyme, a potential regulator of TRH signals at adenohypophyseal target sites, transcripts were significantly expressed only in the TRH-producing adenomas. We conclude that the TRH-signaling elements examined are, in general, not directly involved in the mechanisms causing paradoxical GH secretion in acromegalic patients.

Acromegaly↗
PubMed2000

Serum protein 90K/Mac-2BP is an independent predictor of disease severity during hepatitis C virus infection.

The serum protein designated 90K/Mac-2BP has been found at elevated concentrations in the sera of patients with various types of cancer and viral infections. The importance of the 90K/Mac-2BP serum concentrations in predicting the response towards interferon-alpha treatment for hepatitis C virus (HCV) infection prompted us to utilize a new ELISA for soluble human 90K/Mac-2BP to monitor the serum concentrations of this protein in our HCV-positive patients. Seventy HCV-PCR and anti-HCV antibody positive patients were analyzed for their serum levels of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, cholinesterase, HCV-viral load, viral subtypes, and 90K/Mac-2BP. On correlation of age and 90K/Mac-2BP levels, we found an apparent correlation that was proved rather to be a strong dependence of 90K/Mac-2BP concentrations on disease severity/duration, which increases with age. Multiple correlation analysis demonstrated the independent nature of 90K/Mac-2BP concentrations, underscoring the potential high utility of this new marker. Our data corroborate the potential of the scavenger receptor family protein 90K/Mac-2BP as an independent predictor of disease severity during HCV infection.

Adolescent↗
PubMed2000

Effects of proinflammatory cytokines on anterior pituitary 5'-deiodinase type I and type II.

Cytokines are locally produced in the anterior pituitary and act through para-/autocrine mechanisms to modulate cell growth and hormone production. 5'-Deiodinases type I (D1) and type II (D2) are also expressed in the anterior pituitary and play an integrative role in the regulation of hormone production and pituitary feedback. D1 activity is known to be regulated by proinflammatory cytokines in liver and thyroid. Therefore, we examined the effects of IL-1 beta, IL-6 and TNF alpha on 5'-deiodinase activities in reaggregates of rat anterior pituitaries and rat somatomammotroph GH3 cells cultured alone, or in a bicameral culture system together with the murine folliculo-stellate (FS) cell line TtT/Gf. In reaggregate cultures of rat anterior pituitaries IL-1 beta stimulated D1 and D2 dose-dependently and D2 activity was increased by TNF alpha. When GH3 cells were cocultured with TtT/Gf cells, D2 activities were 2.3-fold lower than in GH3 cells cultured alone. TNF alpha (50 ng/ml) and IL-6 (100 U/ml) stimulated D2 in GH3 cells when the cells were cultured alone and treated with these cytokines for 24 h. When TtT/Gf cells in the coculture model were treated with IL-1 beta, TNF alpha and IL-6, no effect on D1 or D2 activities in GH3 cells was observed. In male, adult rats a single LPS injection (i.p.) stimulated D2 and D1 activities in the anterior pituitary, and decreased liver D1 activities and serum TSH levels. In vitro, LPS stimulation of the coculture model of GH3 and FS cells also increased D1 activity. Electrophoretic mobility shift assays (EMSAs) revealed that IL-1 beta and TNF alpha activate the transcription factor NF kappa B in reaggregates of rat anterior pituitaries and in TtT/Gf cells cultured alone or cocultured with GH3 cells. Taken together, these findings imply that in anterior pituitary cells 5'-deiodinase activities are stimulated by locally produced cytokines in a para-/autocrine manner but cell types other than FS cells seem to mediate some of the effects.

Animals↗
PubMed2000

Modulation of clinical drug resistance in a B cell lymphoma patient by the protein kinase inhibitor 7-hydroxystaurosporine: presentation of a novel therapeutic paradigm.

Emerging evidence suggests that apoptosis is an important mechanism of tumor cell death from antineoplastic therapy. 7-hydroxystaurosporine (UCN-01) is a novel protein kinase inhibitor that increases chemotherapy-induced apoptosis in vitro and is in early phases of clinical development. In this report, we present a 68-year-old patient with chemotherapy-resistant lymphoma treated with UCN-01 and chemotherapy. He had a stage IV plasmacytoid lymphoma that failed to enter remission with high-dose EPOCH II (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin) chemotherapy. Due to disease progression and transformation to large cell lymphoma in the liver and bone marrow, he received UCN-01. Four weeks later, he received "standard-dose" EPOCH because of progression, developed severe neutropenia for 9 days, and expired from Candida sepsis on day 23. At autopsy, there was no histological evidence of residual lymphoma, although PCR for immunoglobulin gene rearrangement analysis revealed a faint clonal band in two of six nodes but none in the liver. Significantly, no B cells were detected by immunohistochemistry in lymph nodes, and a polyclonal ladder pattern associated with the presence of normal B cells was not seen in the immunoglobulin gene rearrangement PCR assay. Profound peripheral lymphopenia (50 cells/microliter) was also observed. Pharmacokinetics showed UCN-01 salivary concentrations, a surrogate for free drug concentrations, to be within an effective range in vitro (45 nmol/L) as a modulator of DNA-damaging agent cytotoxicity. In vitro, UCN-01 is synergistic with multiple cytotoxic agents and increases fludarabine-induced apoptosis in a human breast cell line. These results suggest that UCN-01 sensitized the lymphoma to the cytotoxic effects of EPOCH, possibly by modulating the "threshold" for apoptosis, and may illustrate a new paradigm for reversal of drug resistance.

Aged↗
PubMed2000

Biosynthesis, release, and uptake of carnosine in primary cultures.

Biosynthesis, release, and uptake of carnosine (beta-alanyl-L-histidine) in highly enriched primary cell cultures of skeletal muscle and CNS tissue have been investigated. The synthesis is restricted to muscle cells, oligodendrocytes, and ensheathing cells of olfactory bulb and increases during differentiation of these cells. Astrocytes, in contrast, do not synthesize carnosine but are equipped with a dipeptide transporter by which carnosine is taken up very efficiently.

Animals↗
PubMed2000

[Laboratory diagnosis of hepatitis C virus infection].

In Austria, the prevalence of hepatitis C virus infections is 0.7% (17). Exclusion of a putative infection as well as diagnosis and continuous monitoring of HCV-disease produce considerable costs for the health system. How many and which patients with HCV infection will acquire life-threatening complications is by far not clear. Also, the causes for viral persistence and liver-complications remain obscure. For certain, complex interactions of viral and immunological mechanisms will determine the individual outcome of the disease (1). These considerations pose decisive demands on clinical diagnostics for HCV infections to be dealt with in detail: methods for qualitative detection of an infection as well as for analysis of subtypes and for quantitative determination of viral copies; monitoring of therapy; estimation of the progress of the disease and/or efficacy of therapy.

Antiviral Agents↗
PubMed2000

[Serum soluble CD44 isoform variant 5 level in patients with seropositive rheumatoid arthritis treated with cyclosporin A].

CD44 is a widely expressed cell surface glycoprotein which is involved in many cell-cell and cell-matrix interactions. Expression of soluble CD44 splice variants is strictly regulated and is linked to a high rate of cell division. Serum levels of soluble CD44 variant 5 (sCD44v5) were determined in 14 patients with erosive RA. Patients were divided into two groups. In group 1 cyclosporin A treatment (CYA) was initiated after the first visit. In group 2 preliminary CYA was continued. Controls were performed after 6 months. We found a significant decrease of swollen joint count (SJC) and sCD44v5 in group 1. No effect of CYA was found on c-reactive protein, erythrocyte sedimentation rate and IgM-rheumatoid factor (IgM-RF). In group 2 a significant decrease of CRP was found. Therefore we conclude that measurement of sCD44v5 might be useful in monitoring RA+ patients with CYA.

Adult↗
PubMed1999

Development of a continuous, fluorometric coupled enzyme assay for thyrotropin-releasing hormone-degrading ectoenzyme.

Thyrotropin-releasing hormone degrading-ectoenzyme (TRH-DE) (EC 3.4. 19.6), removes the N-terminal pyroglutamyl residue of thyrotropin-releasing hormone (TRH). Discontinuous assays have been used to measure TRH-DE activity; however, a continuous assay is needed to make reliable measurements of initial rates and facilitate kinetic studies. Presented is a continuous, coupled enzyme assay for TRH-DE in which TRH-DE hydrolyzed the substrate, pyroglutamyl-histidyl-prolylamido-4-methyl coumarin (TRHMCA), to give His-ProMCA, which was then cleaved by dipeptidyl peptidase IV (EC 3.4.14.5) to give 7-amino-4-methyl coumarin (MCA). Reaction progress was monitored continuously by measuring the increase in MCA fluorescence. This assay should be especially useful for rapid screening of potential TRH-DE inhibitors. A previously reported discontinuous assay, where nonenzymatic cyclization at 80 degrees C was used to liberate MCA from His-ProMCA, was found to underestimate the amount of product formed. A modified procedure that avoids this is presented. Initial rates and kinetic parameters for TRHMCA hydrolysis by TRH-DE determined using this modified assay correspond with those determined by the continuous assay. Discontinuous and continuous assays gave K(m) values for TRHMCA of 3.4 +/- 0.7 microM (n = 5) and 3.8 +/- 0.5 microM (n = 5), respectively. K(i) values determined by the discontinuous assay for TRH and TRH-OH were 35 +/- 4 microM (n = 3) and 311 +/- 31 microM (n = 5), respectively.

Aminopeptidases↗
PubMed1999

Human TRH-degrading ectoenzyme cDNA cloning, functional expression, genomic structure and chromosomal assignment.

Thyrotropin-Releasing Hormone (TRH) is an important extracellular signal substance that acts as a stimulator of hormone secretion from adenohypophyseal target cells and fulfills many criteria for the function of a neuromodulator/neurotransmitter within the central and peripheral nervous systems. The inactivation of TRH-signals is catalysed by a highly specific ectoenzyme. Here, we characterize the human TRH-degrading ectoenzyme (TRH-DE) by primary sequence, functional expression, genomic structure and chromosomal assignment. By screening a cDNA-library constructed from human lung, 5.7 kb of cDNA were identified. The longest open reading frame predicts a type II integral membrane protein of 117 kDa. The extracellular domain contains the HEXXH + E motif that is characteristic of a certain family of Zn-dependent aminopeptidases. Within this family, the sequences of human and rat TRH-DE reveal an unusual high degree of conservation (96% identical residues). Specific enzymatic activity was observed after transfecting COS-7 cells with human TRH-DE cDNA yielding a Km for TRH hydrolysis of 29.7 microM. Northern blot analysis demonstrated a restricted tissue distribution with highest transcript levels in the brain. Using fluorescent in situ hybridization with the cDNA and a genomic lambda clone, respectively, we localized the TRH-DE gene to the long arm of human chromosome 12. Five independent P1 artificial chromosome clones were required to span the complete cDNA sequence and revealed that it is distributed on 19 exons. Interspecies Southern analysis suggests that the gene is present as a single copy in human, monkey, rat, mouse, dog, bovine, rabbit and chicken DNA. All of these data further the notion that the TRH-DE is not an ordinary enzyme but a specific neuropeptidase that has been highly conserved among species.

Amino Acid Sequence↗