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U Klein

Publications and source records attributed to U Klein.

At least 73 records · Page 4Linked to original sources

The key role of the H+ V-ATPase in acid-base balance and Na+ transport processes in frog skin.

Frogs are faced with various osmoregulatory problems, such as compensation of salt and water loss or metabolic acidification. Being exposed both to air and to pond water of low salinity in their natural habitat, the epithelium of the frog skin serves as one of the major organs for body fluid homeostasis. For years, the frog skin has been the guiding model for ion transport processes in animal cells energized by a Na(+)-motive force. Meanwhile, however, it was demonstrated that under natural conditions Na+ uptake is electrically coupled to active H+ secretion, mediated by an electrogenic H+ pump. A proton-motive force generated at the apical membrane of the mitochondria-rich cells (MR cells) energizes Na+ entry via apical Na+ channels. The basolateral Na+/K+ P-ATPase then pumps Na+ out of the cell into the body fluid. Thus, there are two pumps functioning in series, both involved in transepithelial Na+ transport. Our recent investigations provided conclusive evidence that the H+ pump of the frog skin is an H+ V-ATPase. In transport studies, Na+ absorption and H+ secretion were blocked by micromolar concentrations of bafilomycin A1 or concanamycin A, two highly specific inhibitors of H(+)-V-ATPases. Using immunofluorescence microscopy, H(+)-V-ATPase-like immunoreactivity was found in MR cells in the region of their apical membrane foldings and intracellularly in the apical portion of the cell at so far unidentified locations. Besides the definition of its molecular nature, these results also confirmed the localization of the H+ pump in the apical membrane of the MR cells. These cells were already candidates for H(+)-V-ATPase localization mostly from correlations between their morphological features and their epithelial H+ secretion capacity. So far, there is evidence for only one type of MR cell serving both H+ and HCO3- secretion through an apical Cl-/HCO3- antiporter. H(+)-V-ATPase-mediated H+ secretion and thus Na+ absorption can be modulated by complementary mechanisms. Changes in intracellular H+ concentration linked to the animal's acid-base status will directly influence H+ V-ATPase activity. Acute acidification increases H+ current, probably as a result of the insertion of H(+)-V-ATPase-bearing vesicles by exocytotic processes, while alkalization causes the reverse effects. Chronic metabolic acidosis induces an increase in MR cell number in response to hormonal signals.

Acid-Base Equilibrium↗

Reduction of ischemia-reperfusion syndrome after abdominal aortic aneurysmectomy by N-acetylcysteine but not mannitol.

BACKGROUND: Abdominal aortic aneurysmectomy results in a general ischemia-reperfusion syndrome accompanied by an acute rise in mean pulmonary artery pressure (MPAP). Severe and sometimes fatal postoperative cardiopulmonary complications have been described. METHODS: This pilot study examined whether N-acetyl-cysteine (NAC), a precursor of the most important physiological antioxidant glutathione (reduced form: GSH; oxidized form: GSSG), or the hydroxyl radical scavenger mannitol (MAN) modifies these events. The patients received 150 mg/ kg b.m.NAC (n = 9) 30 minutes before infrarenal aortic clamping or 500 mg/kg b.m. MAN (n = 10) 10 minutes before declamping. 11 patients had no additional treatment (control). RESULTS: In the control group, a significant increase in plasma levels of oxidized glutathione and lipid peroxides was observed after declamping. Additionally, a significant increase in plasma levels of the stable metabolites of thromboxane (TXB2) and prostacyclin (6-keto-PGF1 alpha) was measureable after declamping. There was a transient increase in MPAP and pulmonary vascular resistance (PVR), both of which returned to normal values within 20 minutes. Six hours after surgery, pulmonary dysfunction was manifest by increase in the intrapulmonary shunt fraction. Relative to the control group, NAC pretreatment led to a complete lack of changes in plasma lipid peroxide, thromboxane and prostacyclin levels after declamping; there was a significant increase in plasma GSH concentration persisting over a period of 12 hours. MPAP, PVR and Qs/QT values were unchanged. MAN pretreatment showed similar effects on the parameters obtained in the acute phase after declamping like the control group. CONCLUSIONS: Pretreatment with NAC, but not mannitol, may help prevent ischemia-reperfusion syndrome following aortic clamping.

Acetylcysteine↗

Temporal and spatial distribution of V-ATPase and its mRNA in the midgut of moulting Manduca sexta.

The spatial and temporal distribution of the plasma membrane V-ATPase and its encoding mRNA in the midgut of Manduca sexta were investigated during the moult from the fourth to the fifth larval instar. Digoxigenin-labelled RNA probes were used for in situ hybridization of V-ATPase mRNA of both peripheral and integrated subunits; monoclonal antibodies to subunits of the peripheral sector of the purified plasma membrane V-ATPase were used for immunocytochemistry. Extensive mRNA labelling was found in both mature columnar and goblet cells of intermoult and moulting larvae. Hybridization screening in several tissues suggested that only cells with increased V-ATPase biosynthesis were labelled by our hybridization method. Mature goblet cells contain a large amount of V-ATPase in the apical plasma membrane and were therefore expected to contain V-ATPase mRNA. The intense mRNA signal found in mature columnar cells was unexpected. However, after refining the techniques of tissue preparation, immunolabelling in apical blebs of columnar cells was demonstrated. Since this immunoreactivity did not appear to be membrane-associated, it suggested a cytosolic localization of peripheral V1 subunits. The mRNA encoding subunit A of the peripheral V1 sector was distributed unevenly in columnar cells with a strong apical preference, whereas the mRNA for the proteolipid of the integral V0 sector was evenly distributed in the cytosol. This spatial pattern reflected the distribution of free ribosomes and rough endoplasmic reticulum in the cell, supporting the view that V1 subunits are synthesized at free ribosomes, whereas the V0 subunits are synthesized at the rough endoplasmic reticulum. All undifferentiated cells exhibited intense mRNA signals for V-ATPase subunits of both holoenzyme sectors from the start of proliferation and thus precursors of columnar and goblet cells could not be distinguished.

Animals↗

[Effect of rocuronium in sufentanil/isoflurane and sufentanil/propofol anesthesia].

It is a well-known fact that the duration of the effect of non-depolarizing neuromuscular blocking agents is influenced by other anaesthetics. Etomidate, propofol and nitrous oxide have no influence on the duration of effect of rocuronium, but a prolongation of the rocuronium effect under the influence of isoflurane has been described. In this study, we investigated the onset time, duration of effect and recovery index of rocuronium in isoflurane/N2O/sufentanil anaesthesia compared with these parameters in propofol/N2O/sufentanil anaesthesia. Forty patients (10 men and 30 women aged 47.5 +/- 13.0 years, ASA 1-2) were included in the investigation. Anaesthesia was induced by intravenous application of 20 mg etomidate, 0.15-0.2 micrograms/kg sufentanil and 0.9 mg/kg rocuronium. In 20 patients, anaesthesia was supplemented with 0.3-0.8 Vol.% (endexpiratory) isoflurane in a mixture of 30% O2 and 70% N2O (group A). In another 20 patients breathing 30% O2 and 70% N2O propofol (2-6 mg/kg/h) was infused continuously (group B). Onset time, duration of action and recovery index of rocuronium were measured with a nerve stimulator (train-of-four mode). The patients were intubated 60 seconds after completing the rocuronium injection and the intubation conditions were estimated clinically. The onset time amounted to 47.6 +/- 17.8 seconds (group A) and 58.1 +/- 13.6 seconds (group B). In group A, a mean duration of action of 54.3 +/- 13.8 minutes and in group B a mean duration of action of 53.9 +/- 18.9 minutes were measured. The recovery index amounted to 21.2 +/- 10.6 minutes in group A and 17.6 +/- 5.8 minutes in group B. The differences in onset time and recovery times were not significant. Significant changes in arterial blood pressure or heart rate were not observed. The intubation conditions after 60 seconds were excellent in 34 patients (85%) and good in 6 patients (15%). It can be concluded that in comparison with anesthesia maintained by propofol/sufentanil and nitrous oxide/oxygen, the relatively low but necessary supplementation with isoflurane instead of propofol does not lead to a clinically relevant amplification of the relaxing effect of rocuronium.

Adjuvants, Anesthesia↗

Alteration of the myometrial plasma membrane cholesterol content with beta-cyclodextrin modulates the binding affinity of the oxytocin receptor.

To investigate the effect of cholesterol on the oxytocin receptor function in myometrial membranes, we developed a new method to alter the membrane cholesterol content. Using a methyl-substituted beta-cyclodextrin, we were able to selectively deplete the myometrial plasma membrane of cholesterol. Vice versa, incubating cholesterol-depleted membranes with a preformed soluble cholesterol-methyl-beta-cyclodextrin complex restored the cholesterol content of the plasma membrane. Binding experiments showed that, with the removal of cholesterol from the membrane, the dissociation constant for [3H]oxytocin is enhanced 87-fold (from Kd = 1.5 nM to Kd = 131 nM), therefore shifting the oxytocin receptor from high to low affinity. Increasing the cholesterol content of the cholesterol-depleted membrane again restored the high-affinity binding (Kd = 1.2 nM). The presence of 0.1 mM GTP gamma S did not significantly change the number of high-affinity binding sites for [3H]oxytocin in native plasma membranes, in membranes depleted of cholesterol, and in plasma membranes with restored cholesterol content. The number of high-affinity binding sites for the oxytocin antagonist [3H]PrOTA was dependent in the same way on the cholesterol content as for [3H]oxytocin. Substitution of the membrane cholesterol with other steroids showed a strong dependence of the oxytocin receptor function on the structure of the cholesterol molecule. The detergent-solubilized oxytocin receptor was not saturable with [3H]oxytocin even at concentrations up to 10(-6) M of radioligand. Addition of the cholesterol-methyl-beta-cyclodextrin complex to the detergent-solubilized oxytocin receptor induced a saturation of the solubilized binding sites (Bmax = 0.98 pmol/mg) for oxytocin (Kd = 16 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Expression of the human oxytocin receptor in baculovirus-infected insect cells: high-affinity binding is induced by a cholesterol-cyclodextrin complex.

We have expressed a c-myc epitope-tagged human oxytocin receptor in the baculovirus/Sf9 cell system. The receptor was identified by SDS-PAGE and subsequent immunoblot as a approximately 50 kDa protein which decreased to about 44 kDa upon treatment with tunicamycin. Binding studies showed that the human oxytocin receptor was expressed in a low-affinity state (Kd = 215 nM; Bmax = 1.66 pmol/mg). After addition of cholesterol in the form of a soluble cholesterol-methyl-beta-cyclodextrin complex to the membranes, we obtained part of the human oxytocin receptor in its high-affinity state for oxytocin (Kd = 0.96 nM and Bmax = 318 fmol/mg of protein). In subsequent studies, we added the cholesterol-methyl-beta-cyclodextrin complex to the Sf9 cell culture medium at various times post infection. Binding analysis showed that this results in a more than 3-fold further increase in functional receptor binding sites of high-affinity state (Bmax = 1.08 pmol/mg). The cholesterol effect was dose-dependent, with an EC50 of about 50 microM cholesterol. Due to these findings, we determined the cholesterol and phospholipid content in purified Sf9 plasma membranes. The untreated naturally cholesterol auxotroph insect cells grown in medium with 2% fetal calf serum had a molar cholesterol/phospholipid ratio of about 0.04, which is approximately 20-fold lower than normally found in plasma membranes of higher eukaryotic cells. The high-affinity binding of the oxytocin receptor increased in parallel with the cholesterol levels present in the corresponding plasma membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Regulation of plasma membrane V-ATPase activity by dissociation of peripheral subunits.

The plasma membrane V-ATPase of Manduca sexta larval midgut is an electrogenic proton pump located in goblet cell apical membranes (GCAM); it energizes, by the voltage component of its proton motive force, an electrophoretic K+/nH+ antiport and thus K+ secretion (Wieczorek, H., Putzenlechner, M., Zeiske, W., and Klein, U. (1991) J. Biol Chem. 266, 15340-15347). Midgut transepithelial voltage, indicating net active K+ transport, was found to be more than 100 mV during intermoult stages but was abolished during moulting. Simultaneously, ATP hydrolysis and ATP-dependent proton transport in GCAM vesicles were found to be reduced to 10-15% of the intermoult level. Immunocytochemistry of midgut cryosections as well as SDS-polyacrylamide gel electrophoresis and immunoblots of GCAM demonstrated that loss of ATPase activity paralleled the disappearance of specific subunits. The subunits missing were those considered to compose the peripheral V1 sector, whereas the membrane integral V0 subunits remained in the GCAM of moulting larvae. The results provide, for the first time, evidence that a V-ATPase activity can be controlled in vivo by the loss of the peripheral V1 domain.

Adenosine Triphosphate↗

A new tritiated oxytocin receptor radioligand--synthesis and application for localization of central oxytocin receptors.

A new tritiated oxytocin antagonist radioligand was synthesized by introducing a tritiated propionic acid residue into the free amino group of ornithine in position 8 of the parent peptide [1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid), 2-(O-methyl)-tyrosine, 4-threonine, 8-ornithine, 9-tyrosylamide]vasotocin (OTA), that was previously described. The tritiated compound [3H][1-(beta-mercapto-beta,beta-cyclo-pentamethylene propionic acid), 2-(O-methyl)-tyrosine, 4-threonine, 8-(N6-propionyl)-ornithine, 9-tyrosylamide]vasotocin ([3H]PrOTA) was obtained in good yield with high specific activity (100 Ci/mmol). [3H]PrOTA exhibited the same affinity (Kd = 0.8 nM) and selectivity for the myometrial oxytocin receptor as the iodinated antagonist [125I]OTA. Autoradiographic localization of oxytocin receptors in the rat brain showed specific binding sites for [3H]PrOTA within regions of the limbic system, the neocortex, and hypothalamus, which is consistent with the binding pattern obtained with [125I]OTA. The high specific activity in combination with the long half-life of tritium and its low radiotoxicity as compared to iodine-125 makes the new tritiated antagonist a valuable tool for pharmacological studies.

Animals↗

[Capnography for bronchoscopy with rigid technique using high frequency jet ventilation (HFJV)].

OBJECTIVES AND METHODS: Rapid bronchoscopy in general anaesthesia still has its precise indications, where the high frequency jet ventilation technique offers several advantages. The monitoring of ventilation, however, has been rather unsatisfactory up to date. We therefore studied capnography in 60 bronchoscopies during HFJV (rate: 100/min; I:E = 0.33; driving pressure: 0.08-0.14 MPa) using a rigid bronchoscope with a distally located sampling port. Continuous capnograms were recorded. End-tidal partial pressures of carbon dioxide (petCO2), however, were obtained from 2-3 single breaths by intermittently reducing the jet-frequency to 10-12/min. After 6 min (MP1: whole group; n = 60) and 18 min of HFJV (MP2: n = 34 of this group) petCO2 values were regularly obtained and compared to pCO2 in synchronously drawn capillary blood samples (pcCO2). The jet driving pressure initially adjusted to body weight, however, was only corrected according to petCO2, aiming at 34 mmHg. RESULTS: During HFJV, sinusoidal capnograms permitted the identification of every single jet impulse. With instruments being passed through the bronchoscope, however, these curves were substantially distorted. Mean pcCO2 at MP1 (37.8 +/- 6.7 mmHg) and MP2 (37.2 +/- 6.7 mmHg) demonstrated normal ventilation to light hyperventilation. Differences from mean petCO2 obtained during low frequency breathing were 3.3 mmHg at MP1 and 4.4 mmHg at MP2 (p < 0.05). There were strong correlations between the individual pairs of pcCO2 and petCO2 from MP1 (r = 0.80) and MP2 (r = 0.75) as well as between the pairs of dpcCO2 and dpetCO2 from both MPs (r = 0.77). The accuracy of the ventilator setting according to petCO2 with reference to pcCO2 was 73% for MP1 and 74% for MP2 (sensitivity: 75%/79%; specificity: 72%/67%). CONCLUSIONS: Capnography in rigid bronchoscopy during HFJV proved a clinically applicable addition to monitoring. Its routine use is strongly recommended in interventional bronchoscopy. The true petCO2 values obtained by intermittent single low frequency jet breathing permit estimates of gas exchange sufficiently exact for clinical purposes and for adjustment of the ventilator setting. Wave forms of the continuously recorded capnogram during HFJV are a warning of impeded ventilation or airway obstruction and, thus, of the danger of barotrauma or hypoventilation. Besides contributing to patient safety, this monitoring method might improve the acceptance of HFJV for bronchoscopy. Furthermore, it can also be applied to rigid bronchoscopy with common ventilation.

Adult↗

Tissue- and subtype-specific modulation of angiotensin II receptors by chronic treatment with cyclosporin A, angiotensin-converting enzyme inhibitors and AT1 antagonists.

We wished to determine whether chronic treatment of rats with cyclosporin A (CSA), an angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor antagonists modulates the angiotensin receptor density. In rats treated chronically with CSA, the vasoconstrictor response to angiotensin II (AII) is increased and this increase is modulated by ACEI and angiotensin receptor antagonists. Rats were treated for 6 weeks orally with CSA (15 mg/kg/day), the ACE inhibitor lisinopril (10 mg/kg/day), the angiotensin receptor antagonists DUP 753 (10 mg/kg/day), and D 8731 (10 mg/kg/day) and the combinations CSA + lisinopril, CSA + DUP 753, and CSA + D 8731. Olive oil was used as a control. The number of total AII receptors (Bmax) was determined by Scatchard analysis of [125I]Sar1 Ile8 AII binding in kidney, liver, adrenal cortex, and adrenal medulla. The receptor subtypes were analyzed with the specific antagonists DUP 753 (subtype 1) and PD 123319 (subtype 2). CSA upregulated angiotensin receptors in all organs studied. Lisinopril alone downregulated angiotensin receptors and abolished the effect of CSA in liver and adrenal cortex and medulla, but not in the kidney, where it had no effect. DUP 753 alone downregulated the angiotensin receptor subtype 1 in kidney, liver and adrenal cortex; its effect on the adrenal medulla in which 89% of angiotensin receptors are subtype 2, did not quite reach significance. The combination of DUP 753 and cyclosporin CSA abolished the CSA-induced increase in angiotensin receptor density in all four organs. The angiotensin receptor antagonists D 8731 downregulated the angiotensin receptors (subtype 1) in liver and kidney and upregulated angiotensin receptors (subtype 2) in the adrenal medulla.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗

Subpopulations of alveolar macrophages in smokers and nonsmokers: relation to the expression of CD11/CD18 molecules and superoxide anion production.

We were previously able to show that the number of alveolar macrophages (AM) expressing CD11/CD18 molecules is increased in smokers compared with nonsmokers and related to the superoxide anion (O2-) production of these cells. Since it has been demonstrated that AM are a heterogeneous cell population that can be separated by density, we performed this study to investigate the expression of CD11/CD18 molecules and O2- production in relation to cell density of AM from smokers and nonsmokers. AM were obtained by bronchoalveolar lavage (BAL) from smokers (n = 32) and nonsmokers (n = 20). Subpopulations were isolated using discontinuous Percoll density-gradient centrifugation with four densities (fraction 1: 1.030; fraction 2: 1.040; fraction 3: 1.050; and fraction 4: 1.070 g/ml). Expression of CD11/CD18 on freshly isolated cells and on AM before and after density centrifugation was studied using peroxidase-antiperoxidase staining. The contribution of AM subpopulations to O2- production in smokers was determined by monitoring the reduction of ferricytochrome C to ferrocytochrome C. We obtained 0.92 +/- 0.1 x 10(5) AM/ml BAL in nonsmokers and 2.4 +/- 0.3 x 10(5) AM/ml in smokers. Recovery after density centrifugation was > or = 72%. The absolute number of AM in smokers was significantly increased in fractions 3 and 4 (median 4.37 x 10(6) and 2.05 x 10(6), respectively) compared with nonsmokers (median 1.26 x 10(6) and 0.7 x 10(6), respectively) (p < 0.05). In both smokers and nonsmokers, fractions 3 and 4 showed a comparable increase in the percentage of CD11/CD18-positive AM compared with fractions 1 and 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

[Not Available].

E. F. Geoffroy's table of different relations ("rapports") between different chemical substances is mainly based on empirical knowledge accumulated in 16th and 17th century metallurgy and pharmacy. The substances listed in the left half of the table were basic for the formation of salts which were produced for medical ends in the chemical-pharmaceutical practice of the 17th century. The right half of the table refers to substances and operations of metallurgy which had already been described in the metallurgical writings of the 16th century. Even Geoffroy's ordering of the substances within the columns of his table has its origin in metallurgical and pharmaceutical practice. The key concept of the conceptual framework underlying the table and its commentary is the concept of chemical compound which emerged at the end of the 17th century. Geoffroy extends the range of application of this concept, which was first limited to chemical artefacts, to include natural bodies. Eliminating the peripatetical distinction between natural bodies and chemical artefacts he formulates a new research program, consisting in the determination of the "laws" of the relations ("rapports") between different chemical substances.

Chemistry↗

Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.

BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents. MATERIALS AND METHODS: Rearranged V kappa region genes from 10 kappa-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three kappa-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined. RESULTS: All BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged V kappa genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines). CONCLUSIONS: The detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for mu chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher.

B-Lymphocytes↗

[Follow-up results of laparoscopic cholecystectomy].

Following a laparoscopic cholecystectomy 400 patients have been interviewed with a questionnaire on the late results of the surgery (15 to 40 months postop.). The cholecystectomies had been performed within the time from March 7, 1990 to April 30, 1992 in Tübingen. After a mean of 16.8 days the patients returned to work, while they themselves felt reduced for an average of 10.6 days. 11.9% of the patients complained of slight wound healing problems and in 3.1% wound infections have been registered. Although 97% of the patients were satisfied with the results of surgery, 8.7% still complained of upper abdominal trouble. Slight persistent problems like light pain or flatulence have been reported by 19%.

Absenteeism↗

Conversion of the myometrial oxytocin receptor from low to high affinity state by cholesterol.

Reconstitution experiments with the myometrial oxytocin receptor from guinea pig showed a strong requirement of the oxytocin receptor binding function for the presence of cholesterol in preformed liposomes. To investigate the effect of cholesterol on the oxytocin receptor in the plasma membrane, a new method to modify the membrane cholesterol content was developed. With substituted cyclodextrins we were able to selectively deplete the myometrial plasma membrane of cholesterol. Vice versa, incubation of cholesterol-depleted membranes with a soluble cholesterol cyclodextrin complex restored the cholesterol content of the plasma membrane. Binding experiments showed, that with the removal of cholesterol from the membrane, the oxytocin receptor was converted from high to low affinity state. Increasing the cholesterol content of the membrane again restored the high binding affinity. Substitution of membrane cholesterol with other steroids showed a strong dependence of the oxytocin receptor function on the structure of the cholesterol molecule. Experiments with the detergent solubilized oxytocin receptor and with oxytocin receptors expressed in the Baculovirus/Sf9 cell system provided further evidence for a direct interaction of the oxytocin receptor with cholesterol.

Animals↗