Search PubMed⌕ Search

Biomedical subjects

U Klein

Publications and source records attributed to U Klein.

At least 109 records · Page 6Linked to original sources
PubMed1991

A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.

In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.

Adenosine Triphosphate↗
PubMed1990

Cell-specific accumulation of Drosophila proteasomes (MCP) during early development.

The proteasome (MCP) is a high relative molecular mass multicatalytic proteinase complex composed of nonidentical protein subunits. We have investigated the cellular distribution of the enzyme complex during Drosophila embryogenesis using the proteasome specific antibodies N19-35 and N19-28 for immunocytology. Antibody staining of whole-mount embryos shows that during embryogenesis proteasomes are present in proliferating cells and that their accumulation and turnover is differentially regulated. Our data suggest that the proteasome may serve different proteolytic processes and that the enzyme may be involved in cell-specific proteolytic events required for cell proliferation and morphogenesis during early Drosophila development.

Animals↗
PubMed1990

Transcriptional analysis of endogenous and foreign genes in chloroplast transformants of Chlamydomonas.

Transcription from modified chloroplast genes has been studied in vitro, but only with the recently developed ability to stably introduce foreign DNA into Chlamydomonas reinhardtii chloroplast chromosomes in situ has it become possible to do so in vivo. Cloned chloroplast DNA sequences, into which had been inserted chimeric genes composed of the GUS coding sequence reporter under transcriptional control of chloroplast promoters for the C. reinhardtii atpA, atpB, and rbcL genes, were introduced into the cells on microprojectiles. These constructs become integrated into chloroplast chromosomes by homologous recombination. RNA gel blot analyses demonstrated that a single major beta-glucuronidase (GUS)-hybridizing transcript accumulates in each chloroplast transformant. We have found that: (1) Transcription of the chimeric gene begins at the same site as in the corresponding endogenous chloroplast gene; (2) the rates of transcription in vivo of the atpA:GUS and atpB:GUS genes relative to one another and to other genes are the same as those for the endogenous atpA and atpB genes, respectively, indicating that these promoters are fully functional despite being fused to a foreign gene and being at an alien location on the chloroplast chromosome; (3) in contrast to the atpA and atpB promoters, the rbcL promoter directs transcription of the rbcL:GUS gene at only 1% of the expected rate, suggesting that other features are required for optimal activity of this promoter; and (4) 22 base pairs upstream of the 5' end of the atpB:GUS transcript in the atpB promoter element is sufficient to confer wild-type levels of promoter activity.

Animals↗
PubMed1990

Developmental expression of Drosophila melanogaster small heat-shock proteins.

We have investigated the developmental expression of the small heat-shock proteins (hsps) during embryogenesis and in adult flies by immunocytology using an antibody that specifically identifies the small hsps. Antibody staining of unstressed early embryos reveals a predominantly cytoplasmic, homogeneous distribution of the small hsps throughout the embryo. At 6h of development small hsp expression can be identified in large, neuroblast-like cells within the extended germ band and in the brain of the embryo. During germ band contraction these cells appear to migrate to the midline where they align pairwise in a segmental pattern. After germ band contraction is complete a high level of small hsp expression can be observed in the midline glia (MECs) and in a cluster of six non-neuronal cells within the midline. In contrast to several other genes that are known to be important for embryogenesis and are expressed in the central nervous system (CNS) of embryos, CNS-specific expression of the small hsps is not restricted to the embryo but is also observed in the adult fly. In adult flies strong small hsp expression is observed in the brain, the thoracic ganglion and the leg nerves. Since the small hsps seem to be expressed predominantly in the glia of the nervous system, our data suggest a protective or stabilizing function of the small hsps within the nervous system during normal fly development, which is independent of the stress response.

Animals↗
PubMed1990

[Functional scintigraphic studies of perfusion and ventilation during high frequency jet ventilation (HFJV)].

In a controlled study, functional-scintigraphic investigations into perfusion and ventilation were performed on 10 dogs with non-damaged and extremely severely damaged lungs. The pulmonary damage was produced by injecting oleic acid (OA) into the right atrium of the heart under controlled ventilation (IPPV). The scintigraphic examinations were carried out using 133Xenon. The study compared HFJV (HFJV100, HFJV300) with IPPV in the non-damaged lung as well as HFJV300 with IPPV and CPPV (PEEP 1 kPa) after damage by OA. With the aid of the present radionuclide investigations, new insights can be gained into the largely unclear regional conditions of the gas exchange under HFJV in both the healthy and the damaged lung. Results from controlled studies on the distribution of ventilation and perfusion under HFJV have not been reported to date. The functional-scintigraphic examination with 133Xe on dogs shows, based on specific conditions of the gas exchange and special anatomic conditions of the lungs, a ventilation distribution that differs fundamentally from all other forms of ventilation, including HFOV, preference being given to apical pulmonary segments. This refers to the normal and the damaged lungs alike. However, ventilation-specific changes in pulmonary perfusion do not occur. The resulting deviating regional VA/Q relationship are obviously not of crucial influence upon the gas exchange. Rather, it is influenced and determined by damage-induced intraregional functional-structural alterations in the lung.

Animals↗
PubMed1990

[Circular intrathoracic tracheal replacement by a devitalized artery in the dog].

Prepared artery (origin: human, calf, dog) with external or internal teflon-spiral-supporting was implanted in dogs (n = 10) as a circular intrathoracic tracheal substitute after cross-resection. Human arteries were rejected (n = 2). Calf arteries (n = 5) and dog arteries (n = 3) were incorporated fibrously. Central granulations appeared. The longest survival time was 36 days.

Animals↗
PubMed1990

[High-frequency jet ventilation and the function of the right heart--an animal experimental study].

In a controlled study, 10 dogs with normal and severely damaged lungs were subjected to nuclide angiocardiographic investigations into the function of the right ventricle. The pulmonary injury was produced by infusion of oleic acid (OA) into the right atrium during controlled ventilation (IPPV). The angiocardiographic examinations were performed using 133Xenon in the first pass technique. The study compared HFJV (HFJV 100, HFJV 300) with IPPV in the non-damaged lung as well as HFJV 300 with IPPV and CPPV (PEEP 1.0 kPa) after injury by OA. Whereas for the non-damaged lung no different right ventricular (RV) function between IPPV and HFJV was observed, the RV functional parameters after OA injury under HFJV showed, as opposed to CPPV, more favourable values on the whole. This became clear in particular in the significantly higher ejection fraction (RVEF) during HFJV. The RV function which is influenced during CPPV in terms of a favourable oxygenation is the consequence of an increased mean pressure in the airways with subsequent rise in the RV afterload and decrease in the RV preload. In contrast, the more favourable RV haemodynamics during HFJV are associated with a comparatively lower mean airway pressure and a significantly worse oxygenation. As the RV function also during HFJV has to be seen in direct dependence on the mean airway or intrapulmonary pressure necessary for sufficient oxygenation, the employment of this form of ventilation in the presence of an acute respiratory insufficiency has no impact on the RV haemodynamics different from the other compared forms of ventilation.

Animals↗
PubMed1989

A vacuolar-type proton pump in a vesicle fraction enriched with potassium transporting plasma membranes from tobacco hornworm midgut.

Mg-ATP dependent electrogenic proton transport, monitored with fluorescent acridine orange, 9-aminoacridine, and oxonol V, was investigated in a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. Proton transport and the ATPase activity from the goblet cell apical membrane exhibited similar substrate specificity and inhibitor sensitivity. ATP and GTP were far better substrates than UTP, CTP, ADP, and AMP. Azide and vanadate did not inhibit proton transport, whereas 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide were inhibitors. The pH gradient generated by ATP and limiting its hydrolysis was 2-3 pH units. Unlike the ATPase activity, proton transport was not stimulated by KCl. In the presence of 20 mM KCl, a proton gradient could not be developed or was dissipated. Monovalent cations counteracted the proton gradient in an order of efficacy like that for stimulation of the membrane-bound ATPase activity: K+ = Rb+ much greater than Li+ greater than Na+ greater than choline (chloride salts). Like proton transport, the generation of an ATP dependent and azide- and vanadate-insensitive membrane potential (vesicle interior positive) was prevented largely by 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide. Unlike proton transport, the membrane potential was not affected by 20 mM KCl. In the presence of 150 mM choline chloride, the generation of a membrane potential was suppressed, whereas the pH gradient increased 40%, indicating an anion conductance in the vesicle membrane. Altogether, the results led to the following new hypothesis of electrogenic potassium transport in the lepidopteran midgut. A vacuolar-type electrogenic ATPase pumps protons across the apical membrane of the goblet cell, thus energizing electroneutral proton/potassium antiport. The result is a net active and electrogenic potassium flux.

Adenosine Triphosphatases↗
PubMed1989

A vacuolar-type ATPase, partially purified from potassium transporting plasma membranes of tobacco hornworm midgut.

An azide- and vanadate-insensitive, N-ethylmaleimide-sensitive ATPase has been partially purified from a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. The properties of the membrane-bound ATPase activity were identical to those of the ATPase activity of highly purified goblet cell apical membranes (Wieczorek, H., Wolfersberger, M. G., Cioffi, M., and Harvey, W. R. (1986) Biochim. Biophys. Acta 857, 271-281). 90% of the azide- and vanadate-insensitive ATPase activity was solubilized by C12E10, leaving 90% of the contaminating azide-sensitive mitochondrial ATPase activity in the pellet after centrifugation at 100,000 x g for 1 h. After discontinuous sucrose gradient centrifugation of the supernatant at 220,000 x g for 1 h nearly all of the azide- and vanadate-insensitive ATPase activity was found in the 30% sucrose fraction without contaminating azide- or vanadate-sensitive ATPase activity. Two prominent bands with relative molecular masses (Mr) of about 600,000 and 900,000, both displaying azide-insensitive and N-ethylmaleimide-sensitive ATPase activity, were found in native microgradient polyacrylamide gel electrophoresis of the 30% sucrose fraction. The two bands could not be separated by anion exchange chromatography. Denaturation of both bands resulted in the same polypeptide pattern (five major bands with Mr 70,000, 57,000, 46,000, 29,000 and 17,000) in sodium dodecylsulfate-polyacrylamide gel electrophoresis, indicating that they represented oligomers of the same protein unit. Substrate and inhibitor specificities of the partially purified ATPase were similar to those of the membrane-bound ATPase activity, whereas salt selectivity differed partly. Altogether, structural and functional properties of the ATPase strongly resemble those of vacuolar-type ATPases.

Adenosine Triphosphatases↗
PubMed1989

[Objectivization of the methods for recording the total contact surface and the number of occlusal contacts in static occlusion].

The occlusal contacts of teeth in a dentition have been analysed metrically with the aid of a new method. The outstanding advantage of this method is offered by the fact that the contacts of teeth are registered immediately in the subject and then evaluated with the aid of the teleplanimetric measuring variant. From error computation it follows that the method leads to reproducible and largely objective results. In 84 subjects with an average age of 16.9 +/- 3.7 years the total contacting area and the number of contacts in static occlusion were determined and examined with respect to specific groups.

Adolescent↗