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B Kottwitz

Publications and source records attributed to B Kottwitz.

7 recordsLinked to original sources
PubMed1998

Subplasmalemmal Ca-stores in Paramecium tetraurelia. Identification and characterisation of a sarco(endo)plasmic reticulum-like Ca(2+)-ATPase by phosphoenzyme intermediate formation and its inhibition by caffeine.

Considering increasing interest in calcium stores in protozoa, including parasitic forms, and specifically in subplasmalemmal stores in higher eukaryotes, we have isolated subplasmalemmal calcium stores (alveolar sacs) from the ciliated protozoan, Paramecium tetraurelia. Using antibodies against established sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCAs) we detected in Western blots of subcellular fractions a band of approximately 106 kDa size selectively in alveolar sacs--but not, for example, in plasma membranes--and concomitant restriction of immunofluorecence labelling to the cell cortex of permeabilised cells. These results are the same as with ABs against a peptide derived from a cloned SERCA-like gene from Paramecium [Hauser K., Pavlovic N., Kissmehl R., Plattner H. Molecular characterization of a sarco(endo)plasmic reticulum Ca(2+)-ATPase gene from Paramecium tetraurelia and localisation of its gene product to subplasmalemmal calcium stores. Biochem J 1998; 334: 31-38]. When such isolated alveolar sacs were now tested for phosphoenzyme intermediate (EP) formation, a phosphoprotein of the same apparent molecular mass (approximately 106 kDa) as in blots could be identified in gel autoradiograms. This EP corresponds to that formed in the reaction cycle of different SERCA-types, with dependency on Ca2+ and Mg2+, sensitivity to La3+ or insensitivity towards calmodulin, calmodulin antagonists and vanadate. However, EP formation in alveolar sacs is not inhibited by established SERCA inhibitors (e.g. thapsigargi[ci]n tested up to 100 microM). Surprisingly, caffeine, which is frequently used to mobilise Ca2+ from intracellular stores, strongly inhibits EP formation. In parallel experiments, we did not find any similar effect with sarcoplasmic reticulum isolated from skeletal muscle. We conclude that the approximately 106 kDa protein of alveolar sacs in Paramecium may represent a SERCA-like Ca(2+)-ATPase with some unorthodox features, which might be relevant also for some other protozoan systems. In this case, the established Ca(2+)-mobilizing effect of caffeine may be amplified by inhibiting store refilling.

Animals↗
PubMed1997

Occurrence of a para-nitrophenyl phosphate-phosphatase with calcineurin-like characteristics in Paramecium tetraurelia.

Using para-nitrophenyl phosphate (pNPP) as a substrate for enzymatic activity, we sought to identify CaN in Paramecium. We isolated three different pNPP-phosphatases from the soluble fraction of Paramecium cells by anion-exchange and affinity column chromatographies. One, pNPP-phosphatase Peak I, is very similar to mammalian CaN. Divalent cation dependency, inhibition by calmodulin (CaM) antagonists (trifluoperazine, calmidazolium), and insensitivity to various phosphatase inhibitors (heparin, okadaic acid, sodium vanadate, etc.) show similarity to mammalian CaN rather than to any other Paramecium pNPP-hydrolyzing enzymes tested. Polyclonal antibodies against bovine brain CaN recognizing subunits A (61 or 58 kDa) and B (17 kDa) of brain CaN cross-reacted with a 63-kDa protein in fractions containing Peak IpNPP-phosphatase activity and coeluted calmodulin. Overlay assays using biotinylated brain calmodulin indicated Ca2+-dependent CaM-binding by the 63-kDa protein. A Ca2+-binding protein with the same electrophoretic mobility as CaN B (17 kDa) was also present, though in other fractions from DEAE-cellulose chromatography. This finding strongly suggests that, in the absence of Ca2+, both subunits, A and B, were separated either before or during chromatographic processing. Our data support the existence of both subunits of a CaN-like phosphatase in Paramecium cells.

4-Nitrophenylphosphatase↗
PubMed1993

Single amino acid substitutions affecting the substrate specificity of the Escherichia coli K-12 nucleoside-specific Tsx channel.

The Tsx protein from the Escherichia coli outer membrane is a channel-forming protein containing a nucleoside-specific binding site. The antibiotic albicidin enters the cell via this substrate-specific channel. Because albicidin is toxic for E. coli at a very low external substrate concentration, the Tsx channel is likely to contain a binding site for this antibiotic. To identify residues involved in the Tsx substrate-specific channel activity, we devised a selection scheme to isolate albicidin-resistant tsx mutants synthesizing Tsx proteins with defects in their nucleoside uptake function. We recovered seven distinct albicidin-resistant tsx alleles, six point mutations and a 39-base pair duplication. The mutants with a duplication of residues 21-33 of Tsx or with single amino acid substitutions of residue Gly28 (to Arg) and Ser217 (to Arg) are completely deficient in nucleoside uptake at a low substrate concentration. Substitutions of Phe27 to Leu, Gly28 to Glu, Gly239 to Asp, and Gly240 to Asp result in a Tsx protein partially defective in nucleoside transport. These mutant proteins still permit nonspecific diffusion of serine indicating that the mutations do not result in a block of the Tsx channel. Our results are discussed in terms of a model for the topological organization of the Tsx protein within the outer membrane of E. coli.

Amino Acids↗
PubMed1993

Identification of a segment of the Escherichia coli Tsx protein that functions as a bacteriophage receptor area.

The Escherichia coli outer membrane protein Tsx functions as a nucleoside-specific channel and serves as the receptor for colicin K and a number of T-even-type bacteriophages, including phage T6. To identify those segments of the Tsx protein that are important for its phage receptor function, we devised a selection and screening procedure which allowed us to isolate phage-resistant strains synthesizing normal amounts of Tsx. Three different Tsx-specific phages (T6, Ox1, and H3) were employed for the selection of phage-resistant derivatives of a strain expressing a tsx(+)-lacZ+ operon fusion, and 28 tsx mutants with impaired phage receptor function were characterized. Regardless of the Tsx-specific phage used for the initial mutant selection, cross-resistance against a set of six different Tsx phages invariably occurred. With one exception, these mutant Tsx proteins could still serve as a colicin K receptor. DNA sequence analysis of 10 mutant tsx genes revealed the presence of four distinct tsx alleles: two point mutations, an 18-bp deletion, and a 27-bp tandem duplication. In three isolates, Asn-249 was replaced by a Lys residue (tsx-504), and in four others, residue Asn-254 was replaced by Lys (tsx-505). The deletion (tsx-506; one isolate) removed six amino acids (residue 239 to residue 244) from the 272-residue Tsx polypeptide chain, and the DNA duplication (tsx-507; two isolates) resulted in the addition of nine extra amino acids (residue 229 to residue 237) to the Tsx protein. In contrast to the wild-type Tsx protein and the other mutant Tsx proteins the Tsx-507 protein was cleaved by trypsin when intact cells were treated with this protease. The Tsx proteins encoded by the four tsx alleles still functioned in deoxyadenosine uptake in vivo, demonstrating that their nucleoside-specific channel activity was not affected by the alterations that caused the loss of their phage receptor function. HTe changes in the Tsx polypeptide that confer resistance against the Tsx-specific phages are clustered in a small region near the carboxy terminus of Tsx. Our results are discussed in terms of a model for the topological organization of the carboxy-terminal end of the Tsx protein within the outer membrane.

Amino Acid Sequence↗
PubMed1990

[Feline immunodeficiency virus in Switzerland: clinical aspects and epidemiology in comparison with feline leukemia virus and coronaviruses].

Serum samples from 1421 domestic cats (561 healthy, 860 sick) were tested for FIV-, FeLV- and coronavirus infection. The results were stored in a computer data base and compared with epidemiologic data and clinical findings. All 3 infections were significantly more prevalent in sick than healthy cats: FIV was found in 0.7% of healthy and 3.4% of sick cats. For FeLV the prevalence was 3.0% and 13.0% and for coronavirus 21% and 36.2%, respectively. FIV-infected cats were mostly male (73%); no sex predilection was observed in FeLV- and coronavirus infection. In sick cats FIV-infection was significantly more prevalent in cats greater than 2 years of age; no age-dependence was found in FeLV- and coronavirus infections. The prevalence of FIV-infection increased significantly with the number of animals per household. In contrast, the frequency of FeLV infection decreased with the number of animals per household. Prevalence of coronavirus infection did not vary with group size or living conditions. The following clinical symptoms were associated with infection: FIV: general depression, diseases of the urinary tract; FeLV: general depression, fever, rough hair coat, lymphadenopathy, impaired functions of heart and circulation and muscle atrophy; coronavirus: lymphadenopathy and alterations in the abdomen. It was concluded that based on the clinical symptoms alone FIV-infection could not be diagnosed nor differentiated from the other 2 infections.

Animals↗
PubMed1987

[ST elevation in the initial phase of myocardial infarct--a sequela of acute coronary occlusion of an expression of neurohumorally-induced metabolic processes?].

The artificial coronary artery occlusion results immediately in ST-segment elevation (ST-E), therefore the ST-E in acute myocardial infarction is considered to be the result of acute coronary occlusion. But in the early phase of myocardial infarction a significant correlation between ST-E and occlusion of the coronary artery does not exist. The early ST-E seems to be predominantly the consequence of sympathico-adrenergic induced metabolic processes in the ischemic myocardium. The beta receptor blockade during the first 90 minutes after the onset of infarction decreased the ST-E by 73 +/- 6% within one hour. Treatment with beta receptor blockers in the acute phase in all patients with first anterior wall infarcts accompanied with ST-E leads to a favorable prognosis. Early ST-E can therefore be considered as a sign of sympathico-adrenergic induced changes in myocardial metabolism. Thus the beta receptor blockade in the acute phase (of anterior wall infarctions) seems to be therapeutically indicated.

Adrenergic beta-Antagonists↗