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Biomedical subjects

M Kott

Publications and source records attributed to M Kott.

14 recordsLinked to original sources
PubMed2000

Smooth-muscle contraction without smooth-muscle myosin.

Here we have used gene-targeting to eliminate expression of smooth-muscle myosin heavy chain. Elimination of this gene does not affect expression of non-muscle myosin heavy chain, and knockout individuals typically survive for three days. Prolonged activation, by KCl depolarisation, of intact bladder preparations from wild-type neonatal mice produces an initial transient state (phase 1) of high force generation and maximal shortening velocity, which is followed by a sustained state (phase 2) characterized by low force generation and maximal shortening velocity. Similar preparations from knockout neonatal mice do not undergo phase 1, but exhibit a normal phase 2. We propose that, in neonatal smooth muscle phase 1 is generated by recruitment of smooth-muscle myosin heavy chain, whereas phase 2 can be generated by activation of non-muscle myosin heavy chain. We conclude that phase 1 becomes indispensable for survival and normal growth soon after birth, particularly for functions such as homeostasis and circulation.

Animals↗
PubMed1999

Signaling from beta-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target possessing similarities to AHNAK.

A novel calcium channel-associated protein of approximately 700 kDa has been identified in mammalian cardiomyocytes that undergoes substantial cAMP-dependent protein kinase (PKA) phosphorylation. It was therefore designated as phosphoprotein 700 (pp700). The pp700 interacts specifically with the beta(2) subunit of cardiac L-type calcium channels as revealed by coprecipitation experiments using affinity-purified antibodies against different calcium channel subunits. It is surprising that amino acid sequence analysis of pig pp700 revealed homology to AHNAK-encoded protein, which was originally identified in human cell lines of neural crest origin as 700-kDa phosphoprotein. Cardiac AHNAK expression was assessed on mRNA level by reverse transcriptase-polymerase chain reaction. Sequence-directed antibodies raised against human AHNAK recognized pp700 in immunoblotting and immunoprecipitation experiments, confirming the homology between both proteins. Anti-AHNAK antibodies labeled preferentially the plasma membrane of cardiomyocytes in cryosections of rat cardiac tissue and isolated cardiomyocytes. Sarcolemmal pp700/AHNAK localization was not influenced by stimulation of either the PKA or the protein kinase C pathway. In back-phosphorylation studies with cardiac biopsies, we identified distinct pp700 pools. The membrane-associated fraction of pp700 underwent substantial in vivo phosphorylation on beta-adrenergic receptor stimulation by isoproterenol, whereas the cytoplasmic fraction of pp700 was not accessible to endogenous PKA. It is important that in vivo phosphorylation occurred in that pp700 fraction which coprecipitated with the calcium channel beta subunit. We hypothesize that both phosphorylation of pp700 and its coupling to the beta subunit play a physiological role in cardiac beta-adrenergic signal transduction. Haase, H., Podzuweit, T., Lutsch, G., Hohaus, A., Kostka, S., Lindschau, C., Kott, M., Kraft, R., Morano, I. Signaling from beta-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target that has similarities to AHNAK.

Amino Acid Sequence↗
PubMed1998

A new efficient method for transfection of neonatal cardiomyocytes using histone H1 in combination with DOSPER liposomal transfection reagent.

Although cationic lipids are successfully used for gene transfer in vitro, primary cells such as neonatal cardiomyocytes frequently resist efficient transfection. We show here that the polycationic lipid DOSPER in combination with histone H1 was much more efficient in transfection of neonatal cardiomyocytes than DOSPER alone or other cationic lipids. This has been shown for transfection with the reporter plasmids pSV beta-gal and pCMV luc. If viral transfections are not possible, this mild method is an alternative to transfect cardiomyocytes.

Animals↗
PubMed1998

Influence of different culture conditions on sarcoplasmic reticular calcium transport in isolated neonatal rat cardiomyocytes.

This study investigates sarcoplasmic reticulum (SR) calcium-(Ca2+) transport ATPase (SERCA2a) and phospholamban (PLB) in cultured spontaneously contracting neonatal rat cardiomyocytes (CM) to ascertain the function of both SR proteins under various culture conditions. The two major SR proteins were readily detectable in cultured CM by immunofluorescent microscopy using specific anti-SERCA2 and anti-PLB antibodies. Double labeling technique revealed that PLB-positive CM also labeled with anti-SERCA2. Coexpression of SERCA2 and PLB in CM was supported by measurement of cell homogenate oxalate-supported Ca2+ uptake which was completely inhibited by thapsigargin and stimulated by protein kinase A-catalyzed phosphorylation. Under serum-free conditions, incubation of CM with the SERCA2a expression modulator 3,3', 5-triiodo-L-thyronine (100 nM, 72 h) resulted in elevated Ca2+ uptake of +33%. Specific Ca2+ uptake activity was not altered if insulin was omitted from the serum-free culture medium but total SR Ca2+ transport activity was reduced under this culture condition. The results indicate that primary culture of spontaneously contracting neonatal rat CM can be employed as a useful model system for investigating both short- and long-term mechanisms determining the Ca2+ re-uptake function of the SR under defined culture conditions.

Animals↗
PubMed1995

Differential influences of carnitine palmitoyltransferase-1 inhibition and hyperthyroidism on cardiac growth and sarcoplasmic reticulum phosphorylation.

To characterize interventions resulting in 'physiological' growth of the heart, Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) had hyperthyroidism induced (0.05 mg.kg-1.day-1 triiodothyronine for 6 days) or were treated with a high dose of the carnitine palmitoyltransferase-1 inhibitor, etomoxir (15 mg.kg-1.day-1 for 5 weeks). Etomoxir increased cardiac growth evenly, but hyperthyroidism resulted in an over-proportional higher right ventricular weight. Both interventions increased the proportion of the myosin isozyme V1. The rate of sarcoplasmic reticulum (SR) Ca2+ uptake was increased to a greater extent in hyperthyroid rats than in etomoxir-treated rats (P < 0.05). Left ventricular levels of immunoreactive phospholamban (semiquantitative ELISA) were moderately decreased (P < 0.05) in hyperthyroid rats but not in etomoxir-treated rats. The protein kinase A-catalyzed in vitro 32P-incorporation into the SR Ca2+ pump modulator phospholamban was greatly reduced (P < 0.05) in hyperthyroid rats, indicating an increased in vivo phosphorylation. Etomoxir did not affect phospholamban phosphorylation in WKY rats. Thus, both a higher in vivo phospholamban phosphorylation state and a greater number of active Ca2+ pumps contributed to an increased rate of SR Ca2+ uptake in hyperthyroidism. The etomoxir treatment primarily increased the number of active Ca2+ pumps. A scheme is proposed focusing on long-term vs short-term regulation of the SR Ca2+ pump/phospholamban system in diseased states.

Animals↗
PubMed1994

[Surgical treatment of mediastinal tumors from personal material].

90 patients with mediastinal tumors treated surgically was analysed. It was find, that tumor mostly was localized in anterior superior mediastinum, had benign character and in over half of cases was derived from thymus. 75% of mediastinal tumors were primary. In over 50% patients longitudinal sternotomy was performed. Radical excision of the tumor was possible nearly in 90% cases.

Adolescent↗
PubMed1990

Structure of the gene for human butyrylcholinesterase. Evidence for a single copy.

We have isolated five genomic clones for human butyrylcholinesterase (BChE), using cDNA probes encoding the catalytic subunit of the hydrophilic tetramer [McTiernan et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6682-6686]. The BChE gene is at least 73 kb long and contains four exons. Exon 1 contains untranslated sequences and two potential translation initiation sites at codons -69 and -47. Exon 2 (1525 bp) contains 83% of the coding sequence for the mature protein, including the N-terminal and the active-site serine, and a third possible translation initiation site (likely functional), at codon -28. Exon 3 is 167 nucleotides long. Exon 4 (604 bp) codes for the C-terminus of the protein and the 3' untranslated region where two polyadenylation signals were identified. Intron 1 is 6.5 kb long, and the minimal sizes of introns 2 and 3 are estimated to be 32 kb each. Southern blot analysis of total human genomic DNA is in complete agreement with the gene structure established by restriction endonuclease mapping of the genomic clones: this strongly suggests that the BChE gene is present in a single copy.

Base Sequence↗
PubMed1987

Brain cDNA clone for human cholinesterase.

A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase (EC 3.1.1.8). Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum. The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase (EC 3.1.1.8) rather than acetylcholinesterase (EC 3.1.1.7). It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes, coded for cholinesterase.

Amino Acid Sequence↗
PubMed1983

Utmost cytoplasmic location of catalytic center in Na,K-motive ATPase disfavors Mitchell's phosphate-cation symport mechanism of Na/K transport across plasma membrane.

A combined electron microscopic and graded tryptic dissection analysis of the catalytic protein of pure Na,K-motive ATPase reveals that the aspartyl phosphate residue-carrying catalytic center is located at the utmost radius of the hydrophilic cytoplasmic domain. This makes the cation-phosphate symport mechanism of Na/K transport across plasma membrane as proposed by Mitchell topologically unrealistic, and indirectly favors the mechanism of conformational coupling between the catalytic and ionophoric function of the transport system.

Animals↗
PubMed1982

Reactive lymph node hyperplasia with giant follicles.

Twenty-two cases are presented of patients whose solitary painless, enlarged lymph nodes demonstrated an extreme degree of reactive hyperplasia with giant follicles which were misdiagnosed as lymphoma in 11 (50%) instances. Parotid or submandibular nodes were involved most frequently, and the mass was thought clinically to be a salivary gland tumor in at least one-third of the cases. Occurrence in men predominated (6.3:1) with a median age of 24.5 years (mean, 26.8 years). There were no recurrences during follow-up periods which ranged from 6 months to 15 years. No specific etiologic factors were identified. Microscopically, the enlarged giant follicles involved the entire surface area of the lymph node, frequently assuming a serpentine configuration resulting from coalescence. The description of findings in these exemplary cases is followed by discussion of the histologic differential diagnosis.

Adolescent↗
PubMed1975

Identification of the ouabain-binding peptide of (nak)-atpase).

The [3H]-ouabain-(NaK)-ATPase complex when treated in the cold with sodium dodecyl sulfate (SDS) dissociates into a larger and smaller peptide called alpha and beta, resp.. Analysis of the released peptides by SDS-polyacrylamide gel electrophoresis reveals that [3H]-ouabain co-migrates with the alpha-peptide only, being apparently identic with the ouabain receptor molecule. The percentage occupancy of the receptor peptide with [3H]-ouabain can be increased up to 90% evidencing the stabilization of the [3H]-ouabain-alpha-peptide complex by SDS-exposure and release from the oligomeric enzyme. A hypothetic explanation for the seemingly paradoxical stabilising effect of SDS on the complex is offered.

Adenosine Triphosphatases↗
PubMedDate not supplied

Developmental changes in the complexity of the electrocortical activity in foetal sheep.

The foetal sheep brain develops organised sleep states from 115-120 d gestational age (dGA, term 150 dGA) alternating between REM and NREM sleep. We aimed to investigate whether maturation of REM or NREM sleep generating structures leads to the development of distinct sleep states. The electrocorticogram (ECoG) was recorded from five unanaesthetised chronically instrumented foetal sheep in utero and was analysed every 5th day between 115-130 dGA by two different non-linear methods. We calculated a non-linear prediction error which quantifies the causality of the ECoG and applied bispectral analysis which quantifies non-linear interrelations of single frequency components within the ECoG signal. The prediction error during REM sleep was significantly higher than during NREM sleep at each investigated age (P<0.0001) coincidental with poor organisation of the rhythmic pattern in the ECoG during REM sleep. At 115 dGA, organised sleep states defined behaviourally were not developed yet. The prediction error, however, showed already different states of electrocortical activity that were not detectable using power spectral analysis. The prediction error of the premature NREM sleep ECoG decreased significantly during emergence of organised sleep states between 115 and 120 dGA and continued to decrease after the emergence of distinct sleep states (P<0.05). The prediction error of the premature REM sleep ECoG did not change until 120 dGA and began to increase at 125 dGA (P<0.05). Using bispectral analysis, we showed couplings between delta waves (1.5-4 Hz) and frequencies in the range of spindle waves (4-8 and 8-12 Hz) during NREM sleep that became closer during development. The results show that maturation of ECoG synchronisation mediating structures is important for the development of organised sleep states. The further divergence of the prediction error of NREM and REM sleep after development of organised sleep states reveals continuous functional development. Thus, complementary application of non-linear ECoG analysis to power spectral analysis provide new insights in the collective behaviour of the neuronal network during the emergence of sleep states.

Algorithms↗