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G Zürcher

Publications and source records attributed to G Zürcher.

At least 37 records · Page 2Linked to original sources
PubMed1994

Characterization of a novel murine testis-specific serine/threonine kinase.

Using degenerate oligos corresponding to two highly conserved motifs within the protein kinase catalytic domain and a PCR-based cloning strategy, we have isolated a cDNA fragment encoding a new member of the Ser/Thr (serine/threonine) family of protein kinases. Expression analysis revealed that the fragment recognized two transcripts (1.6 and 1.4 kb) exclusively in testis. Using this fragment as a probe, we have cloned a full-length cDNA from a mouse testis cDNA library. The sequence has a 1092-bp open reading frame encoding a protein of 364 amino acids. The N-terminally localized kinase catalytic domain has all the conserved motifs found in other Ser/Thr kinases. Northern blot analysis using the full-length sequence as a probe revealed that the cloned gene corresponds to the 1.6-kb transcript, suggesting the existence of at least two testis-specific novel Ser/Thr kinases. We propose the name testis-specific kinase-1 (TSK-1) for the gene described here. A GenEMBL databank search revealed highest homology to the human gene encoding rac protein kinase-beta and the group of yeast Ser/Thr kinases encoded by SNF-1, nim-1, KIN-1 and KIN-2.

3T3 Cells↗
PubMed1994

Expression of two novel eph-related receptor protein tyrosine kinases in mammary gland development and carcinogenesis.

To investigate the involvement of protein tyrosine kinases (PTKs) in the growth control of mammary epithelial cells, we have used PCR based cloning to identify PTKs expressed in a mouse mammary epithelial cell line. This approach led to the isolation of two receptor PTKs of the eph-related subfamily; myk-1, a novel member expressed predominantly in lung, heart and mammary gland and myk-2, a close relative of the human eck gene. Northern blot analysis of RNA from mouse mammary glands at different stages of development revealed that myk-1 and myk-2 expression is induced at puberty and differentially regulated during the estrus cycle. myk-1 and myk-2 expression was down-regulated during the pregnancy induced differentiation of the mammary gland. Over-expression of myk-1 and myk-2 was found in the undifferentiated and invasive mammary tumors of transgenic mice expressing the Ha-ras oncogene. In contrast, no elevated expression of either gene could be detected in the well differentiated and non-metastatic mammary tumors of c-myc expressing transgenic mice. These results indicate that myk-1 and myk-2 expression is induced during the proliferation of the mammary gland and down-regulated by its differentiation.

3T3 Cells↗
PubMed1992

Tyramine content of preserved and fermented foods or condiments of Far Eastern cuisine.

The tyramine content of foodstuffs typical of the Far East was analysed: the items included fermented food and condiments as well as seven menus from different Far Eastern restaurants. The results of the present analysis extend to Far Eastern foods our previous conclusion that no severe dietary restrictions are needed in patients treated with moclobemide, a novel RIMA anti-depressant.

Asia↗
PubMed1992

Expression of functional membrane-bound and soluble catechol-O-methyltransferase in Escherichia coli and a mammalian cell line.

Human catechol-O-methyltransferase (hCOMT) cDNA was used to express the recombinant hCOMT enzyme in sufficient quantities in prokaryotic as well as in eukaryotic cells to allow kinetic studies. When human membrane-bound catechol-O-methyltransferase (MB-COMT; amino acids 1-271) and the soluble catechol-O-methyltransferase COMT (S-COMT; delta membrane anchor hCOMT; amino acids 27-271), with the latter lacking the first 26 hydrophobic amino acids, were expressed in Escherichia coli, a relatively high-level synthesis of catalytically active enzymes was obtained. Insertion of the human MB-COMT-coding sequence into an eukaryotic expression vector under transcriptional control of the cytomegalovirus (CMV) promoter and enhancer yielded large quantities of hCOMT in human kidney 293 cells. Subcellular fractionation of 293 cells transfected with pBC12/CMV-hCOMT showed hCOMT to be located predominantly in the membrane fraction. The catechol-O-methyltransferase (COMT) activity was measured in cytosolic and membrane fractions at 37 degrees C, giving values of 33 and 114 units/mg of protein, respectively (1 unit produces 1 nmol of guaiacol/h). Km values were 10 microM for MB-COMT and 108 microM for S-COMT, indicating that recombinant MB-COMT exhibits a higher affinity for catechol as the substrate than the soluble form. RNA blot analysis of human hepatome cells (Hep G2), kidney, liver, and fetal brain revealed only one species of hCOMT mRNA of approximately 1.4 kb. Its level in these various tissues was similar to those of COMT protein in each tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver.

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.

Amino Acid Sequence↗
PubMed1991

Two novel protein-tyrosine kinases, each with a second phosphotransferase-related catalytic domain, define a new class of protein kinase.

The protein-tyrosine kinases (PTKs) are a burgeoning family of proteins, each of which bears a conserved domain of 250 to 300 amino acids capable of phosphorylating substrate proteins on tyrosine residues. We recently exploited the existence of two highly conserved sequence elements within the catalytic domain to generate PTK-specific degenerate oligonucleotide primers (A. F. Wilks, Proc. Natl. Acad. Sci. USA 86:1603-1607, 1989). By application of the polymerase chain reaction, portions of the catalytic domains of several novel PTKs were amplified. We describe here the primary sequence of one of these new PTKs, JAK1 (from Janus kinase), a member of a new class of PTK characterized by the presence of a second phosphotransferase-related domain immediately N terminal to the PTK domain. The second phosphotransferase domain bears all the hallmarks of a protein kinase, although its structure differs significantly from that of the PTK and threonine/serine kinase family members. A second member of this family (JAK2) has been partially characterized and exhibits a similar array of kinase-related domains. JAK1 is a large, widely expressed membrane-associated phosphoprotein of approximately 130,000 Da. The PTK activity of JAK1 has been located in the C-terminal PTK-like domain. The role of the second kinaselike domain is unknown.

Amino Acid Sequence↗
PubMed1991

Tyrosine kinases: from viral oncogenes to developmental regulators.

Protein tyrosine kinases (PTKs) play a central role in cellular regulation by virtue of their participation in, and control of, signal transduction pathways; they act as a molecular interface between the cell's environment and intercellular metabolism. The mammary gland, unlike most organs, undergoes most of its morphogenesis in juvenile and adult life. The epithelium goes through hormonally controlled cycles of proliferation and regression, the fully differentiated state only being reached at the end of pregnancy. These features make the mammary gland an amenable tissue to study the involvement of PTKs in epithelial cell development and differentiation. We have used a PCR-based molecular cloning strategy to identify PTKs from murine mammary gland cells. Amongst 70 kinase clones characterized we found 3 PTKs previously undescribed in mouse, 4 known PTKs and 5 serine threonine kinases. Expression studies revealed differential tissue specificity and developmental regulation of the 3 previously undescribed PTKs. These results substantiate the view that PTKs are involved in the regulation of cellular differentiation.

Amino Acid Sequence↗
PubMed1990

Simple automated high-performance liquid chromatographic column-switching technique for the measurement of dopa and 3-O-methyldopa in plasma.

A reliable assay procedure for the determination of 3,4-dihydroxyphenyl-L-alanine (dopa) and its O-methylated metabolite 3-O-methyldopa (3-OMD) is described. Supernatants from deproteinized plasma samples were directly injected into a column-switching system, allowing on-line prepurification of the samples by cation-exchange chromatography followed by reversed-phase high-performance liquid chromatography with electrochemical detection. With this method, the detector response was linear from endogenous levels of dopa and 3-OMD up to microgram concentrations. This technique combines the simplicity of direct injection methods with advantages of procedures using a sample clean-up. It represents a valid tool for the rapid and accurate measurement of large numbers of plasma samples in animals treated with levodopa and in clinical trials with new levodopa formulations.

Animals↗
PubMed1990

Monoclonal antibodies recognizing both soluble and membrane bound catechol-O-methyltransferase.

Both cytosolic, soluble and membrane-bound catechol-O-methyl-transferase (COMT) from pig and rat liver or kidney were recognized by mouse monoclonal antibodies (MAbs) raised against soluble COMT isolated from pig liver. In ELISA, the MAbs Co 16 and Co 54 reacted better with the pig than with the rat enzyme. The MAb Co 60 showed good reactivity with both pig and rat COMT. In addition, all three MAbs recognize the soluble (23 kDa) as well as the membrane-bound (26 kDa) forms of the COMT enzyme.

Animals↗
PubMed1990

Ro 40-7592: inhibition of COMT in rat brain and extracerebral tissues.

A random biochemical screening followed by lead optimization culminated in the discovery of Ro 40-7592 3,4-dihydroxy-4'-methyl-5-nitrobenzophenone). Ro 40-7592 was found to inhibit COMT in a competitive fashion both in the CNS and in the periphery (Ki for the liver enzyme = 30 nM). Ro 40-7592 (30 mg/kg p.o.) combined with benserazide (15 mg/kg p.o.) and a low dose of L-DOPA (10 mg/kg p.o.) almost completely blocked (for about 6 h) the formation of 3-O-methyldopa (3-OMD) in brain and plasma, producing a long-lasting increase of L-DOPA in plasma and a parallel marked increase of L-DOPA and dopamine in the brain. Ro 40-7592, combined with peripheral decarboxylase inhibitors and L-DOPA (as in Madopar and Sinemet), will offer substantial advantages in the therapy of Parkinson's disease, i.e. enhanced bioavailability and prolonged plasma half-life of L-DOPA, pronounced DOPA sparing effect and blockade of 3-OMD formation.

Animals↗
PubMed1988

On tyramine, food, beverages and the reversible MAO inhibitor moclobemide.

The pathways for the biosynthesis and metabolism of tyramine are described as a basis for the discussion of the interaction between MAO inhibitors and tyramine. While a role of endogenous tyramine in the antidepressant action of MAO inhibitors remains purely hypothetical at this time, the mechanisms leading to the potentiation of the tyramine pressor effect ("cheese effect") are well known. Experiments in animals and man have provided concordant quantitative information on the effect of irreversible and some novel reversible MAO inhibitors on the presystemic disposition of orally ingested tyramine and on the noradrenaline-releasing action of tyramine in noradrenergic nerve terminals. There is a profound difference in the magnitude of tyramine potentiation between the irreversible inhibitor tranylcypromine and the reversible inhibitor moclobemide. A systematic analysis of the tyramine content of current European food and beverage is reported and serves as a rational basis for providing advice to patients on moclobemide. Most of the food and beverages analyzed contain less tyramine than previously reported and a few rules concerning rare cheeses with high tyramine content are sufficient to eliminate the risk of hypertensive crises.

Animals↗
PubMed1987

Inhibition of decarboxylase and levels of dopa and 3-O-methyldopa: a comparative study of benserazide versus carbidopa in rodents and of Madopar standard versus Madopar HBS in volunteers.

The combinations of benserazide and levodopa (1:4, Madopar) and of carbidopa and levodopa (1:10 and 1:4, Sinemet) are currently the most effective treatment of Parkinson's disease. In the present comparative study some effects of the peripheral aromatic L-amino acid decarboxylase (AADC) inhibitors benserazide and carbidopa administered alone or in combination with levodopa by the oral route were investigated in two animal species (rat and mouse) and in healthy volunteers. Benserazide is about 10 times more potent than carbidopa as inhibitor of peripheral AADC both in animals and man. Even at relatively high doses (up to 60 mumol/kg p.o.) benserazide is shown in animals to inhibit the decarboxylation of levodopa only in the extracerebral tissues, thus permitting the formation of dopamine in the striatum and in the hypothalamus. As benserazide is the most potent peripheral AADC inhibitor presently available, is well tolerated and relatively nontoxic even when used chronically, it appears to be the peripheral AADC inhibitor of choice for the development of controlled-release formulations in which Dopa is combined with a peripheral AADC inhibitor. When administered to healthy subjects the pharmacokinetics of the new drug delivery system named Madopar HBS (hydrodynamically balanced system) was characterized by lower and delayed plasma peak concentrations but a longer-lasting concentration of Dopa than after Madopar standard. Therefore, this new controlled-release system may reduce the clinical fluctuations occurring in patients with 'wearing-off' and 'on-off' phenomena.

Administration, Oral↗
PubMed1987

Bioavailability of L-dopa after Madopar HBS administration in healthy volunteers.

Eight healthy male and fasted volunteers received alternatively either one HBS capsule of Madopar 125 or two HBS capsules of Madopar 125 or one standard capsule of Madopar 125 at weekly intervals with and without benserazide pretreatment (50 mg t.i.d. for 6 days). In this trial the Madopar formulations were administered 9.5 h after the last dose of benserazide. Serial blood samples were collected at various time intervals up to 12 h after Madopar dosing. Levodopa plasma concentrations were measured by high-performance liquid chromatography with electrochemical detection. After Madopar HBS without benserazide pretreatment the peak concentration (Cmax) of levodopa was lower and occurred at later times (tmax) than after standard Madopar. The mean values for tmax were 2.4, 2.8 and 0.8 h, whereas those for Cmax were 0.25, 0.56 and 1.38 micrograms/ml for one HBS capsule, two HBS capsules and standard Madopar, respectively. The mean relative bioavailability (versus standard Madopar) was 58 and 67% (value normalized to dose) for one and two HBS capsules, respectively. The parameter of half-value duration (= time span where plasma concentrations are equal to or higher than the half Cmax) was on average 3.5, 3.8 and 0.8 h for one HBS capsule, two HBS capsules and standard Madopar, respectively. Following benserazide pretreatment the mean tmax values for levodopa were 2.8, 2.3 and 0.8 h and the mean Cmax values were 0.35, 0.60 and 1.33 micrograms/ml, respectively, for one HBS capsule, two HBS capsules and standard Madopar. The relative bioavailability (versus standard Madopar) was 57 +/- 14 and 63 +/- 21% (value normalized to dose) for one and two HBS capsules, respectively. The mean values of the half-value duration were 3.6, 4.2 and 1.3 h for one HBS capsule, two HBS capsules and standard Madopar, respectively. For most of the parameters measured, the interindividual variability after Madopar HBS was less pronounced than after standard Madopar. In conclusion, according to these kinetic data, Madopar HBS shows the characteristics of a controlled-release formulation. The reduced bioavailability of the HBS form (60% of that of the standard form) suggests that a higher daily dose of Madopar HBS should be used for clinical practice.

Administration, Oral↗
PubMed1985

Conversion of the neurotoxic precursor 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine into its pyridinium metabolite by human platelet monoamine oxidase type B.

MPTP (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine) causes selective and irreversible degeneration of the substantia nigra of human and non-human primates. In the central nervous system, the oxidative metabolism of MPTP to 1-methyl-4-phenyl-pyridinium (MPP+) by monoamine oxidase type B (MAO-B) seems to be a critical feature in the neurotoxic process. We now report that [3H]MPTP is rapidly converted in vitro into [3H]MPP+ by human platelet MAO-B. The formation of [3H]MPP+ in human platelets is prevented by specific MAO-B but not by MAO-A or by 5-hydroxytryptamine uptake inhibitors.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1985

Simultaneous determination of histamine and N alpha-methylhistamine in biological samples by an improved enzymatic single isotope assay.

A modified highly sensitive and specific radioenzymatic assay for the simultaneous determination of histamine (HA) and N alpha-methylhistamine (N alpha-MH) in tissues and body fluids is described. In the presence of the enzyme histamine-N-methyltransferase and of the methyl donor [3H]-S-adenosylmethionine, the transmethylation process was about seven times more effective for HA than for N alpha-MH. In the same conditions only very low amounts of N alpha, N alpha-dimethylhistamine (N alpha, N alpha-DMH) were converted into its 1-methylated derivative. The high degree of specificity attained by this method is due to the rapid quantitative extraction of the biological fluids, to the partially purified enzyme preparation and to the thin-layer chromatography system used which allows an excellent separation of the 3H-1-methyl products of HA, N alpha-MH and N alpha, N alpha-DMH. This method is highly sensitive for the assay of HA and N alpha-MH (detection limit 10 and 50 picograms, respectively), but due to lack in sensitivity, it cannot be extended to the measurement of N alpha, N alpha-DMH. The HA content of 20-30 samples can be determined in duplicate by one person in a working day. The concentrations of HA measured by this method in different biological samples (human whole blood, plasma, urine, gastric juice and skin biopsies) were in good agreement with the values reported in the literature. The presence of minute amounts of N alpha-MH in the human gastric juice was established by rigorous checking.

Body Fluids↗