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Biomedical subjects

J E Schultz

Publications and source records attributed to J E Schultz.

At least 19 recordsLinked to original sources
PubMed1996

Cloning and expression of a bovine adenylyl cyclase type VII specific to the retinal pigment epithelium.

A cDNA of a type 7 adenylyl cyclase isoform was cloned from a bovine retinal pigment epithelium cDNA library using oligonucleotides developed to conserved regions common to mammalian adenylyl cyclases. A 6.7 kb mRNA of very high abundance was uniquely present on Northern blots containing mRNA or total RNA from the pigment epithelium. This transcript was undetectable in all other tissues examined. The cDNA encoded a protein of 1,097 amino acids and exhibited the known doublet of 6 transmembrane-spanning regions in a hydrophobicity plot. The novel member of the type 7 adenylyl cyclase isoform was expressed in COS-1 cells. It was stimulated 10- and 20-fold by 10 microM GTP gamma S and 100 microM forskolin, respectively. The high expression rate exclusively in the retinal pigment epithelium suggests that this adenylyl cyclase isoform is involved in processes specific to this functionally exceedingly important subretinal cell layer.

Adenylyl Cyclases↗
PubMed1995

Evidence for involvement of opioid receptors in ischemic preconditioning in rat hearts.

The purpose of the present study was to investigate a possible role of opioid receptors in ischemic preconditioning (PC). To test this hypothesis, anesthetized, open-chest, male Wistar rats were subjected to five different protocols. In group I, the control group was subjected to 30 min of left coronary artery occlusion and 2 h of reperfusion. In group II, ischemic PC was elicited by three 5-min occlusion periods interspersed with 5 min of reperfusion. In group III, naloxone (NL, 3 mg/kg iv), a nonselective opioid antagonist, was given to nonpreconditioned rats 10 min before the 30-min occlusion period. Finally, NL was administered 10 min before preconditioning (NL + PC, group IV) or immediately after the last 5-min preconditioning period (PC + NL, group V). Infarct size (IS) as a percentage of the area at risk (AAR) (IS/AAR) was determined by 2,3,5-triphenyltetrazolium chloride staining. PC resulted in a marked reduction in myocardial IS from 45 +/- 5 to 8 +/- 1 (P < 0.05). NL treatment before or immediately after PC abolished this protective effect; however, NL had no effect on IS in non-PC rats. These results are the first to support the hypothesis that activation of opioid receptors may play an important role in ischemic PC in the rat myocardium.

Animals↗
PubMed1994

A membrane-bound protein phosphatase type 2C from Paramecium tetraurelia. Purification, characterization, and cloning.

We isolated the first membrane-bound type 2C serine/threonine protein phosphatase from the ciliated protozoan Paramecium tetraurelia (PtPP2C). Three isozymes of 33, 32, and 31 kDa with a specific activity of 1 mumol.min-1.mg1 were purified from the ciliary membrane. All enzymatic properties including (a) insensitivity toward inhibitors of other protein phosphatase families such as okadaic acid and microcystin, (b) absolute requirement for divalent cations, and (c) substrate specificity tested with synthetic phosphopeptides were identical to mammalian PP2C enzymes and identified the PtPP2C as a canonical PP2C in spite of it being about 25% smaller. The NH2-terminal was blocked. Microsequencing of six tryptic peptides established a relationship to other PP2C enzymes. The PtPP2C gene was obtained using degenerate oligonucleotide primers and the polymerase chain reaction. The gene coded for a 33-kDa protein with 300 amino acids and had an (A+T) content of 62%, typical for this protozoan. Nine of 15 Gln residues are encoded by TAA, a universal stop codon which codes for Gln in Paramecium. A large truncation at the COOH-terminal is responsible for the smaller size of the PtPP2C. Only a single transcript of 1 kilobase was detected with a Northern blot indicating that the 32- and 31-kDa proteins were proteolytic products of the 33-kDa enzyme. Sequence comparisons with PP2C enzymes from rat, rabbit, yeast, Arabidopsis, and Leishmania defined a highly diverged enzyme family which shares three conserved domains, I, II, and III, accounting for about 25% of the primary structure. We demonstrated further that the distances between domains I/II and II/III are very similar in all PP2C enzymes (9-13 and 74-80 amino acids, respectively). However, the amino acid sequences of the spacer regions are unrelated. In addition, the COOH-terminal ends of 100-200 amino acids which comprise 30-50% of the enzyme, display no identity. A dendrogramm shows that PtPP2C surprisingly is most closely related to the mammalian PP2C, and enzymes from Leishmania, Arabidopsis, and yeast are more distant relatives.

Amino Acid Sequence↗
PubMed1994

Cyclic AMP formation in Tetrahymena pyriformis is controlled by a K(+)-conductance.

Responses of the cAMP generating system of Tetrahymena to changes in the concentrations of external [K+] or [Ca2+] ions were examined. When Tetrahymena are equilibrated in high [K+] buffers, intracellular levels of cAMP decreased to 40% within 2 h. Hyperpolarization of the cells by dilution of external [K+] to one-eighth of its original concentration instantly stimulated intracellular cAMP formation. Manipulations of the K+ resting conductance of Tetrahymena by equilibration in buffers of different K+ content greatly affected the responsivity of the adenylyl cyclase. Hyperpolarization of the cell by addition of Ca2+ also resulted in a rapid generation of cAMP. Blockade of K+ conductances by the K+ channel blockers tetraethylammonium, quinine, and Cs+, dose-dependently inhibited hyperpolarization-stimulated cAMP formation. The data indicate that a hyperpolarization-activated K+ current is directly coupled to adenylyl cyclase regulation.

Animals↗
PubMed1994

Molecular cloning of a protein serine/threonine phosphatase containing a putative regulatory tetratricopeptide repeat domain.

Two novel protein serine/threonine phosphatases were cloned from a rat fat cell library with probes generated by a polymerase chain reaction-based cloning approach. One of these cDNAs encoded a protein presumably representing the rat homologue of PPV from Drosophila (75% identity of amino acids). The other novel cDNA encoded a protein phosphatase of 499 amino acids and was designated PPT. Its catalytic domain contains motifs typical for protein phosphatases but is only distantly related with PP1, PP2A, and PP2B (38-42% identical amino acids). When expressed in Escherichia coli, the catalytic domain of PPT exhibited protein phosphatase activity (dephosphorylation of phosphorylase a) that was inhibitable by okadaic acid. As a unique feature among other members of this gene family, PPT has an amino-terminal extension of 200 amino acids harboring three tandemly arranged tetratricopeptide repeat (TPR) motifs. This domain has previously been found in other proteins involved in the regulation of RNA synthesis or mitosis. mRNA of PPT was predominantly found in brain and, in lower levels, in testis, but was nearly undetectable in spleen, lung, skeletal muscle, kidney, and liver. It is suggested that the TPR domain of PPT may be involved in the regulation of the function of this novel protein phosphatase.

3T3 Cells↗
PubMed1993

In vivo development of teicoplanin resistance in a VanB Enterococcus faecium isolate.

Acquired vancomycin resistance in enterococci may be associated with teicoplanin susceptibility (VanB) or teicoplanin resistance (VanA). This paper characterizes the first instance of in vivo emergence of teicoplanin resistance in an Enterococcus faecium strain of VanB phenotype. Vancomycin-resistant (MIC, 256/512 micrograms/mL) E. faecium was isolated intermittently from a patient's blood over 4 months. The MIC of teicoplanin for the first 5 isolates was 1.0 micrograms/mL; it was 64 micrograms/mL for the final 2. Analysis of plasmid and chromosomal DNA revealed the isolates to be of clonal origin. Conjugal transfer of vancomycin resistance was not obtained. A vanB DNA probe hybridized with both teicoplanin-susceptible and resistant isolates, but a vanA probe failed to hybridize with any isolate. SDS-PAGE of membrane proteins from a teicoplanin-resistant isolate revealed constitutive production of a normally inducible 41-kDa protein. These findings challenge the ultimate utility of teicoplanin for treatment of infections caused by vancomycin-resistant enterococci.

Bacterial Proteins↗
PubMed1992

Purification, characterization and structure of protein phosphatase 1 from the cilia of Paramecium tetraurelia.

A type 1 serine/threonine protein phosphatase (PP1) which is mostly localized in the excitable ciliary membranes from the protozoan Paramecium, was purified to homogeneity. Approximately 4 micrograms enzyme of 37 kDa was isolated from 100 l axenic culture. The enzymic properties were characterized using phosphorylase a from rabbit skeletal muscle as a substrate and several known effectors of mammalian PP1. The protozoan PP1 was enzymically indistinguishable from its mammalian congener. The amino acid sequence of the Paramecium PP1 was deduced from its cDNA. The full-length clone was obtained in several steps starting with a pair of degenerate primers made according to the two most conserved peptides of rabbit PP1 and PP2A. The gene encodes a protein of 36,392 Da. The identity of the cloned gene and the isolated ciliary PP1 was unequivocally established by microsequencing of four tryptic and cyanogen-bromide peptides which were generated from the purified protein. Paramecium PP1 shows 75% amino-acid-sequence identity with rabbit PP1 alpha. Areas of major differences are the C-termini and N-termini and a sequence between residues 219-242.

Amino Acid Sequence↗
PubMed1992

Metabolism of inositol phosphates in the protozoan Paramecium. Characterization of a novel inositol-hexakisphosphate-dephosphorylating enzyme.

Basal and stimulated levels of inositol phosphates were determined in the protozoan Paramecium labelled with myo-[3H]inositol. Under resting conditions, intracellular InsP6 (phytic acid), InsP5 and InsP4 concentrations were 140, 10 and 2 microM, respectively. InsP5 was comprised of 56% Ins(1,2,3,4,5)P5 and/or Ins(1,2,3,5,6)P5, 40% Ins(1,2,4,5,6)P5 and/or Ins(2,3,4,5,6)P5 and small amounts of Ins(1,3,4,5,6)P5 and Ins(1,2,3,4,6)P5. InsP4 was mainly Ins(1, 4, 5, 6)P4 and/or Ins(3, 4, 5, 6)P4. Other inositol phosphates were not detected at a detection limit of 50-85 nM. Using various depolarizing and hyperpolarizing stimuli, no significant changes in level of inositol phosphates were observed in vivo, indicating that in the ciliate a contribution of inositol phosphates to signal-transduction mechanisms is unlikely. In homogenates prepared from myo-[3H]inositol-labelled cells, a marked relative increase in InsP3 and InsP4 over the concentrations in vivo was observed. These inositol phosphates were identified as degradation products of endogenous InsP6. A novel separation methodology for inositol phosphates was established to allow unequivocal assignment of phosphate locations of all dephosphorylated InsP6-derived products. The dephosphorylation was catalyzed by a phytase-like enzyme with a molecular mass of 240 kDa, most likely of a hexameric structure. The enzyme had a pH optimum of 7.0 and did not require divalent cations for activity. Substrate concentrations above 300 microM were inhibitory. Dephosphorylation of InsP6 by the Paramecium enzyme differs from that of phytases from plants in that it proceeds via a sequential release of phosphate groups from positions 6, 5, 4 and 3 of the myo-inositol ring or/and positions 4, 5, 6 and 1.

Animals↗
PubMed1992

Regulation of adenylyl cyclase from Paramecium by an intrinsic potassium conductance.

Hyperpolarization of the cell membrane of Paramecium stimulates adenosine 3',5'-monophosphate (cAMP) formation. Manipulations of the K+ resting conductance of the ciliate by adaptation in different buffers affected excitability of the cAMP generating system. Blockade of K+ channels inhibited hyperpolarization-stimulated cAMP formation. A mutant of Paramecium that is unable to control its K+ resting conductance had a defect in cAMP formation. Purified adenylyl cyclase, when incorporated into an artificial lipid bilayer membrane, revealed properties of a voltage-independent K+ channel. This indicates that the adenylyl cyclase of Paramecium has a secondary function as carrier of the K+ resting conductance. A hyperpolarization-activated K+ efflux appears to directly regulate adenylyl cyclase activity in vivo.

Adenylyl Cyclases↗
PubMed1992

Metabolic products of microorganisms. 261. Obscurolides, a novel class of phosphodiesterase inhibitors from streptomyces. I. Production, isolation, structural elucidation and biological activity of obscurolides A1 to A4.

A novel class of butyrolactones, named obscurolides, was isolated from the culture filtrate of Streptomyces viridochromogenes by chemical screening methods. The structural elucidation of the obscurolides A1 to A4 (1 approximately 4) is described. The carboxy group of the 4-aminobenzoic acid moiety of obscurolide A1 (1) is reduced in the other compounds. The isolated natural products have been proved to be diastereomeric mixtures by a partial racemization at C-7 which belongs to an allylic alcohol system. The obscurolides showed a weak inhibitory activity against calcium/calmodulin-dependent and independent phosphodiesterases from bovine.

4-Butyrolactone↗
PubMed1991

Identification of a 42 kDa protein as a substrate of protein phosphatase 1 in cilia from Paramecium.

Okadaic acid, a specific inhibitor of protein phosphatase 1 in Paramecium causes sustained backward swimming in response to depolarising stimuli (S. Klumpp et al. (1990) EMBO J. 9, 685). Here, we employ okadaic acid, tautomycin, microcystin LR and inhibitor 1 as phosphatase inhibitors to identify a 42 kDa protein in the excitable ciliary membrane that is dephosphorylated by protein phosphatase 1. Identification of the 42 kDa protein was facilitated by the finding that the protein kinase responsible for its phosphorylation uses Ca-ATP as a substrate just as effectively as Mg-ATP. Notably, dephosphorylation of the 42 kDa protein is specifically inhibited by cyclic AMP; cyclic GMP has no effect.

Animals↗
PubMed1991

Molecular and functional characterization of a carbon starvation gene of Escherichia coli.

Escherichia coli induces the synthesis of at least 30 proteins at the onset of carbon starvation, two-thirds of which are positively regulated by the cyclic AMP (cAMP) and cAMP receptor protein (CRP) complex. Two of the cAMP-CRP-dependent genes mapped to 14 and 93 minutes of the chromosome and are designated cstA and cstB, respectively. The cstA promoter region was cloned and localized to a 600 base-pair fragment downstream from the iron-regulated entCEBA-P15 operon. Carbon starvation-inducible transcription initiated at three sites spaced one turn of the DNA helix apart. All had--10 sequences similar to consensus E sigma 70 promoters and poor--35 sequences. Deletion of a putative CRP binding site abolished carbon starvation-mediated induction. Sequence analysis of the cstA coding region revealed the presence of three sequential open reading frames potentially encoding two hydrophobic proteins of 60,223 Da and 15,201 Da and a hydrophilic protein of 7467 Da. Overexpression of the cstA region produced starvation-inducible proteins of the expected sizes. Suggestive evidence was obtained that cstA is involved in peptide utilization.

Amino Acid Sequence↗
PubMed1991

Differential effects of ovarian steroid hormones on beta-adrenoceptor downregulation caused by the antidepressants imipramine and rolipram.

Downregulation of beta-adrenoceptors in response to repeated administration of imipramine or rolipram was investigated in the brain of pregnant rats and ovariectomized animals treated with estrone, progesterone or a combination of both hormones. Ovariectomy alone was without influence on drug responses. Pregnancy and complete hormone replacement of animals prior to drug treatment halved the response to imipramine and obliterated that to rolipram. Administration of only estrone to ovariectomized rats did not affect the extent of adrenergic downregulation caused by imipramine, but fully suppressed the rolipram action. Castrated males with or without treatment with estrone and progesterone were used as further controls. Castration alone diminished the effects of both antidepressants. Treatment of gonadectomized rats with both ovarian steroid hormones voided the actions of rolipram. The data indicate that imipramine and rolipram share a common pathway for their mechanism of action which can be attenuated by ovarian steroid hormones. In addition, imipramine has a second major site of action which is not subject to modulation by female steroids.

Animals↗
PubMed1990

Chromatographic separation of four Ser/Thr-protein phosphatases from solubilized ciliary membranes of Paramecium tetraurelia by heparin-sepharose.

Chromatography of deoxycholate-solubilized proteins from Paramecium ciliary membranes on heparin-Sepharose resolved three peaks of protein phosphatase activities: one type 2A-like and a type 2C phosphatase in the flow-through fractions, another type 2A-like enzyme in the 0.1 M NaCl eluate and type 1 protein phosphatase in the 0.5 M NaCl eluate. The differential sensitivity of the two type 2A-like phosphatases to heparin and protamine further substantiated the existence of distinct isozymes. Once solubilized, none of these ciliary phosphatases required detergent to remain soluble. The molecular mass as determined by chromatography on Superose 6 was in the range 30,000-45,000 dalton for all four protein phosphatases.

Animals↗
PubMed1990

Guanylate cyclase in olfactory cilia from rat and pig.

A guanylate cyclase was identified in cilia from rat and pig olfactory epithelia. Enzyme activities were 200-250 and 90-100 pmol/min.mg-1, respectively. Activity required the presence of non-ionic detergents, e.g., 0.1% Lubrol PX. MnGTP, not MgGTP was used as a substrate. Furthermore, 0.9 mM free Mn2+ was necessary for optimal activity indicating a regulatory site for a divalent cation. The guanylate cyclase displayed sigmoidal Michaelis-Menten kinetics suggesting cooperativity between MnGTP and enzyme. S0.5 was 160 microM MnGTP. The Hill coefficient of 1.7 indicates that more than one class of substrate-binding sites interact in a positive cooperative manner. ATP inhibited the enzyme and linearized plots of substrate kinetics with MnGTP. SH-Blocking agents reversibly inhibited enzyme activity. Sodium azide and nitroprusside were without effect as were several odorants. A guanylate cyclase activity in cilia from tracheal tissue had properties similar to the olfactory enzyme.

Adenosine Triphosphate↗