Preparation of soluble guanylyl cyclase from bovine lung by immunoaffinity chromatography.
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Amino Acid Sequence↗
Biomedical subjects
G Schultz
Publications and source records attributed to G Schultz.
Assessment of biological activity of synthetic fragments of transforming growth factor alpha.
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Amino Acid Sequence↗
Long-term morbidity of adjuvant infradiaphragmatic irradiation in patients with testicular cancer and implications for the treatment of stage I seminoma.
Long-term abdominal or urological morbidity and second malignancies in 289 surviving patients with infradiaphragmatic adjuvant radiotherapy (RT) for testicular cancer between 1950 and 1988 are analysed by retrospective chart review. After RT with single doses between 1.5 and 2.0 Gy and a total dose between 30 and 35 Gy, we did not observe any peptic ulcer nor other abdominal or urological long-term morbidity, except second tumours. The cumulative incidence of 16 second extratesticular malignancy was (in years after RT): 0.4% (4 years), 1.3% (9 years), 4.5% (14 years), 6.3% (19 years), 7.5% (24 years), 15.6% (29 years) and 23.6% (35 years). The ratio of observed to expected incidence of extratesticular malignancies did not differ significantly from unity; only the frequency of penile cancer (n = 2) was somewhat higher than expected. The cumulative risk of bilateral testicular cancer was 4.8% with no difference between patients with seminoma or non-seminomatous germ cell tumours. In a recent group of 128 patients with a stage I seminoma staged and treated between 1978 and 1988 by modern standard, there was no recurrence.
Diaphragm↗
Electrophysiological properties of rat calcitonin-secreting cells.
The spontaneous electrical activity of calcitonin-secreting cells (C-cells) appears to play an important role in the coupling of fluctuations in the extracellular Ca2+ to changes in the intracellular Ca2+ concentration and thus for calcitonin secretion. Using the patch clamp technique, we have investigated the spontaneous electrical activity and the underlying ionic currents in C-cells of the rMTC 44-2 cell line. With 1.2 mM external Ca2+, the membrane potential was -46.1 +/- 1.7 mV (n = 58) and about 30% of the cells spontaneously fired action potentials. Rising the external Ca2+ to 1.8 mM caused the cells to depolarize to -42.1 +/- 2.1 mV (n = 56) and spontaneous electrical activity was seen in about 70% of cells. Under voltage clamp conditions, tetrodotoxin-sensitive voltage-dependent Na+ currents, outward-rectifying K+ currents and isradipine-, omega-conotoxin-sensitive as well as isradipine- and omega-conotoxin-insensitive Ca2+ currents were observed. These voltage-dependent currents appear to be the major ionic currents contributing to action potentials in C-cells and to participate in calcitonin secretion.
Action Potentials↗
Ras proteins activate calcium channels in neuronal cells.
Ras (p21) proteins are involved in the control of cell growth and differentiation, but the mechanism by which they exert these effects is not yet known. Here we present evidence that c-Ha-ras (p21(Gly-12)) and its oncogenic mutant T24-ras (p21(Val-12)) selectively induce omega-conotoxin and dihydropyridine-sensitive Ca2+ currents within a few hours after introduction into the cytoplasm of neuroblastoma x glioma hybrid cells. Whereas control cells exhibited a mean Ca2+ current of 250 pA, it amounted to 730 pA in cells pretreated with ras protein. In cells loaded with p21(Gly-12), the effect occurred after 2 hours and was terminated after 8 hours. In contrast, introduction of p21(Val-12) resulted in a prolonged delay (6 hours) of the effect which lasted for more than 24 hours. When ras proteins were preactivated with the non-hydrolysable GTP analog GppNHp, the time courses of both p21(Gly-12) and p21(Val-12) effects were fast and sustained, suggesting that in intact cells (i) the GDP/GTP exchange is faster for p21(Gly-12) compared to p21(Val-12) and (ii) inactivation of p21(Gly-12) is mediated by GAP-induced GTPase activity. T-type Ca2+ currents and K+ currents were unaffected by ras proteins.
Animals↗
Differential regulation of chemoattractant-induced superoxide formation in human neutrophils by cell-permeant analogues of cyclic AMP and cyclic GMP.
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Bucladesine↗
The Charles Bonnet syndrome: 'phantom visual images'.
The Charles Bonnet syndrome is a condition in which individuals experience complex visual hallucinations without demonstrable psychopathology or disturbance of normal consciousness. An analysis of the sixty-four cases described in the literature reveals that the syndrome can occur at any age though it is more common in elderly people. Reduction in vision, due to peripheral eye pathology as well as pathology within the brain, is associated with the syndrome. Individual hallucinatory episodes can last from a few seconds to most of the day. Episodes can occur for periods of time ranging from days to years, with the hallucinations changing both in frequency and in complexity during this time. The hallucinations may be triggered or stopped by a number of factors which may exert their effect through a general arousal mechanism. People, animals, buildings, and scenery are reported most often. These images may appear static, moving in the visual field, or animated. Emotional reaction to the hallucinations may be positive or negative. Several theories have been proposed to account for the hallucinations. This paper highlights the sensory deprivation framework, with particular emphasis on the activity in the visual system after sensory loss that produces patterns of nerve impulses that, in turn, give rise to visual experience.
Brain Damage, Chronic↗
Guanylyl cyclases, a growing family of signal-transducing enzymes.
Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other signal-transducing enzymes such as protein tyrosine-kinases and protein tyrosine-phosphatases. So far, three isoforms of mammalian membrane-bound guanylyl cyclases (GC-A, GC-B, GC-C), which are stimulated by either natriuretic peptides (GC-A, GC-B) or by the enterotoxin of Escherichia coli (GC-C), have been identified. These proteins belong to the group of receptor-linked enzymes, with different NH2-terminal extracellular receptor domains coupled to a common intracellular catalytic domain. In contrast to the membrane-bound enzymes, the heme-containing soluble guanylyl cyclase is stimulated by NO and NO-containing compounds and consists of two subunits (alpha 1 and beta 1). Both subunits contain the putative catalytic domain, which is conserved in the membrane-bound guanylyl cyclases and is found twice in adenylyl cyclases. Coexpression of the alpha 1- and beta 1-subunit is required to yield a catalytically active enzyme. Recently, another subunit of soluble guanylyl cyclase was identified and designated beta 2, revealing heterogeneity among the subunits of soluble guanylyl cyclase. Thus, different enzyme subunits may be expressed in a tissue-specific manner, leading to the assembly of various heterodimeric enzyme forms. The implications concerning the physiological regulation of soluble guanylyl cyclase are not known, but different mechanisms of soluble enzyme activation may be due to heterogeneity among the subunits of soluble guanylyl cyclase.
Amino Acid Sequence↗
Inhibition of Ca2+ channels via alpha 2-adrenergic and muscarinic receptors in pheochromocytoma (PC-12) cells.
Biochemical studies have suggested a voltage-dependent dihydropyridine-sensitive catecholamine release in adrenal chromaffin cells. This release is inhibited by activation of alpha 2-adrenergic and muscarinic receptors; the underlying molecular mechanism is not known. We used undifferentiated PC-12 cells to study the effect of epinephrine and carbachol on transmembranous currents. Applying the patch-clamp technique in the whole cell configuration and using Ba2+ as charge carrier, we identified a high voltage-activated Ca2+ channel current. Both epinephrine (10 microM, in the presence of 1 microM propranolol) and carbachol (10 microM) reversibly inhibited the Ca2+ channel current by 30-40%. Yohimbine abolished and clonidine mimicked the effect of epinephrine. Phenylephrine failed to inhibit the Ca2+ channel current. The effect of carbachol was abolished by atropine. Epinephrine and carbachol did not affect the Ca2+ channel current reduced by the dihydropyridine, PN 200-110 (1 microM), suggesting a selective inhibition of dihydropyridine-sensitive Ca2+ channels. The Ca2+ channel current and its inhibition by receptor agonists were not influenced by intracellularly applied adenosine 3',5'-cyclic monophosphate (cAMP; 100 microM). Pretreatment of cells with pertussis toxin or intracellular infusion of the GDP analogue guanosine-5'-O-(2-thiodiphosphate) was without effects on the control Ca2+ channel current but abolished its hormonal inhibition. Four pertussis toxin-sensitive G proteins were identified in membranes of PC-12 cells: two members of the Gi family, Gi1 and Gi2, and two members of the Go family, Go2 and another Go subtype (possibly Go1). The present data indicate that activated alpha 2-adrenergic and muscarinic receptors inhibit dihydropyridine-sensitive Ca2+ channels via pertussis toxin-sensitive G proteins without the involvement of a cAMP-dependent intermediate step.
Adrenal Gland Neoplasms↗
Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart.
Morphological, electrophysiological, and biochemical properties of H9c2 cells, a permanent cell line derived from rat cardiac tissue, were studied. Although the lectin binding pattern revealed similar sugar residues in the surface coat of H9c2 cells and isolated rat cardiocytes, heart-specific morphological structures could not be detected in H9c2 cells. Under physiological ionic conditions, H9c2 cells exhibited an outwardly rectifying, transient K+ current. When this current component was blocked by Ba2+ and Cs+, we observed an inward current through Ca2+ channels (15.8 +/- 2.2 pA/pF, n = 18, measured as Ba2+ current) that showed all characteristics of cardiac L-type currents. The activation kinetics were fast, and the current was stimulated by isoproterenol. The effect of isoproterenol was mimicked by forskolin or intracellularly applied cAMP. In radioligand binding experiments, we identified a specific, saturable, stereoselective and reversible high-affinity [3H]-(+)PN 200-110 binding with a dissociation constant Kd = 0.53 +/- 0.28 nM and a maximal specific binding of Bmax = 129.3 +/- 16.1 fmol/mg protein. There was an additional low-affinity/high-capacity binding site, which is unlikely to be related to a Ca2+ channel protein. Signal-transducing G proteins in membranes were characterized by [32P]ADP-ribosylation catalyzed by bacterial toxins and by the use of various antibodies. Cholera toxin substrates of 42 and 45 kd were identified that apparently correlated to Gs alpha-subunits. Pertussis toxin substrates of 40-41 kd were tentatively identified as Gi alpha-subunits. The G protein Go was absent or at least extremely low in concentration.
Adrenergic beta-Agonists↗
Inhibition of voltage-dependent Ca2+ currents and activation of pertussis toxin-sensitive G-proteins via muscarinic receptors in GH3 cells.
In the rat pituitary cell line GH3, carbachol inhibits PRL secretion in a pertussis toxin-sensitive manner. For elucidation of the underlying mechanisms, we studied the effect of carbachol on voltage-dependent Ca2+ currents. Under voltage-clamp conditions, carbachol inhibited whole-cell Ca2+ currents by about 25%. This inhibitory action of carbachol was not observed in cells treated with pertussis toxin, indicating the involvement of a pertussis toxin-sensitive G-protein. In membranes of GH3 cells, carbachol stimulated a pertussis toxin-sensitive high-affinity GTPase. In immunoblot experiments with peptide antisera, we identified two forms of the Gi alpha-subunit (41 and 40 kDa) and two forms of the Go alpha-subunit (40 and 39 kDa). The 40-kDa Gi alpha-subunit was recognized by an antibody specific for the Gi2 alpha-subunit, and the 39-kDa Go alpha-subunit was detected by an antibody specific for the Go2 alpha-subunit. Incubation of membranes with the photoreactive GTP analog [alpha-32P]GTP azidoanilide resulted in photo-labelling of 40- and 39-kDa pertussis toxin substrates comigrating with G-protein alpha-subunits of the corresponding molecular masses. Carbachol dose-dependently stimulated incorporation of the photoreactive GTP analog into the 39-kDa pertussis toxin substrate and, to a lesser extent, into 40-kDa pertussis toxin substrates. The data indicate that muscarinic receptors of GH3 cells couple preferentially to Go, which is likely to be involved in the inhibition of secretion, possibly by conferring an inhibitory effect to voltage-dependent Ca2+ channels.
Affinity Labels↗
Improved purification and characterization of membraneous and cytosolic inositol phospholipid-specific phospholipases C from porcine brain cortex.
A phospholipase C was solubilized and purified from membranes of porcine brain cortex. Simultaneously, a phospholipase C was purified from a cytosolic fraction of porcine brain cortex. The enrichment of phospholipase C from either fraction was about 1000-fold as determined by hydrolysis of phosphatidylinositol 4,5-bisphosphate. Phospholipases C purified from membranes or from cytosol were indistinguishable with regard to the following properties: The enzyme activities copurified with a protein of 145 kDa. The standard sedimentation coefficients (s20,w values) of the purified enzymes were 6.2 S in the absence or presence of 0.3% (w/v) sodium cholate; Stokes' radii, estimated by gel filtration on a Superose 6 HR 10/30 column in the presence of 0.3% sodium cholate, were 4.5 nm; calculated molecular masses were about 120 kDa; no significant hydrolysis of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine by preparations of purified phospholipase C was observed; adenine and guanine nucleotides affected the activity of purified enzymes in a complex manner. Thus, the enzymes purified from membraneous and from cytosolic fractions exhibited properties of the phospholipase C-beta form. The enzymes purified from either fraction required Ca2+ at a low concentration (100 nM to 10 microM) for maximal activity. The advantage of the present purification procedure is that the purified enzymes were free of phosphatidylinositol 4,5-bisphosphate 5-phosphatase, inositol 1,4,5-trisphosphate 5-phosphatase and guanine nucleotide-binding proteins after three chromatographic steps. The purified enzymes may, therefore, prove useful for studying the hormonal regulation of phospholipase C in reconstituted systems and for the preparation of [5-32P]inositol 1,4,5-trisphosphate from [5-32P]phosphatidylinositol 4,5-bisphosphate.
Animals↗
The superoxide-forming NADPH oxidase of phagocytes. An enzyme system regulated by multiple mechanisms.
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Animals↗
Stimulation of healing of chronic wounds by epidermal growth factor.
We evaluated the effect of topical epidermal growth factor treatment on healing of chronic wounds in a prospective, open-label, crossover trial. Five males and four females who ranged in age from 40 to 72 years (average 57 +/- 9 years) were enrolled. Four patients had adult-onset diabetes mellitus, two had rheumatoid arthritis, two had old burn scars, and one had a failed abdominal incision. The average duration of the ulcers prior to treatment with epidermal growth factor was 12 +/- 5 months (range 1 to 48 months). Following failure of the wounds to heal with conventional therapies, including debridement, skin graphs, and vascular reconstruction, wounds were treated twice daily with Silvadene alone for periods ranging from 3 weeks to 6 months. No evidence of healing was observed in any of the patients' wounds during Silvadene treatment, and patients were crossed over to twice a day treatment with Silvadene containing 10 micrograms epidermal growth factor per gram. Wounds of eight patients healed completely with epidermal growth factor-Silvadene treatment in an average of 34 +/- 26 days (mean +/- SD, range 12 to 92 days) and did not reoccur for periods ranging from 1 to 4 years. One patient failed therapy. These results suggest that topical treatment of chronic wounds with epidermal growth factor may stimulate healing.
Administration, Cutaneous↗
A slowly inactivating calcium current works as a calcium sensor in calcitonin-secreting cells.
Calcitonin (CT)-secreting cells (C-cells) are remarkably sensitive to changes in the extracellular Ca2+ concentration. In order to detect the mechanism by which C-cells monitor Ca2+, we compared a C-cell line responding to Ca2+ (rMTC cells) with another one known to have a defect in this Ca2+ signal transduction (TT cells). Rises of the Ca2+ concentration caused rMTC cells to depolarize and/or elicited spontaneous action potentials. Under voltage-clamp conditions, rMTC cells showed a slowly decaying Ca2+ inward current which was sensitive to dihydropyridines but not to Ni2+ at a low concentration. In contrast, the 'defective' TT cells neither depolarized nor fired action potentials with high Ca2+; they only exhibited an Ni2(+)-sensitive, transient Ca2+ current. The data strongly suggest that the slowly inactivating Ca2+ current is a prerequisite for Ca2(+)-sensitivity of C-cells and that fast inactivating channels are not sufficient to act as sensors of the extracellular Ca2+ concentration.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Expression of soluble guanylyl cyclase. Catalytic activity requires two enzyme subunits.
Purified soluble guanylyl cyclase consists of two subunits (70 and 73 kDa) whose primary structures were recently determined. The availability of cDNA clones coding for either subunit allowed to study the question of the functional roles of the two subunits in expression experiments. Enzyme subunits were expressed in COS-7 cells by transfection with expression vectors containing the coding region for the 70 of 73 kDa subunit of the enzyme. No significant elevation in the activity of soluble guanylyl cyclase was observed in cells transfected with cDNA coding for one of the subunits. In contrast, transfection of cells with cDNAs coding for both subunits resulted in a marked increase in activity of soluble guanylyl cyclase. Enzyme activity was stimulated about 50-fold by sodium nitroprusside. The results indicate that formation of cyclic GMP by soluble guanylyl cyclase requires both 70 and 73 kDa subunits.
Animals↗
Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method.
Soluble guanylyl cyclase was purified from bovine lung by an immunoaffinity chromatographic method using IgG fractions of antisera against a synthetic peptide of the C-terminus of the 70-kDa subunit of the enzyme. After anion-exchange chromatography, the enzyme was bound to an immunoaffinity column and was eluted with the synthetic peptide. This method allowed the convenient isolation of 2 mg of apparently homogeneous enzyme from 40 g cytosolic proteins. The enzyme had an apparent molecular mass of about 150 kDa and consisted of two subunits (70 kDa and 73 kDa) as determined by gel permeation fast protein liquid chromatography and SDS/PAGE. The basal activities determined in the presence of Mg2+ and Mn2+ were 10-20 nmol.min-1.mg-1 and 80-100 nmol.min-1.mg-1, respectively. The enzyme exhibited an ultraviolet-visible absorption spectrum typical for hemoproteins, with a Soret band at 430 nm. The purified enzyme was stimulated by NO-containing compounds. Maximal enzyme activities measured in the presence of sodium nitroprusside were 1.2-2.4 mumol.min-1.mg-1 (half-maximal effect of sodium nitroprusside at 1.3-1.9 microM) and 0.9-1.8 mumol.min-1.mg-1 (half-maximal effect at 0.28-0.41 microM sodium nitroprusside) in the presence of Mg2+ and Mn2+, respectively. The method developed for the large-scale purification of soluble guanylyl cyclase by immunoaffinity chromatography, using synthetic peptides for the elution of the enzyme, appears to be superior to previously described methods. As antibodies against synthetic peptides corresponding to deduced amino acid sequences of the respective protein are easily obtained, the described method may be suitable for a convenient large-scale purification of various proteins.
Animals↗
The primary structure of the larger subunit of soluble guanylyl cyclase from bovine lung. Homology between the two subunits of the enzyme.
The primary structure of the larger subunit of the soluble guanylyl cyclase from bovine lung, which catalyzes the formation of cyclic GMP from GTP, has been determined. Two clones, isolated from two bovine libraries yielded a total of 3261 bp with a coding region of 2073 bp. The open reading frame encodes a protein of 691 amino acids and a molecular mass of 77,500. The deduced amino acid sequence reveals regions which are, to a large extent, homologous to the sequence of the smaller subunit of the enzyme as well as to the sequences of other gyanylyl and adenylyl to a large extent, homologous to the sequence of the smaller subunit of the enzyme as well as to the sequences of other gyanylyl and adenylyl cyclases.
Amino Acid Sequence↗