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M Svoboda

Publications and source records attributed to M Svoboda.

At least 91 records · Page 5Linked to original sources
PubMed1986

Rapid purification of Bordetella pertussis toxin by alternating affinity and hydrophobic chromatography.

Pertussis toxin was purified to homogeneity from a 2-day culture supernatant of Bordetella pertussis by stepwise elution from three columns of, consecutively, Blue Sepharose, phenyl Sepharose, and hydroxyapatite. The toxin was eluted from Blue Sepharose and hydroxyapatite by high ionic strength and from phenyl Sepharose with low ionic strength and with 17% glycerol. Toxin fractions from one chromatographic column were immediately charged on the next column, saving laborious and time-consuming concentration or dialysis steps. Based on peptide composition (after sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and on HPLC profile (under nondenaturing conditions), the toxin was already practically pure after two steps, the third hydroxyapatite column serving only to separate the whole native toxin from any free S1 subunit. Recovery was estimated from the capacity of the preactivated toxin (and any preexisting free S1 subunit) to catalyze the ADP-ribosylation of the guanine nucleotide binding protein Ni in rat pancreatic plasma membranes: of the total capacity initially present in the culture medium, 23% could be recovered as pure native toxin with the present procedure. Besides, the nondenaturing HPLC method used to check the purity of the native toxin appeared to be superior to classical acidic polyacrylamide gel electrophoresis.

Adenosine Diphosphate Ribose↗
PubMed1986

Identification of two pro-VIP forms in a human neuroblastoma cell line.

The immunoreactivity of VIP and PHI standards, immobilized on a nitrocellulose membrane, was first assayed with various detection procedures. For VIP, the double bridge peroxidase-antiperoxidase (PAP) method was the most sensitive procedure, giving a detection limit of 0.1-0.3 pmol per mm2 with the 4 rabbit anti-VIP antisera tested. By contrast, the detection limit of immobilized PHI was 100 times higher with the 4 rabbit anti-PHI/PHM antisera tested presumably because major antigenic sites were masked in the immobilized peptide. With this information at hand, the VIP and PHI immunoreactivity of human neuroblastoma NB-OK-1 cells was tested after extraction, SDS-PAGE, electrotransfer, and PAP immunodetection. Two faint immunoreactive bands corresponding to two pro-VIP forms with an Mr of, respectively, 19 kDa and 18 kDa, were detected in undifferentiated cells. These distinct bands increased progressively and markedly during differentiation in the presence of dibutyryl cyclic AMP. In addition, two intermediary VIP forms of lower Mr (11 kDa and 6 kDa) and 3 kDa VIP itself were also present after 2 days of differentiation. The 19 kDa and 18 kDa pro-VIP forms were detected with a sensitivity several times higher than that of VIP and their staining was specific for VIP epitopes. By contrast, when using 4 rabbit anti-PHI/PHM antisera, we observed essentially the strong unspecific staining of a 17 kDa polypeptide. VIP immunoreactivity was also visualized by immunocytochemistry in neuroblastoma cells cultured on glass coverslips and fixed in situ. Specific VIP staining using the PAP method was present in 10 percent of the cells in the undifferentiated state.(ABSTRACT TRUNCATED AT 250 WORDS)

Bucladesine↗
PubMed1986

Effector mechanisms of peptides of the VIP family.

The present review is focused on the exocrine pancreas and liver where the only known effector mechanism of VIP is the activation of adenylate cyclase in plasma membranes. A two-state model of activation-deactivation of the enzyme visualizes the participation of VIP receptors and Ns, the guanyl nucleotide stimulatory protein of adenylate cyclase. In the rat pancreas, VIP and GRF receptors are indistinguishable and disulfide bridges influence their functional integrity. The antagonism of VIP and somatostatin perhaps requires, at the adenylate cyclase level, the contribution of Ni, the guanyl nucleotide inhibitory protein. The potentiation of VIP by various stimulants acting on Ca2+ movements may rely on later events, e.g., on a concerted activation of protein kinases. When comparing quantitatively peptide binding to receptors with adenylate cyclase activation, cyclic AMP levels and amylase secretion, a tool is at hand to tailor synthetic agonists and antagonists of VIP, with appropriate changes in the N-terminal moiety of the peptide (a good agonist allows efficient coupling of receptors to the adenylate cyclase system). Apart from stimulus-secretion coupling, VIP may influence protein synthesis in the rat pancreas, through the phosphorylation of ribosomal protein S6, and may alter the activity of the endoplasmic reticulum via the phosphorylation of Mr = 21 kDa and Mr = 25 kDa proteins. In rat liver membranes, high affinity VIP receptors are specifically labelled with 125I-helodermin and are coupled to adenylate cyclase (at variance with low affinity VIP receptors). These receptors are highly responsive to divalent cations and to guanyl nucleotides.

Adenylyl Cyclases↗
PubMed1986

[Incidence of Toxoplasma gondii oocysts in cat feces].

Within two years and a half, the faeces of 620 cats coming from Brno and the area around the city were subjected to parasitological examination with special regard to the occurrence of the oocysts of Toxoplasma gondii. Sucrose solution at the specific weight of 1,150 was used as flotation medium. Oocysts of Toxoplasma gondii were eliminated by eight cats (1.29%) at the age from 16 days to 1.5 years. Six of the eight cats were younger than seven months. The Toxoplasma gondii oocysts were eliminated by the cats for 1-16 days while exhibiting signs of short-term scours and swelling of lymph nodes. In all cases the oocysts of Toxoplasma gondii were produced in the summer and autumn seasons (June-December). During the patent period, other coccidia (Isospora felis and Isospora rivolta) were also present in the cats.

Animals↗
PubMed1985

Rapid electrotransfer of proteins from polyacrylamide gel to nitrocellulose membrane using surface-conductive glass as anode.

A new type of inexpensive horizontal apparatus for the electrophoretic transfer of proteins from a gel to an immobilization membrane has been developed. In this system, gel and membrane were directly pressed between two flat electrodes. A surface-conductive glass was used as anode and a stainless-steel plate as cathode. Proteins could be transferred from polyacrylamide gel to nitrocellulose sheet, with a yield of at least 90% in 60-90 min, without overheating, using a voltage gradient of 30-40 V/cm. Moderate volumes of separate anodic (with 20% methanol) and cathodic (with 0.1% sodium dodecyl sulfate) buffers were suited for optimal transfer of proteins with relative molecular mass (Mr) in the 14,000-94,000 range.

Animals↗
PubMed1985

Presence of helodermin-like peptides of the VIP-secretin family in mammalian salivary glands and saliva.

Helodermin is a biologically active peptide isolated from the venom of the Gila monster lizard (Heloderma suspectum) whose structure is related to that of vasoactive intestinal peptide and secretin. Using a specific radioimmunoassay based on antisera prepared by immunizing rabbits with natural helodermin, we demonstrated the presence of helodermin-like material in mammalian salivary glands, including parotid, submaxillary and sublingual glands from rat and dog, and parotid and submaxillary glands from man. All helodermin-like materials had an apparent molecular mass of 4-12 kDa. Dog saliva, collected after pilocarpine stimulation, revealed similar immunoreactivity with a major component around 6 kDa.

Amino Acid Sequence↗
PubMed1985

Solubilization from rat pancreatic plasma membranes of a cholecystokinin (CCK) agonist-receptor complex interacting with guanine nucleotide regulatory proteins coexisting in the same macromolecular system.

Using the non-denaturing detergent 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate (Chaps), cholecystokinin (CCK) receptors were solubilized from rat pancreatic membranes as a reversible complex with the CCK 31-39 nonapeptide 125I-labelled by the Bolton and Hunter reagent. Bound ligand dissociation from this soluble complex was similar to that from the membranous receptors of origin and the marked increase in the rate of dissociation induced by GTP was preserved in the soluble state, indicating that the solubilized CCK receptors remained functionally coupled with the guanine nucleotide regulatory site modulating the affinity for CCK. In fact, two guanine nucleotide regulatory proteins, Ns and Ni, coexisted in the soluble complex as established by identifying the 42-kDa subunit of Ns and the 40-kDa subunit of Ni, after ADP-ribosylation by cholera toxin and Bordetella pertussis toxin, respectively.

Animals↗
PubMed1985

Stimulation by cholera toxin of ADP-ribosylation of membrane proteins, adenylate cyclase and insulin release in pancreatic islets.

In rat pancreatic islet membranes exposed to [alpha-32P]NAD, cholera toxin stimulated the labelling of three peptides with Mr close to 22 000, 42 000 and 48 000, respectively. In the islets, the toxin-stimulated ADP-ribosylation of the heavy form of the Ns alpha-subunit predominated over that of the light form, in mirror image of the situation found in the exocrine pancreas. When intact islets were preincubated with cholera toxin, the adenylate cyclase activity of a subcellular particulate fraction was increased. The responsiveness of adenylate cyclase to GTP was also augmented, but that to NaF was decreased. In intact islets, the production of cyclic AMP and the glucose-stimulated release of insulin were also enhanced after pretreatment with cholera toxin. These findings reveal the presence in pancreatic islets of the guanyl nucleotide regulatory protein of adenylate cyclase, with an unusual predominance of the heavy form of the Ns alpha-subunit.

Adenosine Diphosphate Ribose↗
PubMed1985

[Neonatal toxoplasmosis in kittens].

The occurrence of toxoplasmosis is described in the cat-mother and three kittens. Clinical symptoms were not significant: bristled hair, conjunctivitis, sneezing and hypertrophy of mandibular and popliteal lymph nodes. The titres of antibodies to Toxoplasma gondii were demonstrated in all kittens and also in the cat: Sabin-Feldman reaction titre 4-32, complement-fixing reaction titre 5-40. Microprecipitation in agar gel was positive in all animals. Oocytes of Toxoplasma gondii were eliminated through faeces by the cat and two 16-day-old kittens. An isolation trial on white mice with a negative finding of toxoplasmosis (inoculation of brain, liver and spleen samples) has demonstrated toxoplasmosis in all animals under study. Most probably the kittens were infected transplacentally.

Animals↗
PubMed1984

Cleaving of disulfide bridges and apparent molecular weight of human prolactin variants as revealed by immunoperoxidase electrophoresis.

Five highly purified preparations of human pituitary prolactin, widely used in radioimmunoassays, were analyzed by immunoperoxidase electrophoresis with rabbit antisera raised against ovine and rat prolactin. When unreduced, the prolactin content of these preparations was separated into one major prolactin-like immunoreactive band of Mr 23,000, and four fainter immunoreactive bands of Mr's 16,000, 22,000, 25,000, and 45,000. Increasing the 2-mercaptoethanol concentration modified the relative proportions and the Mr of immunoreactive bands so that, after extensive reduction, only two immunoreactive bands were separated, one major band of Mr 25,000 and one fainter band of Mr 17,000. A direct relationship existed between the load of prolactin submitted to electrophoresis in the 0.25 to 75 ng range and the optical density of the separated bands.

Animals↗
PubMed1984

Effect of Bordetella pertussis toxin on ADP-ribosylation of membrane proteins, adenylate cyclase activity and insulin release in rat pancreatic islets.

Exposure of rat pancreatic islet membranes to [alpha-32P]-NAD+ in the presence of Bordetella Pertussis toxin (islet-activating protein) reveals the ADP-ribosylation of a peptide with a Mr close to 41 kDa, which corresponds to the alpha-subunit of the guanine nucleotide regulatory protein Ni. Islets removed from rats pretreated with the Bordetella Pertussis toxin display a specific increase in adenylate cyclase responsiveness to GTP and are characterized by a resistance to the inhibitory action of alpha2-adrenergic agonists upon either adenylate cyclase activity or glucose-induced insulin release.

Adenosine Diphosphate Ribose↗
PubMed1984

One-step isocratic high-performance liquid chromatographic purification of radioiodinated and radioiodinated-photoactivable derivatives of cholecystokinin.

N-Hydroxysuccinimidyl-3-(4-hydroxy-3-[125I]iodophenyl)propionate (the Bolton-Hunter reagent) was conjugated with (Thr 34, NLeu 37) cholecystokinin (CCK) 31-39 in anhydrous dimethylformamide-pyridine in 20% yield. The radiolabelled peptide was purified from the reaction mixture in one step, by isocratic elution from a C18 high-performance liquid chromatographic (HPLC) column with 35% aqueous acetonitrile-0.13% heptafluorobutyric acid as eluent. The concentration of the radiolabelled peptide was estimated by UV monitoring. The acylating photoactivable radioiodinated reagent N-hydroxysuccinimidyl-N-(4-azido-2-nitrophenyl)-3-[125I]iodotyrosi ne was synthesized, purified on a C18 HPLC column by isocratic elution with 65% acetonitrile-1 mM hydrochloric acid, then conjugated with (Thr 34, NLeu 37) CCK31-39. The resulting photoactivable radioiodinated CCK analogue was purified by isocratic elution on a C18 HPLC column with 39% aqueous acetonitrile-0.1 M triethylamine phosphate (pH 3.5). The binding ability of both tracers and their non-radioactive analogues to CCK receptors was tested on rat pancreatic plasma membranes. As compared to a KD of 4.5 nM for unmodified (Thr 34, NLeu 37) CCK31-39, the KD of the radioiodinated Bolton-Hunter derivative was 3 nM, and that of the photoactivable radioiodinated derivative was 19 nM.

Animals↗
PubMed1984

[Incidence of coccidia in dogs in Brno and the surrounding area].

In the course of two years, 663 dogs from Brno and its vicinity were subjected to clinical and parasitological examination with a special reference to the occurrence of coccidia. A saturated sugar solution with the specific weight of 1150 was used as the flotation medium. Coccidia were found in 2.17% of the dogs examined. The oocysts of the groups Isospora ohioensis, I. burowsi and I. neorivolta were identified most frequently (2.11% of the dogs). Four dogs (0.60%), all alike, eliminated the oocysts of Hammondia heydorni and sporocysts of Sarcocystis species, and two dogs oocysts of Hammondia heydorni and sporocysts of Sarcocystis species, and two dogs (0.30%) excreted oocysts of I. canis. Out of the animals infected, 58% were younger than six months. Clinical signs were observed only at a medium to strong invasion with oocysts of the species I. ohioensis, I. burowsi and I. neorivolta. The digestive tract was affected most severely--the animals suffered from bloodless diarrhoeas lasting two to twelve days, from occasional vomiting and systemic dehydration. Invasion by I. canis, Hammondia heydorni and Sarcoystis sp. did not cause any clinical changes.

Animals↗
PubMed1983

Monomeric pituitary growth hormone and prolactin variants in man characterized by immunoperoxidase electrophoresis.

Immunoperoxidase electrophoresis, combining SDS--ME--PAGE and the 'double bridge' immunoperoxidase staining was applied to crude human pituitary homogenates. With anti-hGH and anti-hPL sera, 4 hGH-related monomers were characterized: a Mr 22 000 peptide corresponding to hGH; a Mr 20 000 peptide corresponding to the known hGH variant and two unknown hGH variants (Mr 65 000 and Mr 75 000). With anti-ovine, rat and human PRL sera, 4 PRL-related monomers were immunostained: one comigrated with purified hPRL (Mr 25 000), and 3 were unknown (Mr 29 000; Mr 45 000; Mr 16 000).

Aged↗
PubMed1982

Specific photoaffinity crosslinking of [125I]cholecystokinin to pancreatic plasma membranes. Evidence for a disulfide-linked Mr 76 000 peptide in cholecystokinin receptors.

In rat pancreatic plasma membranes, preincubated with [125I]cholecystokinin-33 (CCK-33) and washed free of unbound tracer, the irradiation by UV light induced the irreversible binding of radioactivity to high molecular weight peptides as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and autoradiography. This was not observed when the membranes were preincubated in the simultaneous presence of [125I]CCK-33 and of either an excess of unlabelled CCK-8 or of guanosine 5'-(beta, gamma-imido)-triphosphate. The radioactivity was mostly crosslinked with a Mr 96,000 peptide and peptide species of Mr greater than 200,000, after SDS solubilization in the absence of beta-mercaptoethanol. Peptide reduction with beta-mercaptoethanol converted the high molecular weight radioactive species into a Mr 76,000 peptide that contained as much as 65% of the radioactivity crosslinked. The Mr 76,000 peptide appears, therefore, to be a disulfide-linked constituent of rat pancreatic cholecystokinin receptors.

Animals↗
PubMed1981

Determining steps in the regulatory GTPase cycle of rat pancreatic adenylate cyclase.

The time course of activation and deactivation and the degree of activation at steady state [Ea]/[Etot] of adenylate cyclase, in semi-purified rate pancreatic plasma membranes, were compatible with a simple two-state model with three rate constants, so that [Ea]/[Etot] = k+1/(k+1 + k2 + k-1). The hormone CCK-8 increased k+1 with GTP in a dose-dependent manner, from 0.2 to 10.9 min-1; k-1 increased from 0.01 to 0.3 min-1, i.e. in proportion, but k2 was unaltered at 7 min-1, so that [Ea]/[Etot] increased 15-fold, from 4 to 61%. A similar activation was obtained after cholera toxin pretreatment by a different mechanism. The toxin pretreatment exerted a major inhibitory effect on the value of k2 and on the corresponding GTPase activity. A pretreatment at the high cholera toxin concentration (10 micrograms/ml) exerted two additional effects that became evident when p[NH]ppG rather than GTP was used as activating nucleotide: (a) a relatively large increase in k-1 from an unmeasurably low control value to 0.3 min-1, and (b) a four-fold increase in the p[NH]ppG activation rate, k+1. This contrasted with the action of CCK-8, which increased k-1 and k+1 in proportion.

Adenylyl Cyclases↗