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Biomedical subjects

M Svoboda

Publications and source records attributed to M Svoboda.

At least 73 records · Page 4Linked to original sources
PubMed1990

N-terminal sequence determination of polypeptides and peptide mixtures by Edman degradation combined with californium-252 plasma desorption mass spectrometry.

The combination of manual Edman degradation with 252Cf plasma desorption mass spectrometry has been developed as an efficient method of polypeptide sequence determination. Results obtained with a variety of peptides and small proteins demonstrate unequivocal, two-fold sequence data, at each step from molecular weight identification of successively truncated Edman coupling and cleavage products. A rapid, greatly simplified procedure of manual Edman degradation with phenyl isothiocyanate was employed without extraction and purification steps, which was found compatible with plasma desorption mass spectrometry of low picomole amounts of peptide sample, at each cycle. Particular advantages of this combined method are the possibility of obtaining simultaneous sequence information from multicomponent peptide mixtures, such as proteolytic digests, and the direct identification of modified sequences, such as glycosylation sites. With the currently used conditions, plasma desorption mass spectrometry has been found feasible for approximately 30 sequence steps and sensitive to subnanomole amounts of proteins, with Mr up to approximately 10,000 Da.

Amino Acid Sequence↗
PubMed1989

Molecular architecture of secretin receptors: the specific covalent labelling of a 51 kDa peptide after cross-linking of [125I]iodosecretin to intact rat pancreatic acini.

p-Azidophenylglyoxal (APG), a heterobifunctional reagent with one group reacting selectively with arginine residues and another group photoactivable, was used to cross-link [125I]secretin prebound to intact rat pancreatic acini. The best yield was obtained when the [125I]secretin-acini complex was incubated under dim light with 2 mM APG at 37 degrees C and pH 8.0, followed by photolysis at 312 nm. The main secretin binding peptide cross-linked under reducing conditions, when tested by SDS-PAGE and autoradiography: (i) had a molecular mass of 51 kDa and was not a subunit of a larger disulfide-linked structure, and (ii) was distinct from the main VIP binding peptide coexisting in the same preparation.

Animals↗
PubMed1989

Bretylium tosylate binds preferentially to muscarinic receptors labelled with [3H]oxotremorine M (SH or 'high affinity' receptors) in rat heart and brain cortex.

Bretylium tosylate is an antiarrhythmic agent. In guinea pig atria it showed the properties of a competitive muscarinic (cholinergic) antagonist and could distinguish between two muscarinic receptor classes or states in cardiac membranes. We decided to further investigate its binding properties at muscarinic cholinergic receptors of the rat heart and brain (cortex), keeping in mind the recently discovered heterogeneity of muscarinic receptor protein. Bretylium tosylate recognized two receptor classes or states in the heart with Ki values of 0.9 and 11 microM. All cardiac membrane receptors showed a homogeneous (11 microM) Ki value for the drug in the presence of GTP in the incubation medium, or after in vivo pretreatment with islet activating protein (IAP). Bretylium tosylate was able (but only at a high concentration, 1 mM) to slow the dissociation kinetics of the tracer, which suggests that it also bound to an allosteric site on the muscarinic receptor, or that it affected the receptor environment. In the brain cortex, as in the heart, bretylium tosylate displayed a high affinity for receptors labelled with the agonist [3H]oxotremorine M (Ki value: 0.8 microM for the SH-or cardiac-type high-affinity receptors), and a 8- to 10-fold lower affinity for cortex M and L receptors. These data suggest that the antagonist bretylium tosylate had binding properties in rat cardiac membranes analogous to those of the partial agonist pilocarpine and that it interacted with a single type of receptor.

Animals↗
PubMed1989

Synthesis of cholecystokinin-related peptides and their biological properties.

Two tyrosine-O-sulfate containing cholecystokinin-related peptides were synthetized, i.e. the undeca- and the dodecapeptide amide corresponding to the C-terminus of this gut hormone; in the undecapeptide the N-terminal serine was O-substituted with the 2-deoxy-alpha-D-galactosyl group. Within the limits of error of the biological assay systems the two CCK peptides exhibited potencies in stimulating amylase secretion from dispersed rat pancreatic acini comparable to those of the CCK deca-, nona- and octapeptide.

Amino Acid Sequence↗
PubMed1989

[Occurrence of leptospiral antibodies in dogs in Brno and the surrounding area].

A serological examination by the microagglutination reaction of lysis (RMAL) was performed with 12 leptospira strains to investigate 1272 blood serums of dogs coming for various kinds of treatment to the outpatient ward of the veterinary hospital. The higher infestation rate was recorded in the dogs from outside the city (14.08%) than in the city dogs (7.96%). A considerably high infestation was found in the dogs of the service breeds (13.47%). The males were infested more frequently (10.99%) than the bitches (7.22%). As for age, young dogs until the age of one year were found to be infested more frequently. Separate significant titres were demonstrated with the leptospira of the Grippotyphosa, Icterohaemorrhagiae, Sejroe, and Australis (L. bratislava) serological groups. Reactions with the leptospira of other groups were recorded only at low titres: they were as coagglutinations with parallel high-titre reactions with the leptospira of the Grippotyphosa and Icterohaemorrhagiae groups.

Animals↗
PubMed1989

[Czechoslovak norms of the Harvard group scale of hypnotic susceptibility, form A].

The author presents Czechoslovak normal of the Harvard group scale of hypnability type A. Two different groups of experimental subjects were examined: 1. a sample of 564 secondary school students aged 14-20 years and 2. a group of 642 people attending a course of hypnosis, doctors and psychologists aged 23-57 years (mean age 31.5 years). The mean score of hypnability in the first group was 5.76 and the standard deviation 2.42. In the second sample the mean score was 4.30 and the standard deviation 2.33. The author presents also the distribution of different levels of hypnability, the order of difficulty of items and reliability of different items. The results of the secondary school students are comparable with data from abroad, the group attending courses on hypnosis differs in some areas. Experience assembled with the Czech translation of the Harvard scale justifies its use.

Adolescent↗
PubMed1988

Molecular characteristics and evidence for internalization of vasoactive-intestinal-peptide (VIP) receptors in the tumoral rat-pancreatic acinar cell line AR 4-2 J.

1. Vasoactive intestinal peptide (VIP) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas [125I]Iodo-VIP binding to cell membranes showed the following IC50 values for unlabeled peptides: VIP, 0.3 nM; peptide His-IleNH2, 2 nM; helodermin, 30 nM; secretin, 100 nM. After incubation with 20 nM dexamethasone, the binding capacity increased twofold but affinities were unchanged. External [125I]iodo-VIP binding to intact cells reached steady state after 5 min at 37 degrees C, while the sequestration-internalization of the [125I]iodo-VIP-receptor complex (tested by cold acid washing) increased progressively, reaching 75% of total binding after 1 h. This phenomenon was blocked at 4 degrees C. Further data with dexamethasone, tunicamycin, cycloheximide, low temperature, and/or phenylarsine oxide, suggested a half-life of 2 days for VIP receptors and the necessity of N-glycosylation for proper translocation. 2. For chemical [125I]iodo-VIP cross-linking bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone gave the best yield when compared with five other bifunctional reagents. In membranes, the main specifically cross-linked peptide had Mr 66,000 under nonreducing conditions, and migrated with lower velocity (-5%) under reducing conditions. Cross-linking was suppressed by VIP, peptide His-IleNH2 and helodermin (competitively) and also by GTP. In intact cells, the Mr of [125I]iodo-VIP-cross-linked peptides depended on the mode of cell solubilization. After direct solubilization, the major cross-linked radioactivity migrated as a smear of Mr 130,000-180,000 but an Mr-66,000 peptide was also detectable. In contrast, the solubilization of cross-linked cells detached by mild trypsinisation gave mainly the Mr-66,000 labeled peptide. This suggests that most VIP receptors in intact, attached cells were in a high-Mr complex and that mild cell treatment was sufficient to disrupt this complex.

Animals↗
PubMed1988

Optimization of fluorescence detection for the determination of labetalol in plasma by high-performance liquid chromatography.

A sensitive reversed-phase high-performance liquid chromatographic method for the determination of labetalol has been developed. A mobile phase consisting of citrate buffer (pH 6.5), acetonitrile and 2-propanol and an RP-8 column were used. The sensitivity of the fluorescence detection was enhanced to 1 ng/ml of labetalol in plasma by optimizing the emitted light. General guidelines for optimization of fluorescence detection are discussed.

Chromatography, High Pressure Liquid↗
PubMed1988

Species differences in the molecular characteristics of vasoactive-intestinal-peptide receptors in the pancreas from rat and guinea-pig.

1. The receptors for vasoactive intestinal peptide (VIP) present in dispersed acini and membranes from rat and guinea-pig pancreas differed in selectivity pattern, i.e. in the displacement of [125I]iodo-VIP by parent peptides, as revealed by a VIP:secretin IC50 ratio at least ten times higher in rat than in guinea-pig preparations. The molecular properties of these VIP receptors were therefore investigated. 2. When comparing six succinimidyl ester cross-linkers, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone proved to be the most universal [125I]iodo-VIP cross-linker for all pancreatic preparations. 3. In intact rat acini the main labeled peptide had an Mr of 80,000, whereas the main labeled peptide in intact guinea-pig acini was a smear of Mr 160,000. In both rat and guinea-pig pancreatic membranes, the main labeled peptide ([125I]iodo-VIP-binding-protein complex) had an Mr of 66,000. In addition, variable proportions of an Mr-80,000 peptide and an Mr-83,000 peptide were visualized in, respectively, rat and guinea-pig membranes. The labeling of all peptides was suppressed by VIP and by GTP. Reducing conditions allowed only a better resolution, making the presence of intermolecular disulfide bridges unlikely. 4. Taking into account the Mr of VIP it is thus plausible that the main native Mr-77,000 VIP-binding site present in rat acini could be easily converted to an Mr-63,000 peptide during membrane preparation, while in guinea-pig acini Mr-80,000 and/or Mr-63,000 VIP-binding sites were often closely associated with another membrane component in the native state. These molecular differences between VIP receptors in intact rat and guinea-pig acini are in keeping with functional differences.

Animals↗
PubMed1988

Identification by immunoblotting of somatostatin proforms in a rat pancreatic cell line.

The immunoblotting technique is applied to the analysis of "somatostatin" compounds secreted by R.I.N. T3 cells. We can confirm that two proforms of 15,300 +/- 750 and 29,000 +/- 1,100 Da accumulate in the extracellular medium. The unexpected form of 29 kDa, probably a dimeric form, disappears in reducing conditions. However, the 15 kDa peptide is characterized by several antibodies directed against either the intramolecular cycle of somatostatin-14 or the N-terminal extension of somatostatin-28. The 15 kDa form presents the same electrophoretic mobility in SDS-PAGE than the prosomatostatin isolated from a hypothalamic extract. Furthermore, this compound corresponds to the calculated mass of 10,388 Da deduced from the cDNA sequence. The detection of an immunoreactive 6 kDa peptide in the gel filtration fractions suggests an intermediate step in the prosomatostatin processing in these cells.

Animals↗
PubMed1988

Vasoactive intestinal peptide receptors in pancreas and liver. Structure-function relationship.

In purified rat pancreatic plasma membranes, (D-Phe4)PHI interacts as a selective VIP agonist for rat pancreatic VIP-preferring receptors, based on binding selectivity and adenylate cyclase activation, therefore allowing us to discriminate between the participation of VIP-preferring and secretin-preferring receptors in VIP stimulation. VIP-preferring receptors also bind GRF. They rely on disulfide bridges for their functional integrity. Their coupling with adenylate cyclase, based on the intrinsic activity of VIP analogues, is poor when compared to that of hepatic VIP receptors. In fresh rat liver plasma membranes, high-affinity VIP receptors are specifically labeled with [125I]helodermin and [125I]His1, D-Ala NLeu27)GRF and are well coupled to adenylate cyclase while low-affinity VIP receptors are not. The first subtype of VIP receptors is highly responsive to guanyl nucleotides and is easily altered by dithiothreitol. Only after freezing and thawing are low-affinity hepatic VIP receptors coupled to adenylate cyclase. Concerning the chemical characterization of VIP receptors, 66- and 35-kDa peptides are detected after specific [125I]VIP cross-linking with double agents in rat pancreatic membranes. In contrast, in intact pancreatic acini, the main source of radioactivity has a molecular mass of 130-180 kDa (with no contribution of intramolecular disulfide bridges), and an 80-kDa peptide is also detectable. The 66-kDa species in membranes can conceivably derive from the 80-kDa species observed in intact cells. Its molecular mass is higher than that of the 56-kDa [125I]VIP cross-linked protein previously observed in rat liver membranes. Besides, species differences between rat and guinea pig pancreas are also evident.

Adenylyl Cyclases↗
PubMed1988

Functional somatostatin receptors on a rat pancreatic acinar cell line.

Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of 125I-[Tyr11]somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites (Kd = 0.55 +/- 0.06 nM) with a maximal binding capacity of 258 +/- 20 fmol/10(6) cells. Somatostatin receptor structure was analyzed by covalently cross-linking 125I-[Tyr11]somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide (Mr 80,000) containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition of cAMP formation (IC50 = 0.4 nM) was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. We conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein Ni to inhibit adenylate cyclase.

Adenylyl Cyclases↗
PubMed1988

[Relation between the occurrence of antibodies against Toxoplasma gondii and clinical findings in cats].

Within a three-year period, 178 clinically healthy and 442 sick cats (patients of the Clinic for Small Animal Diseases of the University of Veterinary Medicine, Brno) were examined for the presence of antibodies to Toxoplasma gondii. Antibodies to T. gondii were detected in 34.8% of the animals without clinical signs of the disease and in 54.3% of the sick cats (P less than 0.01). Compared with the group of clinically healthy cats, the specific antibodies occurred significantly more frequently in the cats suffering from diseases of the digestive tract (P less than 0.01), particularly in those with acute gastroenteritis, and in those having liver disorders (P less than 0.01; P less than 0.05). A statistically significantly higher occurrence of antibodies to T. gondii was also recorded in the cats with disorders of the nervous system (P less than 0.05; P less than 0.01), particularly in those with symptoms of extraordinary aggressivity at the age span from four months to three years. Enlarged lymph nodes were found out in 44% of the cats having antibodies to T. gondii. This is 15% more than the average for the investigated set of animals, which is a statistically significant dependence (P less than 0.01).

Animals↗
PubMed1988

[Incidence of antibodies to Toxoplasma gondii in cats from Brno and the surrounding area].

In the course of three years, 620 cats of all age categories were investigated for the occurrence of antibodies to Toxoplasma gondii. The serological examinations included the Sabin-Feldman reaction (SFR), complement-fixation reaction (CFR) and microprecipitation in agar gel (MPA), according to methods prescribed for use in Czechoslovakia. When the Sabin-Feldman reaction was used, 250 (40.3%) of the examined cats were found to be positive at the titre of 4-128. By the complement-fixation reaction, antibodies were demonstrated in 144 (23.2%) of the cats at the titre of 5-80. Microprecipitation in agar gel was positive in 106 animals, i.e. 17.1%. Antibodies to Toxoplasma gondii were demonstrated by one serological reaction in 150 (24.2%) cats; coincidence of two serological reactions occurred in 104 (16.8%) of the animals, and coincidence of all the three serological reactions was demonstrated in 48 (7.7%) cats. Specific antibodies were detected by minimally one serological reaction (which means one to three positive findings) in 302 (48.7%) of the examined cats. The diagnostic value of all serological reactions is discussed. It is recommended to repeat the examination after two to three weeks.

Animals↗
PubMed1988

[Diagnosis and prevention of feline toxoplasmosis].

At the "Small Animal Clinic of the University of Veterinary Science" in Brno during four years 442 sick and 178 clinically normal cats were examined in regard to incidence and diagnosis of toxoplasmosis. Using the Sabin-Feldman reaction, antibodies against T.gondii were found in 40.3% of the cats (titer 4-128), whereas by means of complement fixation reaction and microprecipitation in agar gel, antibodies were found in 23.2% (titer 5-80) and 17.1% of the cats, respectively. Eight cats (1.3%) excreted T.gondii oocysts. The number of animals having specific antibodies increased statistically significant with age (P less than 0.01) and with particular hunting habits: it was higher in cats which were usually catching small rodents (P less than 0.01). A lower toxoplasmosis incidence was observed in indoor-cats (P less than 0.005) and in cats fed with boiled food only (P less than 0.005). In 35% of the cats showing no clinical symptoms of toxoplasmosis, there were found antibodies against T.gondii. Comparison of clinically normal and sick cats revealed that antibodies against T.gondii occurred significantly more often in cats with enlarged lymph nodes (P less than 0.01), with a disease of the digestive tract (P less than 0.01), of the liver (P less than 0.01), and of the nervous system (P less than 0.01). Several recommendations for diagnosis and prophylaxis of toxoplasmosis in cats are given.

Animals↗
PubMed1987

Chemical, immunological and biological properties of peptides like vasoactive-intestinal-peptide and peptide-histidine-isoleucinamide extracted from the venom of two lizards (Heloderma horridum and Heloderma suspectum).

Having previously isolated helodermin, the major peptide like vasoactive-intestinal-peptide and peptide-histidine-isoleucinamide, from the venom of the lizard Heloderma suspectum, we decided on a systematic exploration of all (VIP-PHI)-like peptides present in the venom of another lizard of the Helodermatidae family: Heloderma horridum. Six (VIP-PHI)-like peptides (PHH1 to 6) were purified to homogeneity from the venom of the lizard H. horridum with PHH3 and PHH4 representing two minor forms. All peptides cross-reacted in radioimmunoassays for helodermin and PHI but not for VIP. They yielded four fragments (T1 to T4) after trypsin digestion. T1, T2 and T3 showed the same retention time by reverse-phase HPLC and the same amino acid composition; the differences were confined to T4, the C-terminal sequence. PHH5 and PHH6 were found to be identical to synthetic helospectins I and II respectively. PHH1 and PHH3 probably resulted from a secondary modification of PHH5, while PHH2 and PHH4 derived from PHH6. Thus, the VIP-like peptides, previously called helospectins, are in fact typical of H. horridum venom. We confirmed that helodermin is the major (VIP-PHI)-like peptide of the venom of H. suspectum and observed its absence in H. horridum venom. Also, we found that positions 8 and 9 of helodermin are occupied by two Glu residues instead of two Gln as previously published. Helospectin-like material was also present in H. suspectum venom but in very small amount. In both venoms all VIP-like peptides were equally potent and efficient when tested for (a) their ability to occupy VIP as well as secretin receptors in rat pancreatic membranes and VIP receptors in rat liver membranes, and (b) the ensuing activation of adenylate cyclase in both membrane preparations.

Adenylyl Cyclases↗
PubMed1987

Mechanisms of lithium-vasopressin interaction in rabbit cortical collecting tubule.

In single cortical collecting tubules (CCT) of the rabbit, guanosine 5'-triphosphate (GTP) increased the arginine vasopressin (AVP)-stimulated adenylate cyclase (AC) by 60% (P less than 0.05). In contrast, guanosine 5' O-(2-thio)-diphosphate (GDP-beta S), a competitive inhibitor of GTP action on the stimulatory guanine regulatory protein (Ns), reduced the AVP-stimulated AC activity by 72% (P less than 0.001), indicating the presence of endogenous GTP in the cells under study. That inhibitory effect was reversed by the addition of GTP to the incubation medium. In isolated perfused CCT, cholera toxin (CT) induced a significant increase in water permeability in the absence of AVP. In contrast, Bordetella pertussis toxin (BPT) did not modify the low AVP-independent water permeability. Lithium, an inhibitor of the hydrosmotic action of AVP, also inhibits the hydrosmotic action of CT by 70% (P less than 0.05) but not that of forskolin. The conclusions of the present study are Ns is required for AVP stimulation of AC in the CCT; Ns is functionally active in this system as evidenced by the hydrosmotic effect of CT; the lack of effect of BPT suggests that the low AVP-independent water permeability in the CCT is not the result of a tonic inhibition of the AC operating through the inhibitory guanine nucleotide regulatory protein; and the inhibition by lithium of the hydrosmotic action of AVP in the CCT appears to involve an interaction with the regulatory proteins (probably Ns) or with their binding to the catalytic unit of AC.

Adenylyl Cyclases↗