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Biomedical subjects

M Svoboda

Publications and source records attributed to M Svoboda.

At least 37 records · Page 2Linked to original sources
PubMed1999

Overexpression of pterin-4a-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 in human colon cancer.

Pterin-4a-carbinolamine dehydratase (PCD) is a bifunctional protein also known as DCoH (dimerization co-factor of hepatocyte nuclear factor 1 (HNF1)). PCD/DCoH modulates the DNA binding specificity of HNF1, thus acting on its transcriptional activity. In addition, it participates in the recycling of tetrahydrobiopterin (BH(4)), an essential cofactor of several metabolic reactions. We investigated colorectal tumors and colorectal tumor cell lines as compared to normal colon samples in search of a potential differential expression of PCD/DCoH. Immunohistochemistry was conducted on 20 human colorectal tumors and 20 normal samples using a specific polyclonal antibody. Immunoblotting and RT-PCR analysis for PCD/DCoH and HNF1 were also performed on both human tissues and CACO-2 and HT-29 cell lines. All of the 20 tumors and both colon cancer cell lines presented a strong and widespread immunoreactivity for PCD/DCoH, contrasting with the absence of expression in the normal epithelia. We thus report the massive overexpression of PCD/DCoH in colon tumors, which is in striking contrast with the absence of staining in normal counterparts. The sharp contrast in the expression of a modulator of transcriptional activity between tumoral and normal cells may have a physiopathological role. PCD/DCoH could potentially be a new marker of malignant colon cells in vivo.

Adenocarcinoma↗
PubMed1999

Screening for drugs in serum by electrospray ionization/collision-induced dissociation and library searching.

Factors influencing in-source collision-induced dissociation (ESI/CID) of organic molecules in a Perkin-Elmer/SCIEX ionspray source have been investigated. Breakdown curves of four drugs and organic compounds were acquired by monitoring the intensities of MH+ and specific fragment ions while ramping the orifice voltage. Haloperidol, diazepam, 1,4-acetamido-acetoxybenzene and diacetamido-1,2-benzene were found to be substances with characteristic breakdown curves, with maximums and points of intersection at orifice voltages between 20 and 70 V. The breakdown curves of haloperidol were used for comparison of ESI/CID with ionspray and turboionspray sources on three PE/SCIEX-API instruments. Using standardized source parameters and mass resolution, very similar fragmentation graphs were obtained for haloperidol with all instruments. Infusion of varying concentrations of haloperidol (0.1 to 10 micrograms/mL with ionspray, and 0.01 to 1 microgram/mL with turboionspray) yielded comparable breakdown curves. With turboionspray, a preconcentration of the aerosol occurred, yielding higher ion abundances. Solvent pH and the ratio of aqueous ammonium formate/acetonitrile had minor effects on the degree of fragmentation of haloperidol in a wide range. With these preconditions, a currently expanding mass spectral library of 400 drugs was set up by liquid chromatography/mass spectrometry with alternating orifice voltages (20, 50, and 80 V, respectively) in a looped experiment. An example of drug identification in a patient's serum with library search is shown.

Chromatography, High Pressure Liquid↗
PubMed1999

Epidemiology of feline leukaemia and feline immunodeficiency virus infections in the Czech Republic.

Commercial serological sets were used for the examination of 727 cats kept in larger towns of the Czech Republic. FeLV antigen and antibodies to FIV were demonstrated in 96 (13.2%) and 42 (5.8%) of the animals, respectively. Seven (0.96%) animals were positive for both FeLV and FIV. Most of the FeLV and/or FIV positive patients were intact rambling males aged 1-4 years. Chronic gastrointestinal and respiratory diseases were found in 54.2% and 43.8% of the FeLV-positive patients, respectively. Chronic urinary tract diseases and generalized lymphadenopathy were found in 47.6% and 45.2% of the FIV-positive patients, respectively. The results of this first survey in the Czech Republic have shown prevalence values and clinical patterns similar to those reported formerly from other European countries.

Animals↗
PubMed1999

Guanine nucleotide binding proteins in cultured renal epithelia: studies with pertussis toxin and aldosterone.

The GTP-binding proteins from cultured A6 epithelia were examined in isolated membrane preparations. Binding of [35S]GTPgammaS revealed a class of binding sites with an apparent Kd value of 100 nM and a Bmax of 220 pmol/mg protein. Short-term aldosterone treatment of the cells did not modify the binding kinetics, whereas pertussis toxin (PTX) decreased Bmax by 50%. The mRNA levels for Galphai-3, Galpha0, Galphas, and Galphaq were not increased after aldosterone. The patterns of small Mr G proteins and of PTX-ribosylated proteins were identical in membranes of both control and aldosterone-treated cells. Cross-linking of [alpha-32P]GTP, in control membranes, showed either no labeling or a faint band of Mr 59.5 kDa. This protein became prominent after aldosterone, and its labeling decreased with spironolactone. Thus short-term aldosterone does not promote increased expression of known heterotrimeric G proteins in epithelial membranes but activates resident PTX-sensitive Gi proteins and stimulates the expression of a specific GTP-binding protein of Mr 59.5 kDa.

Adenosine Diphosphate Ribose↗
PubMed1998

Prevalence of IgG and IgM antibodies specific to Toxoplasma gondii in cats.

IgG and IgM antibodies specific to Toxoplasma gondii were determined simultaneously by IFAT in cats. Group 1 included 357 cats with various diagnoses, but negative for FeLV and FIV. Specific IgG antibodies in titres ranging from 10 to 2560 (mean 247) were detected in 61.3% animals, specific IgM antibodies were found only in one cat (0.28%) in titre 1:40 showing an IgG titre of 160. Oocyst shedding was demonstrated in one cat (0.28%) showing an IgG titre of 1:40. Group 2 included 33 cats positive for FeLV or FIV. IgG antibodies in titres ranging from 10 to 640 (mean 101) were found in 63.6% cats. Neither IgM antibodies nor shedding of T. gondii oocysts were detectable. Clinically unapparent toxoplasmosis, manifested by the presence of IgG antibodies is frequent in cats; the opportunistic character of the infection is important.

Animals↗
PubMed1998

Expression of transforming growth factor beta receptors in normal human colon and sporadic adenocarcinomas.

BACKGROUND & AIMS: An absence or a presence of mutated transforming growth factor (TGF)-beta receptors is a possible hypothesis explaining the resistance of cancer cells to the growth-inhibitory effect of TGF-beta. Mutations involving microsatellite-like regions of the type II TGF-beta receptor have been described in subgroups of colorectal cancers. The aim of this study was to investigate the expression and distribution of TGF-beta receptors in sporadic colorectal cancers and normal tissues. METHODS: Thirty-three sporadic colorectal cancers and 20 normal colonic tissues were explored by immunohistochemistry for the expression of type I and type II TGF-beta receptors. Eighteen tumor and 20 normal samples were used for radioactive thermocycling and sequencing of the two microsatellite-like regions of the type II receptor. RESULTS: Both receptors were overexpressed in tumors compared with normal samples. There was a relationship between the abundance of type II receptor expression and the degree of differentiation of the tumors but not the Dukes' staging or the localization of the neoplasias. No mutation was observed in the microsatellite-like regions of receptor II in any of the samples. CONCLUSIONS: Sporadic colorectal cancers do not show an absence or a presence of mutated TGF-beta receptors that could explain a resistance to TGF-beta-mediated growth inhibition. The pathways to tumorigenesis of sporadic colorectal cancers may be different from those of some hereditary ones.

Adenocarcinoma↗
PubMed1998

Molecular cloning and in vitro properties of the recombinant rabbit secretin receptor.

The rabbit secretion receptor cDNA was cloned from rabbit pancreas using combined polymerase chain reaction (PCR)/rapid amplification of cDNA ends (PCR/RACE) approaches. The rabbit cDNA encoded 445 amino acids and had 80 and 85% homology with rat- and human receptor, respectively, in terms of nucleic and amino acid sequences. Several regions where the rabbit receptor sequence diverged from the rat/human receptor sequences were observed in the putative extracellular domains of the receptor. A cDNA coding for a similar sequence with a 76 bp deletion after the 5th transmembrane domain was also found; it probably encoded an inactive protein. The whole rabbit secretin receptor cDNA was subcloned in expression vector pCR3.1, then stably and transiently transfected in Chinese hamster ovary (CHO) cells. The pharmacological properties of the rat and rabbit secretin receptor studies were compared by radiolabeled secretin binding, binding inhibition, and adenylate cyclase activation (using secretin analogs and fragments). Porcine secretin was equipotent with rabbit secretin on the rabbit secretin receptor, but fivefold more potent than rabbit secretin on the rat receptor. This was due to the serine-->arginine residue replacement, in position 16 of rabbit secretin. Amino terminal modified secretin analogs (secretin (2-27), [E3]secretin, [N3]secretin) and VIP were less potent than secretin on both secretin receptors, but more potent on the rabbit than on the rat receptor. The carboxy-terminally truncated fragment (1-26) had the same reduced potency on rat and rabbit receptors. Thus, the rabbit secretin receptor had original properties, different from those of the rat receptor.

Adenylyl Cyclases↗
PubMed1998

Secondary immunodeficiency in dogs with enteric, dermatologic, infectious or parasitic diseases.

A group of 238 dogs with various infectious and parasitic disease, in which suppressed activity of the immune system could e presumed, was examined using a set of immunological methods. The frequency and depth of immunosuppression and its association with certain infectious or parasitic disease were determined. Marked immunosuppression was found 62 (26%) of the dogs examined. Dogs with distemper, parvovirosis and German Shepherd dog pyoderma (GSP) were the most severely impaired. Dogs in acute phases of distemper or parvovirosis had decreased numbers and activity of lymphocytes and decreased immunoglobulin levels. Dogs with GSP had some of the following immunologic symptoms: inhibition of phagocytosis, reduced activity of lymphocytes, decreased levels of haemolytic complement and increased levels of immunoglobulin and lysozyme. A persistent immunosuppression was found in 12 dogs. These dogs were diagnosed with deep pyoderma, giardiasis, dermatophytosis or neoplasms. Although samples were not taken before the clinical diseases appeared, it can be presumed that some diseases caused immunosuppression (distemper or parvovirosis), and for other diseases immunosuppression was a predisposing factor (dermatophytosis, giardiasis and possibly GSP).

Animals↗
PubMed1997

The influence of strongly acidic groups on the protonation of peptides in electrospray MS.

In order to obtain experimental data on the question of compensation of positive charges by anionic groups in multiply charged ions of polyfunctional molecules in electrospray MS, several pairs of peptides with the same basic structure but differentiated by one or two strongly acidic groups (phosphate or sulfonate) were investigated. It was found that depending on the density of basic centers and the strength of acidic groups present, the observed changes upon introduction of acidic groups ranged from complete elimination of the most highly charged state to absence of any difference.

Amino Acid Sequence↗
PubMed1997

Chronic alcohol consumption does not cause hippocampal neuron loss in humans.

High alcohol consumption for long periods of time causes significant hippocampal neurodegeneration in rodents. A single study using neuronal density measures has reported similar findings in humans. The present study aims to substantiate these findings in human alcoholics using unbiased stereological techniques. Both amnesic (n = 5) and nonamnesic (n = 7) chronic alcoholics were selected and compared with nonalcoholic controls (n = 8) and patients with marked memory loss and hippocampal neurodegeneration caused by Alzheimer's disease (n = 4). Hippocampal volume was significantly reduced in the alcoholics and in patients with Alzheimer's disease. However, in alcoholics the volume reduction occurred exclusively in the white matter, whereas both the gray and white matter were reduced in the patients with Alzheimer's disease. Neuron loss occurred exclusively from the CA1 and subiculum subregions of the hippocampus in Alzheimer's disease. No neuron loss occurred from any subregion of the hippocampus in alcoholics. There were no correlations with age and any of the volume or neuron number measures. Hippocampal volume correlated with brain volume and with the regional gray and white matter volumes within the hippocampus. In addition, hippocampal gray matter volume correlated with the number of CA1 pyramidal neurons. These results do not support the theory that chronic alcohol consumption is neurotoxic to hippocampal pyramidal neurons in humans. Further, the present results suggest that changes observed in rodent models of alcoholism do not parallel those observed in humans, questioning the validity of such models.

Aged↗
PubMed1997

Identification, expression pattern and potential activity of Na/Ca exchanger isoforms in rat pancreatic B-cells.

In the pancreatic B-cell, Na/Ca exchange displays a quite high capacity and participates in the control of cytosolic free Ca2+ concentration. The Na/Ca exchanger was recently cloned in various tissues. Two genes coding for two different exchangers (NCX1 and NCX2) have been identified and evidence for several isoforms for NCX1 shown. To characterize the isoform(s) expressed in pancreatic B-cells, a RT-PCR analysis was performed on mRNA from rat pancreatic islets, purified B-cells and insulinoma B-cells (RINm5F cells). PCR amplification did not yield the expected NCX2 DNA fragment but yielded 2 NCX1 bands, corresponding to NaCa3 and NaCa7, in the three preparations. NaCa3 and NaCa7 were equally expressed in pancreatic islets and purified B-cells. In RINm5F cells, NaCa3 expression did not differ from that in islet and purified B-cells but NaCa7 was 3 times less expressed. This lower expression was accompanied by a 3 times lower Na/Ca exchange activity in RINm5F cells compared to islet cells. Our data indicate the existence of 2 NCX1 isoforms but not of NCX2 in pancreatic B-cells. The difference in both the expression patterns of NCX1 isoforms and the activity of Na/Ca exchange in islet cells and RINm5F cells is compatible with a difference in activity between NaCa3 and NaCa7.

Animals↗
PubMed1997

Identification of motilin mRNA in the brain of man and rabbit. Conservation of polymorphism of the motilin gene across species.

The data regarding the identity of motilin-like immunoreactivity in the central nervous system are controversial. The aim of the present study was to clarify whether motilin mRNA is present in the brain of rabbit and man. Total RNA, prepared from several regions of the rabbit brain, was subjected to RT-PCR aimed at amplifying a 294 bp cDNA fragment of the rabbit motilin precursor. The amplified product was subcloned and sequenced. The sequence showed 7 differences compared to the one reported for the duodenal precursor (1). However the duodenal precursor from the rabbit used in the present study revealed identical substitutions. One of these, involving amino acid -11 of the signal peptide, was shown to be due to gene polymorphism, as has also been described at this site in man. By radioimmunoassay the highest concentration of motilin (fmol/mg protein) was detected in the hippocampus (4788 +/- 295), the lowest in the telencephalon (2127 +/- 221). Using a similar approach, but starting from commercial human brain mRNA, the sequence of a comparable cDNA fragment of the human brain motilin precursor was obtained. Its sequence was identical with the one published for the human intestinal precursor (41). Our study demonstrates that motilin mRNA is present in the brain of man and rabbit. Together with our recent findings of central motilin receptors, they suggest a central role for motilin.

Amino Acid Sequence↗
PubMed1997

Immunosuppression in dogs with pyoderma and/or demodicosis.

The occurrence of decreased activity of the immune system was studied in a group of 66 dogs with various combinations of pyoderma and demodicosis. Our complex examination of the dogs included the following: leukocyte count, differential count, phagocytosis, blastogenic lymphocyte transformation and quantitation of total serum immunoglobulins, lysozyme and haemolytic complement. Immunosuppression was found in 19 (28.8%) cases. Immunosuppression was rare in dogs with demodicosis and did not appear without a concurrent pyoderma. An increase in the neutrophil counts and total serum immunoglobulin levels significant was found in dogs with demodicosis combined with pyoderma. On the contrary, marked immunosuppression was detected in dogs with deep pyoderma. A considerable immunosuppression was present in 7 of 10 German shepherds dog pyoderma (GSP). Significant depressions were found in phagocyte activity and lymphocyte activity. Immunosuppression was observed in 4 of 9 dogs in other breeds with uncomplicated deep pyoderma. All groups of dogs with pyoderma showed a significant increase in total serum immunoglobulins. Conclusion from these findings is that deep pyoderma more than Demodicosis was concerned with immunosuppression. German shepherds with deep pyoderma had more expressed immunosuppression than other breeds.

Animals↗
PubMed1996

Research on antifungal and antimycobacterial agents. Synthesis and activity of 4-alkylthiopyridine-2-carbothioamides.

A series of 4-alkylthiopyridine-2-carbothioamides have been prepared and evaluated in vitro for antimicrobial activity. Chemical structures have been demonstrated by IR and 1H NMR data and by elemental analysis. The antimycobacterial activity of these compounds against Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium avium, and Mycobacterium fortuitum, and the antifungal activity against Candida albicans, Candida tropicalis, Candida krusei, Candida glabrata, Trichosporon beigelii, Trichophyton mentagrophytes, Aspergillus fumigatus, and Absidia corymbifera were determined by the MIC values. Compounds 3 exhibit good antimycobacterial activity compared to isoniazide. A moderate antifungal activity was observed against T. mentagrophytes. Activity is influenced by hydrophobicity of the alkyl group.

Anti-Infective Agents↗
PubMed1996

Characterization of the VIP receptor from SUP T1 lymphoblasts.

The SUP T1 lymphoblasts express an original subtype of VIP receptors characterized by a high affinity for the VIP analogue from lizard venom named helodermin, a preference for the neuropeptide PACAP-38 over PACAP-27 and VIP, and an extremely low affinity for secretin. The molecular cloning of that receptor revealed its identity with the VIP2 receptor subtype first cloned in rat and mouse tissues. The access to selective probes permits the detection of the mRNA coding for the VIP2 receptor by Northern blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. These highly selective and sensitive techniques identify the cell types that are equipped to synthesize the receptor but do not prove that the receptor is indeed efficiently expressed at the cell surface. VIP2 mRNA was detected in selected areas of the brain different from that expressing the classical VIP1 receptor, in pituitary, in pineal, in pancreatic islets, in testes and ovary. It was also detected in the stomach, in the thymus and in spleen and in T lymphoblastic cell lines. A systematic screening of the immunocompetent cells must still be performed.

Amino Acid Sequence↗
PubMed1996

Pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide receptor subtypes are differently expressed in rat transplanted pituitary tumours (SMtTW) and in the normal gland.

The expression of the pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide (PACAP/VIP) receptor subtypes was evaluated in the normal rat pituitary gland and in different rat spontaneous transplantable SMtTW tumours (SMtTW2 which expresses prolactin (PRL), SMtTW10 which expresses GH and SMtTW3 which expresses both PRL and GH) by measurement of PACAP/VIP-stimulated adenylate cyclase activity and detection of the presence of mRNA coding for the different receptor forms. In normal glands, the order of potency of the peptides suggested that adenylate cyclase activity was mediated through interaction with PACAP selective receptors (PACAP I receptors); mRNAs coding for the PACAP I receptor, but also for the PACAP II VIP2 receptor, were detected. In SMtTW2 tumours, the functional response was close to that observed in the presence of PACAP II VIP2 receptors; mRNAs coding for PACAP I and PACAP II VIP1 and PACAP II VIP2 receptors were detected. In the SMtTW10 tumours, the functional response was complex but compatible with the involvement of PACAP I and PACAP II receptors; mRNAs coding for the PACAP I and PACAP II VIP1 receptors were detected. In the SMtTW3 tumour, the profile was similar to that of the normal pituitary gland and the mRNA coding for the PACAP I receptor only was detected. Thus, while the control of normal pituitary gland adenylate cyclase activity by PACAP and VIP was mediated by PACAP-selective receptors, in spontaneous transplantable tumours a variable profile was observed and PACAP, as well as VIP1 and VIP2 receptors, may contribute to the responses.

Adenylyl Cyclases↗