Biomedical subjects

M Rupnik

Publications and source records attributed to M Rupnik.

At least 19 recordsLinked to original sources

PubMed2006

Distinct role of Rab3A and Rab3B in secretory activity of rat melanotrophs.

Members of the Rab3 (A-D) subfamily of small GTPases are believed to play a key role in regulated exocytosis. These proteins share approximately 80% identity at amino acid level. The question of whether isoforms of Rab3 are functionally redundant was the subject of this study. We used RT-PCR analysis, in situ hybridization histochemistry, and confocal microscope-based analysis of immunocytochemistry to show that rat melanotrophs contain about equal amounts of Rab3A and Rab3B transcripts as well as proteins. Therefore, these cells are a suitable model to study the subcellular distribution and the role of these paralogous isoforms in regulated exocytosis. Secretory activity of single cells was monitored with patch-clamp capacitance measurements, and the cytosol was dialyzed with a high-calcium-containing patch pipette solution. Preinjection of antisense oligodeoxyribonucleotides specific to Rab3A, but not to Rab3B, induced a specific blockage of calcium-dependent secretory responses, indicating an exclusive requirement for Rab3A in melanotroph cell-regulated secretion. Although the injection of purified Rab3B protein was ineffective, the injection of recombinant Rab3A proteins into rat melanotrophs revealed that regulated secretion was stimulated by a GTP-bound Rab3A with an intact COOH terminus and inhibited by Rab3AT36N, impaired in GTP binding. These results indicate that Rab3A, but not Rab3B, enhances secretory output from rat melanotrophs and that their function is not redundant.

Animals↗

PubMed2005

KATP-channels in beta-cells in tissue slices are directly modulated by millimolar ATP.

In pancreatic beta-cells, inhibition of K(ATP)-channels plays a pivotal role in signal transduction of glucose-induced insulin release. However, the extreme sensitivity of K(ATP)-channels to its ligand ATP as found in inside-out patches is not directly compatible with modulation of these channels at physiological [ATP](i). We studied K(ATP)-channel sensitivity to ATP in beta-cells in dispersed culture and in fresh pancreatic tissue slices. Physiological [ATP](i) blocks more than 99% of K(ATP)-channels in cultured beta-cells, while only 90% in beta-cells in slices, indicating reduced sensitivity to ATP in the fresh slices. Applying cytosolic factors like ADP, phosphatidylinositol-4,5-bisphosphate (PIP(2)) or oleoyl-CoA did not restore the K(ATP)-channel sensitivity in cultured beta-cells. Our data suggest that interaction between SUR1 and Kir6.2 subunit of the K(ATP)-channel could be a factor in sensitivity modulation. Tissue slices are the first beta-cell preparation to study direct K(ATP)-channel modulation by physiological [ATP](i).

Adenosine Triphosphate↗

PubMed2004

Anaerobic bacteria in the gut of terrestrial isopod Crustacean Porcellio scaber.

Anaerobic bacteria from Porcellio scaber hindgut were identified and, subsequently, isolated using molecular approach. Phylogenetic affiliation of bacteria associated with the hindgut wall was determined by analysis of bacterial 16S rRNA gene sequences which were retrieved directly from washed hindguts of P. scaber. Sequences from bacteria related to obligate anaerobic bacteria from genera Bacteroides and Enterococcus were retrieved, as well as sequences from 'A1 subcluster' of the wall-less mollicutes. Bacteria from the genus Desulfotomaculum were isolated from gut wall and cultivated under anaerobic conditions. In contrast to previous reports which suggested the absence of anaerobic bacteria in the isopod digestive system due to short retention time of the food in the tube-like hindgut, frequent renewal of the gut cuticle during the moulting process, and unsuccessful attempts to isolate anaerobic bacteria from this environment our results indicate the presence of resident anaerobic bacteria in the gut of P. scaber, in spite of apparently unsuitable, i.e. predominantly oxic, conditions.

Bacteria, Anaerobic↗

PubMed2003

Binary toxin producing Clostridium difficile strains.

Clostridium difficile produces three toxins, TcdA, TcdB and CDT. TcdA and TcdB are single-stranded molecules acting as glucosyltransferases specific for small GTPases. CDT is an actin specific ADP-ribosylating binary toxin characteristically composed of two independent components, enzymatic CDTa (48 kDa) and binding CDTb (99 kDa). The cdtA and cdtB genes were sequenced in two CDT-positive strains of C. difficile (CD 196 and 8864) and at least two CDT-negative strains with truncated form of binary toxin genes are known (VPI 10463 and C. difficile genome strain 630). The prevalence of binary toxin producing strains is estimated to be from 1.6% to 5.5%, although a much higher proportion has been reported in some studies. The role of the binary toxin as an additional virulence factor is discussed.

Journal Article↗

PubMed2003

A European survey of diagnostic methods and testing protocols for Clostridium difficile.

OBJECTIVE: To conduct a survey of the methods used in clinical microbiology laboratories in Europe to diagnose infection with Clostridium difficile. METHODS: A questionnaire was devised and sent to a co-ordinating member of the Study Group in each of eight European countries. This co-ordinator was in charge of forwarding the questionnaire to hospital laboratories arbitrarily selected. The number of laboratories in each country was determined on the basis of one laboratory for 10,000 beds of hospitalization. This questionnaire covered different aspects pertaining to Clostridium difficile associated to diarrhea (CDAD) diagnosis such as circumstances of request, criteria used for undertaking C. difficile investigations, methods used for the diagnosis, etc. RESULTS: A total of 212 questionnaires were completed and submitted for analysis: 87.7% of laboratories reported routinely performing C. difficile diagnostic tests. Methods used included toxin detection (93%), culture (55%), and glutamate dehydrogenase (GDH) detection (5.9%). Among the laboratories detecting toxins, different enzyme immunoassays (EIA) and cytotoxicity assays were used in 79% and 17.3% of cases, respectively. Among the different strategies reported, 4.8% were considered suboptimal for the diagnosis of C. difficile infections, but marked discrepancies could be observed between countries. The overall incidence (median) of CDAD was estimated at 1.1 for 1,000 patient admissions. CONCLUSION: The results of this study suggest marked discrepancies between laboratories and also between countries regarding the criteria by which C. difficile is investigated for, and the methods and the strategies that are used for the diagnosis of C. difficile. These discrepancies could be explained by the lack of clear guidelines for C. difficile diagnosis in each country, and by the importance that physicians attach to C. difficile. Precise guidelines for C. difficile diagnosis would be the first step to make possible accurate comparison of the incidence and the epidemiology of CDAD from one hospital to another or from one country to another.

Bacterial Proteins↗

PubMed2002

Synaptotagmin I increases the probability of vesicle fusion at low [Ca2+] in pituitary cells.

Synaptotagmin I (Syt I), a low-affinity Ca(2+)-binding protein, is thought to serve as the Ca(2+) sensor in the release of neurotransmitter. However, functional studies on the calyx of Held synapse revealed that the rapid release of neurotransmitter requires only approximately micromolar [Ca(2+)], suggesting that Syt I may play a more complex role in determining the high-affinity Ca(2+) dependence of exocytosis. Here we tested this hypothesis by studying pituitary cells, which possess high- and low-affinity Ca(2+)-dependent exocytic pathways and express Syt I. Using patch-clamp capacitance measurements to monitor secretion and the acute antisense deletion of Syt I from differentiated cells, we have shown that the rapid and the most Ca(2+)-sensitive pathway of exocytosis in rat melanotrophs requires Syt I. Furthermore, stimulation of the Ca(2+)-dependent exocytosis by cytosol dialysis with solutions containing 1 microM [Ca(2+)] was completely abolished in the absence of Syt I. Similar results were obtained by the preinjection of antibodies against the CAPS (Ca(2+)-dependent activator protein for secretion) protein. These results indicate that synaptotagmin I and CAPS proteins increase the probability of vesicle fusion at low cytosolic [Ca(2+)].

Animals↗

PubMed2002

Cardiovascular physiology: simulation of steady state and transient phenomena by using the equivalent electronic circuit.

By using commercially available software it is readily possible to design electronic circuits and to analyze them. By introducing the concept of equivalent quantities a simulation of various physiological phenomena is possible. This includes the steady state as well as various complex transient phenomena. This paper describes the use of an equivalent electronic circuit in simulating the cardiovascular system. It allows a stepwise upgrading. The first step is a one-ventricle circuit similar to the Starling heart-lung preparation. The final step is an equivalent circuit allowing simulation of various normal as well as pathological states (e.g. effects of heart rate, negative intrathoracic pressure, exercise, hemorrhage, heart failure, and hypertension). The degree of disturbance can be set by adjusting the value of single components. Following this, the optimal type of compensation (e.g. the increase in blood volume in failure of the right ventricle; systemic venoconstriction in failure of the left ventricle) of the basic disturbance can be searched for, activated and the consequences studied. The described approach has been found a useful tool in teaching physiology and pathophysiology for postgraduate medical students.

Cardiovascular Physiological Phenomena↗

PubMed2001

How to detect Clostridium difficile variant strains in a routine laboratory.

Toxin A-negative, toxin B-positive strains (A-/B+) are the best studied examples of Clostridium difficile variant strains. In addition, there are some other groups of variant C. difficile strains that produce both toxins (A+/B+) or are non-cytotoxic (A-/B-) but differ from the reference strain VPI 10463 in their toxin genes. Here we describe two simple methods (amplification of the tcdA gene and amplification of the binary toxin gene cdtA) which can be used in rapid screening for variant C. difficile strains in collections or in routine laboratories.

Bacterial Proteins↗

PubMed2000

Production of actin-specific ADP-ribosyltransferase (binary toxin) by strains of Clostridium difficile.

In addition to the two large clostridial cytotoxins (TcdA and TcdB) certain strains of Clostridium difficile produce an actin-specific ADP-ribosyltransferase, or binary toxin. PCR reactions were developed to detect genes encoding the enzymatic (cdtA) and binding (cdtB) components of the binary toxin and 170 representative strains were tested to assess the prevalence of the toxin. Positive PCR results (n=59) were confirmed by immunoblotting and ADP-ribosyltransferase assay. PCR ribotype and toxinotype (restriction fragment length polymorphism analysis of genes for TcdA and TcdB) correlated with possession of binary toxin genes. All strains with cdtA and cdtB belonged to toxin-variable toxinotypes and five toxin-producing groups of strains have been described according to the presence or absence of TcdA, TcdB and binary toxin. Result indicate that ca. 6.4% of toxigenic isolates of C. difficile referred to the Anaerobe Reference Unit from UK hospitals have cdtA and cdtB genes.

ADP Ribose Transferases↗

PubMed2000

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein.

Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca(2+)-dependent activator protein for secretion), a protein required for Ca(2+)-dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca(2+) elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca(2+) dependencies. A threshold of approximately 10 microM Ca(2+) was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca(2+) activity < 10 microM. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca(2+) and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.

Animals↗

PubMed2000

A chimeric ribozyme in clostridium difficile combines features of group I introns and insertion elements.

CdlSt1, a DNA insertion of 1975 bp, was identified within tcdA-C34, the enterotoxin gene of the Clostridium difficile isolate C34. Located in the catalytic domain A1-C34, Cd/St1 combines features of two genetic elements. Within the first 434 nt structures characteristic for group I introns were found; encoding the two transposase-like proteins tlpA and tlpB nucleotides 435-1975 represent the remainder of a IS605-like insertion element. We show that the entire CdlSt1 is accurately spliced from tcdA-C34 primary transcripts and that purified TcdA-C34 toxin is of regular size and catalytic activity. A search for CdlSt1-related sequences demonstrates that the element is widespread in toxinogenic and non-toxinogenic C. difficile strains, indicating the mobility of CdlSt1. In strain C34, we characterize 10 CdlSt1 variants; all are highly homologous to CdlSt1 (> 93% identity), integrated in bacterial open reading frames (ORFs), show the typical composite structure of CdlSt1 and are precisely spliced from their primary transcripts. CdlSt1-like chimeric ribozymes appear to combine the invasiveness of an insertion element with the splicing ability of a group I intron, rendering transposition harmless for the interrupted gene.

Amino Acid Sequence↗

PubMed1999

A nonsense mutation abrogates production of a functional enterotoxin A in Clostridium difficile toxinotype VIII strains of serogroups F and X.

Clostridium difficile strains of toxinotype VIII from serogroups F and X are described as toxin B-positive, toxin A-negative (TcdB+ A-), although they harbour almost the entire tcdA gene. To identify the reason for the lack of TcdA detection, we analyzed catalytic and ligand domains of TcdA-1470 of the type strain of serogroup F, strain 1470. Using recombinant fragments, the C-terminal immunodominant ligand domain TcdA3-1470, spanning amino acid residues 1694-2711 (corresponding to VPI 10463 sequence), was detected in Western blots. Similar experiments using the recombinant N-terminal catalytic fragment TcdAc1-2-1470 (amino acid positions 1-544) failed. In addition, this fragment showed no glucosylation activity. We determined the size and the position of alterations in the ligand domain tcdA3-1470 by DNA sequencing. Within the N-terminal fragment tcdAc1-2-1470, a nonsense mutation was identified introducing a stop codon at amino acid position 47. Identical mutations were found in the two serogroup X strains 17663 and 10355. The mutation might explain the lack of TcdA production observed in strains of serotypes F and X.

Bacterial Proteins↗

PubMed1999

The heterotrimeric Gi(3) protein acts in slow but not in fast exocytosis of rat melanotrophs.

Besides having a role in signal transduction some trimeric G-proteins may be involved in a late stage of exocytosis. Using immunocytochemistry and confocal microscopy we found that Gi(3)-protein resides mainly in the plasma membrane, whereas Gi(1/2-)protein is preferentially associated with secretory granules. To study the function of trimeric Gi(3)- and Gi(1/2)-proteins, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. We report here that mastoparan, an activator of trimeric G-proteins, enhances calcium-induced secretory activity in rat melanotrophs. The introduction of synthetic peptides corresponding to the C-terminal domain of the (&agr;)-subunit of Gi(3)- and Gi(1/2)-proteins indicated that Gi(3 )peptide specifically blocked the mastoparan-stimulated secretory activity, which indicates an involvement of a trimeric Gi(3)-protein in mastoparan-stimulated secretory activity. Flash photolysis of caged Ca(2+)-elicited biphasic capacitance increases consisting of a fast and a slower component. Injection of anti-Gi(3) antibodies selectively inhibited the slow but not the fast component of secretory activity in rat melanotrophs. We propose that the plasma membrane-bound Gi(3)-protein may be involved in regulated secretion by specifically controlling the slower kinetic component of exocytosis.

Animals↗

PubMed1998

A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates.

Two hundred nineteen Clostridium difficile isolates from 22 serogroups were screened for changes in the genes coding for toxin B (tcdB) and toxin A (tcdA). Parts of the toxin genes were amplified, and the PCR fragments were checked for length polymorphisms and cut with several restriction enzymes to monitor restriction fragment length polymorphisms (RFLPs). For 47 strains (21%), differences in the toxin genes were found compared to the toxin genes of reference strain VPI 10,463. Polymorphisms were usually observed in both toxin genes. RFLPs were more commonly found in the tcdB gene, in which a single restriction enzyme could give up to five different patterns. Restriction sites seemed to be less heterogeneous in the tcdA gene, in which for most enzymes only two different RFLPs were recognized. However, deletions were observed in tcdA, and four new types of shortened tcdA genes are described. According to the changes in their toxin genes, variant strains could be divided into 10 groups (toxinotypes I to X). A toxinotype was characterized by similar patterns of changes in the toxin genes and in other regions of the pathogenicity locus and also similar pulsed-field gel electrophoresis patterns. Variant toxinotypes were found in 9 of the 22 serogroups studied, and some toxinotypes were clearly associated with specific serogroups. Toxinotype VIII is characteristic for all strains of serogroup F. Other serogroups in which variant toxinotypes were commonly found are A1, A15, E, and X. Testing of variability in C. difficile toxin genes not only might be useful as a molecular typing system but also could have implications in diagnostics and pathogenesis.

Bacterial Proteins↗

PubMed1997

Mastoparan and Rab3AL peptide potentiation of calcium-independent secretory activity in rat melanotrophs is inhibited by GDPbetaS.

The whole-cell patch-clamp membrane capacitance measurement was used to monitor secretory activity in rat melanotrophs, while rab3AL, putative effector domain peptides of Rab3 small GTPases (20-30 kDa), were introduced into cytosol. In melanotrophs dialyzed with calcium free solutions membrane capacitance tends to decrease slightly. This decrease is further potentiated with GDPbetaS (500 microM). We found that rab3AL (100 microM) stimulated secretory activity in the absence of calcium. The rab3AL response was qualitatively comparable to the response to mastoparan (1 microM), an activator of certain heterotrimeric GTP-binding proteins. Interestingly, inclusion of GDPbetaS (500 microM) resulted in a blockade of both rab3AL and mastoparan induced responses. We conclude that rab3AL and mastoparan induce calcium-independent stimulation of secretory activity in rat melanotrophs by activation of a downstream heterotrimeric GTP-binding protein.

Amino Acid Sequence↗

PubMed1997

Characterization of polymorphisms in the toxin A and B genes of Clostridium difficile.

We have used six independent polymerase chain reactions (A1-A3 and B1-B3) for amplification of the entire sequence of the two toxin genes tcdA and tcdB of several Clostridium difficile strains. With this approach we have detected (1) restriction site polymorphisms which are distributed all over the genes, and (2) deletions that could be found only in tcdA. Characteristic differences between strains were mainly focused to the 5' third of tcdB (B1 fragment) and/or the 3' third of tcdA (A3 fragment). The possible use of our approach for typing of C. difficile toxin genes is discussed.

Bacterial Proteins↗

PubMed1995

Brefeldin A and a synthetic peptide to ADP-ribosylation factor (ARF) inhibit regulated exocytosis in melanotrophs.

We investigated the role of ADP-ribosylation factor (ARF) in regulated exocytosis in patch-clamped rat melanotrophs. Addition of brefeldin A (BFA) to inhibit activation of endogenous ARF protein was found to attenuate regulated secretory activity monitored as changes in membrane capacitance (Cm). A synthetic peptide to amino acids 46-61 of ARF (P-14) was also found to inhibit Ca(2+)-induced secretory activity in these cells. This inhibition was not apparent with a scrambled amino acid sequence of ARF-P14 peptide. This paper provides the first patch-clamp study to suggest that the small GTP-binding protein ARF is required to trigger release of secretory granules from rat pituitary melanotrophs.

ADP-Ribosylation Factors↗

PubMed1995

Intracellular Cl- modulates Ca2+-induced exocytosis from rat melanotrophs through GTP-binding proteins.

We used the whole-cell patch-clamp technique to monitor changes in membrane capacitance (Cm) to study the influence of cytosolic concentration ([Cl-]i) on the secretory activity of rat melanotrophs. The sensitivity of the secretory machinery to Ca2+ was enhanced in the presence of a high [Cl-]i. The free concentration of Ca2+ required for half-maximal secretory activity was reduced from 3.2microM at 4mM [Cl-]i to 0.7microM at 154mM [Cl-]i. To study whether the modulation of secretory activity by Cl- involves guanosine 5'-triphosphate-(GTP-) binding proteins, cells were dialysed with non-hydrolysable GTP and GDP analogues, fluoroaluminate (AlF4(-)), or were pretreated with pertussis toxin. With guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S], 100microM) the maximal rate of Cm increase (dCm/dt) was enhanced at 4 and 14mM [Cl-]i, but it was not affected at 154mM [Cl-]i. In contrast, the secretory response, measured as a percentage of resting Cm 10min after the start of recordings, was reduced at 154mM [Cl-]i, but not affected at 4mM [Cl-]i. Only with 154mM [Cl-]i did intracellular dialysis of cells with guanosine 5'-O-(2-thiodiphosphate) (GDP[beta-S], 500microM) inhibit dCm/dt as well as relative secretory responses. The presence of AlF4(-) (30microM) or a 7-h pretreatment of cells with pertussis toxin (250ng/ml) significantly reduced both the maximal dCm/dt and relative secretory responses, but only in the presence of 154mM [Cl-]i. Since the effects of GDP[beta-S], AlF4(-), and pertussis toxin pretreatment were only detected with a high [Cl-]i, we conclude that modulation by Cl- of secretory activity of rat melanotrophs is mediated through GTP-binding proteins. Furthermore, the effects of AlF4(-) and pertussis toxin indicate a role of heterotrimeric GTP-binding proteins in the secretory activity of melanotrophs.

Aluminum Compounds↗