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J Tytgat

Publications and source records attributed to J Tytgat.

At least 37 records · Page 2Linked to original sources
PubMed2000

Cannabinoid mimics in chocolate utilized as an argument in court.

A case is presented involving chocolate cannabinoid mimics which have been utilized in court by the defendant's lawyer in order to clear the accused of smoking and dealing in marijuana after he was found positive for cannabis in a routine urine immunoassay screening test. The argumentation in this case was that the accused had supposedly eaten a massive amount of chocolate which contained anandamide-related lipids. These lipids inhibit anandamide hydrolysis in the brain, act as cannabinoid mimics and, according to the lawyer, were the cause of the positive cannabinoid test. To investigate this in detail, we synthesized N-oleoyl- and N-linoleoylethanolamide and spiked these compounds together with N-arachidonoylethanolamide in urine for immunological investigations. None of the samples were found positive, indicating that no cross-reactivity occurs with cannabinoids. As a result, the lawyer's claim could be refuted and the accused was convicted.

Arachidonic Acids↗
PubMed2000

Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channels.

It was the aim of this study to look for a high-affinity and selective polypeptide toxin, which could serve as a probe for the volume-regulated anion channel (VRAC) or the calcium-activated chloride channel (CaCC). We have partially purified chlorotoxin, including new and homologous short chain insectotoxins, from the crude venom of Leiurus quinquestriatus quinquestriatus (Lqq) by means of gel filtration chromatography. Material eluting between 280 and 420 min, corresponding to fractions 15-21, was lyophilized and tested on VRAC and CaCC, using the whole-cell patch-clamp technique. We have also tested the commercially available chlorotoxin on VRAC, CaCC, the cystic fibrosis transmembrane conductance regulator (CFTR) and on the glioma specific chloride channel (GCC). VRAC and the correspondent current, I(Cl,swell), was activated in Cultured Pulmonary Artery Endothelial (CPAE) cells by a 25% hypotonic solution. Neither of the fractions 16-21 significantly inhibited I(Cl,swell) (n=4-5). Ca(2+)-activated Cl(-) currents, I(Cl,Ca), activated by loading T84 cells via the patch pipette with 1 microM free Ca(2+), were not inhibited by any of the tested fractions (15-21), (n=2-5). Chlorotoxin (625 nM) did neither effect I(Cl,swell) nor I(Cl,Ca) (n=4-5). The CFTR channel, transiently transfected in COS cells and activated by a cocktail containing IBMX and forskolin, was not affected by 1.2 microM chlorotoxin (n=5). In addition, it did not affect currents through GCC. We conclude that submicromolar concentrations of chlorotoxin do not block volume-regulated, Ca(2+)-activated and CFTR chloride channels and that it can not be classified as a general chloride channel toxin.

Animals↗
PubMed2000

Interaction of p-fluorofentanyl on cloned human opioid receptors and exploration of the role of Trp-318 and His-319 in mu-opioid receptor selectivity.

In this study, we investigated the interactions of p-fluorofentanyl, an opioid designer drug, fentanyl, sufentanyl, and morphine on cloned human mu-, kappa-, and delta-opioid receptors coexpressed with heteromultimeric G protein-coupled inwardly rectifying K(+) channels (GIRK1/GIRK2) and a regulator of G protein signaling (RGS4) in Xenopus oocytes. We demonstrate that p-fluorofentanyl more potently activates GIRK1/GIRK2 channels through opioid receptors than fentanyl and that the p-fluoro substitution also changes the potency profile from mu > kappa > delta (fentanyl) to mu > delta > or = kappa (p-fluorofentanyl). A comparison of ligand efficacy revealed that morphine, fentanyl, and its analogs less efficiently activate GIRK1/GIRK2 channels through human mu-opioid receptor than [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin. Using site-directed mutagenesis, we investigated whether mutating residues Trp-318 and His-319 to their corresponding residues in kappa- and delta-opioid receptors provides the molecular basis for mu/delta selectivity and mu/kappa selectivity. Changes in EC(50) values for the W318L and W318Y/H319Y mu-opioid receptors show a partial contribution of these residues to the decreased GIRK1/GIRK2 channel activation by fentanyl analogs through kappa- and delta-opioid receptors. The most pronounced effect was observed for p-fluorofentanyl, suggesting that an interaction between the 4-fluorophenylpropanamide moiety of the drug and residues Trp-318 and His-319 is important for the resulting enhanced GIRK1/GIRK2 channel activation through the mu-opioid receptor. Finally, we demonstrate that mutation of W318L confers delta-like potency for morphine on the mutant mu-opioid receptor.

Animals↗
PubMed1999

The dual modulation of GIRK1/GIRK2 channels by opioid receptor ligands.

It is well known that activation of the cloned kappa-opioid receptor by nanomolar concentrations of U50488H (trans-(+/-)-3, 4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl-benzeneacetamide) , a selective kappa-opioid receptor agonist, leads to the opening of GIRK1 channels. In this study, we demonstrate that the cloned kappa-opioid receptor functionally couples to GIRK1/GIRK2 channels (G-protein-coupled inwardly rectifying K(+) channels), mimicking the probable heteromultimeric state of neuronal GIRK channels. We also show that micromolar concentrations of U50488H reduce GIRK1/GIRK2 current through direct GIRK1/GIRK2 channel block in a voltage-independent manner (IC(50)=70.28+/-3.68 microM). Similarly, it was found that propoxyphene, methadone, and naloxone also can block GIRK1/GIRK2 current. In contrast, elevated concentrations of morphine (up to 1 mM) did not cause channel block. The related inwardly rectifying K(+) channel, IRK1, was not affected by elevated concentrations of these drugs. We conclude that nanomolar concentrations of opioid receptor ligands activate GIRK1/GIRK2 channels through a receptor-mediated pathway, while micromolar concentrations of some opioid receptor ligands inhibit GIRK1/GIRK2 channels by direct channel block.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1999

Norpropoxyphene-induced cardiotoxicity is associated with changes in ion-selectivity and gating of HERG currents.

OBJECTIVE: Norpropoxyphene (NP) is a major metabolite of propoxyphene (P), a relatively weak mu-opioid receptor agonist. Toxic blood concentrations ranging from 3 to 180 mumol/l have been reported and the accumulation of NP in cardiac tissue leads to naloxone-insensitive cardiotoxicity. Since several lines of evidence suggest that not only block of INa but also IK block may contribute to the non-opioid cardiotoxic effects of P and NP, we investigated the effects of P and NP on HERG channels. HERG presumably encodes IKr, the rapidly-activating delayed rectifier K+ current, which is known to have an important role in initiating repolarization of action potentials in cardiac myocytes. METHODS: Using the 2-microelectrode voltage clamp technique we investigated the interaction of P and NP with HERG channels, expressed in Xenopus oocytes. RESULTS: Our experiments show that low drug concentrations (5 mumol/l) facilitate HERG currents, while higher drug concentrations block HERG currents (IC50-values of approx. 40 mumol/l) and dramatically shift the reversal potential to a more positive value because of a 30-fold increased Na(+)-permeability. P and NP also alter gating of HERG channels by slowing down channel activation and accelerating channel deactivation kinetics. The mutant S631C nullifies the effect of P and NP on the channel's K(+)-selectivity. CONCLUSION: P and NP show a complex and unique drug-channel interaction, which includes altering ion-selectivity and gating. Site-directed mutagenesis suggests that an interaction with S631 contributes to the drug-induced disruption of K(+)-selectivity. No specific role of the minK subunit in the HERG block mechanism could be determined.

Animals↗
PubMed1999

A unified nomenclature for short-chain peptides isolated from scorpion venoms: alpha-KTx molecular subfamilies.

Peptidyl toxins are used extensively to determine the pharmacology of ion channels. Four families of peptides have been purified from scorpion venom. In this article, the classification of K+-channel-blocking peptides belonging to family 2 peptides and comprising 30-40 amino acids linked by three or four disulfide bridges, will be discussed. Evidence is provided for the existence of 12 molecular subfamilies, named alpha-KTx1-12, containing 49 different peptides. Because of the pharmacological divergence of these peptides, the principle of classification was based on a primary sequence alignment, combined with maximum parsimony and Neighbour-Joining analysis.

Amino Acid Sequence↗
PubMed1999

Scorpion toxins specific for Na+-channels.

Na+-channel specific scorpion toxins are peptides of 60-76 amino acid residues in length, tightly bound by four disulfide bridges. The complete amino acid sequence of 85 distinct peptides are presently known. For some toxins, the three-dimensional structure has been solved by X-ray diffraction and NMR spectroscopy. A constant structural motif has been found in all of them, consisting of one or two short segments of alpha-helix plus a triple-stranded beta-sheet, connected by variable regions forming loops (turns). Physiological experiments have shown that these toxins are modifiers of the gating mechanism of the Na+-channel function, affecting either the inactivation (alpha-toxins) or the activation (beta-toxins) kinetics of the channels. Many functional variations of these peptides have been demonstrated, which include not only the classical alpha- and beta-types, but also the species specificity of their action. There are peptides that bind or affect the function of Na+-channels from different species (mammals, insects or crustaceans) or are toxic to more than one group of animals. Based on functional and structural features of the known toxins, a classification containing 10 different groups of toxins is proposed in this review. Attempts have been made to correlate the presence of certain amino acid residues or 'active sites' of these peptides with Na+-channel functions. Segments containing positively charged residues in special locations, such as the five-residue turn, the turn between the second and the third beta-strands, the C-terminal residues and a segment of the N-terminal region from residues 2-11, seems to be implicated in the activity of these toxins. However, the uncertainty, and the limited success obtained in the search for the site through which these peptides bind to the channels, are mainly due to the lack of an easy method for expression of cloned genes to produce a well-folded, active peptide. Many scorpion toxin coding genes have been obtained from cDNA libraries and from polymerase chain reactions using fragments of scorpion DNAs, as templates. The presence of an intron at the DNA level, situated in the middle of the signal peptide, has been demonstrated.

Amino Acid Sequence↗
PubMed1998

Purification and partial characterization of a 'short' insectotoxin-like peptide from the venom of the scorpion Parabuthus schlechteri.

A disulfide-rich, low-molecular-mass toxin-like peptide has been isolated from Parabuthus schlechteri venom using gel filtration, ion exchange, and reversed phase chromatography. Partial characterization of this peptide reveals a relationship with four-disulfide bridge proteins belonging to the family of 'short' insectotoxins (44% residue identity). In recognition hereof, the peptide was named PBITx1 (sITx10). Our work also reports on the deduced sequences of two other 'short' insectotoxins from Buthus eupeus, I3 and I4, and it provides a consensus sequence and nomenclature for all known 'short' insectotoxins. Finally, sequence similarities with K+ channel blockers (charybdotoxin, kappa-conotoxin), and a Cl- channel blocker (chlorotoxin) are highlighted.

Amino Acid Sequence↗
PubMed1998

Comparison and characterization of the venoms of three Parabuthus scorpion species occurring in southern Africa.

Parabuthus transvaalicus, P. granulatus, and P. villosus are three medically important scorpion species occurring in southern Africa which can cause severe envenoming among people. In contrast to many other genera, no data is available on the venom composition of scorpions belonging to the genus Parabuthus. Here we have investigated the components which may contribute to the venomous potential. The constancy of venom composition within each of the three species and between the three species was investigated by means of gel filtration chromatography. The venoms of the three species each were characterized by a constant and typical elution pattern, resulting in a 'gel filtration fingerprint' which allows distinction between each species. It appears that certain components in the venoms are common to either all three species, or to two of the three species. This points to a clear interspecies relationship within the genus. We also describe the isolation and characterization of some of the polypeptide toxins present in the venoms of P. villosus, P. transvaalicus and P. granulatus by means of reversed phase chromatography and screening of the toxic components on voltage-activated potassium and sodium channels. Our results confirm that toxins which inhibit potassium channels and alter sodium channel gating are present in the venoms studied.

Animals↗
PubMed1997

Alternative splicing of ClC-6 (a member of the CIC chloride-channel family) transcripts generates three truncated isoforms one of which, ClC-6c, is kidney-specific.

ClC-6 is a protein that structurally belongs to the family of ClC-type chloride channels. We now report the identification of three additional ClC-6 isoforms that are truncated because of alternative splicing. We have isolated, from human K562 cells, four types of ClC-6 cDNAs that encode four distinct ClC-6 protein isoforms. ClC-6a (869 amino acids) corresponds to the previously published ClC-6 protein [Brandt and Jentsch (1995) FEBS Lett. 377, 15-20] and it has a canonical ClC structure. However, ClC-6b (320 amino acids), ClC-6c (353 amino acids) and ClC-6d (308 amino acids) are truncated at their C-termini. Hydropathy-plot analysis indicates that the shortened isoforms contain maximally four (ClC-6b and -6d) or seven (ClC-6c) transmembrane domains. Sequence analysis of a human genomic ClC-6 fragment indicates that the cDNA variability arises from alternative splicing at two different positions: the first alternative site consists of an intron flanked by two alternative donor sites and two alternative acceptor sites, the second being due to an exon that is optionally included or excluded. Reverse-transcription-PCR analysis of ClC-6 expression in human cell lines and tissues shows that the majority (83%) of ClC-6 mRNAs consists of ClC-6a or ClC-6c messengers. Furthermore, in a mouse tissue panel, the ClC-6a mRNA has a relatively broad tissue expression pattern, since it could be detected in brain, kidney, testis, skeletal muscle, thymus and pancreas. In contrast, expression of ClC-6c is more restricted, since it was only detected in kidney.

Alternative Splicing↗
PubMed1997

Effect of lanthanum on voltage-dependent gating of a cloned mammalian neuronal potassium channel.

The effect of the trivalent cation lanthanum (La3+) on voltage-dependent gating of a cloned mammalian neuronal Kv1.1 potassium channel was studied under whole-cell voltage-clamp conditions in oocytes of Xenopus laevis. La3+ (100 microM) was found to decrease the potassium currents at all test potentials and to shift the midpoint of the fraction open channels/membrane voltage curve by approximately +20 mV. The opening and closing time constants of Kv1.1 channels were empirically fitted with a 4th power Hodgkin-Huxley formalism, or with mono- and multi-exponentials. It was found that La3+ slowed down the kinetics of activation, speeded up those of deactivation, and shifted the opening kinetics by approximately + 60 mV. Interestingly, all these parameters of channel gating were not affected equally by La3+. Furthermore, amplitudes of the inward tail currents evoked at potentials more negative than the potassium equilibrium potential (E(K+)) were more strongly inhibited by La3+ than those of the outward tail currents evoked at potentials more positive than E(K+). This suggests voltage-dependent block and binding of La3+ to the Kv1.1 channel protein. We conclude that these actions cannot be explained in terms of surface charge considerations alone. Our results provide evidence for a direct interaction with the potassium channel protein, shedding new light on the mechanism of action of this lanthanide.

Animals↗
PubMed1997

Expression of human pICln and ClC-6 in Xenopus oocytes induces an identical endogenous chloride conductance.

pICln is a protein that induces an outwardly rectifying, nucleotide-sensitive chloride current (ICln) when expressed in Xenopus oocytes, but its precise function (plasma-membrane anion channel versus cytosolic regulator of a channel) remains controversial. We now report that a chloride current identical to ICln is induced when Xenopus oocytes are injected with human ClC-6 RNA. Indeed, both the pICln and the ClC-6 induced current are outwardly rectifying, they inactivate slowly at positive potentials and have an anion permeability sequence NO3- > I- > Br- > Cl- > gluconate. Cyclamate, NPPB, and extracellular cAMP block the induced currents. The success rate of current expression is significantly increased when the injected Xenopus oocytes are incubated at a higher temperature (24 or 37 degrees C) prior to the analysis. In addition, the ICln current was detected in 6.2% of noninjected control Xenopus oocytes. We therefore conclude that the ICln current in Xenopus oocytes corresponds to an endogenous conductance that can be activated by expression of structurally unrelated proteins. Furthermore, functional, biochemical, and morphological observations did not support the notion that pICln resides in the plasma membrane either permanently or transiently after cell swelling. Thus, it is unlikely that pICln forms the channel that is responsible for the ICln current in Xenopus oocytes.

Animals↗
PubMed1997

Value of axillary ultrasonography and sonographically guided puncture of axillary nodes: a prospective study in 144 consecutive patients.

The value of axillary sonography in 144 consecutive patients was prospectively studied. Abnormal lymph nodes were demonstrated in 72 axillae, only half (36 of 72) of which were clinically detected. In comparison with intraoperative findings in 47 patients with breast carcinoma, the sensitivity of sonography in detecting malignant nodes was 92%, the specificity 95%, and the positive and negative predictive value 96% and 91%, respectively. In 42 patients a sonographically guided node puncture was performed. Evidence of malignancy was found in 38 (90.5%). In 14 (33%) of them, cyto-histology pointed to a clinically undetected primary malignancy.

Adult↗
PubMed1997

Effect of fluoxetine on a neuronal, voltage-dependent potassium channel (Kv1.1).

1. Fluoxetine (Prozac) is widely used as an antidepressant drug and is assumed to be a selective 5-hydroxytryptamine (5-HT) reuptake inhibitor (SSRI). Claims that its beneficial psychotropic effects extend beyond those in treatment of depression have drawn clinical and popular attention to this compound, raising the question of whether there is anything exceptional about the supposed selective actions. 2. We have used the voltage clamp technique to study the effect of fluoxetine on a neuronal, voltage-dependent potassium (K+) channel (RCK1; Kv1.1), expressed in p6nopus laevis oocytes. This channel subunit is abundantly expressed in the central nervous system and K+ channels containing this subunit are involved in the repolarization process of many types of neurones. 3. Blockade of the K+ currents by fluoxetine was found to be use- and dose-dependent. Wash-out of this compound could not be achieved. Fluoxetine did not affect the ion selectivity of this K+ channel, as the reversal potential was unaltered. 4. Slowing of both activation and deactivation kinetics of the channel by fluoxetine was observed, including tail current crossover upon repolarization. 5. Hodgkin-Huxley type of models and more generalized Markov chain models were used to fit the kinetics of the data. Based upon a Markov kinetic scheme, our data can be interpreted to mean that blockade of fluoxetine consists of two components: a voltage-independent occurring in the last closed, but available state of the channel, and a voltage-dependent occurring in the open state. 6. This study describes the first biophysical working model for the mechanism of action of fluoxetine on a neuronal, voltage-dependent K+ channel, RCK1. Although this channel is not very potently blocked by fluoxetine when expressed in oocytes, this study may help us to understand some of the clinical symptoms seen with elevated serum concentrations of this SSRI.

Animals↗
PubMed1996

Detection and quantification of the hypnotic zopiclone, connected with an uncommon case of drowning.

A fatality, attributed to a suicidal ingestion of zopiclone and subsequent death by drowning, is described. A radioimmunoassay was performed to prescreen for the presence of the analyte. HPLC analysis with fluorescence detection demonstrated a testicular tissue concentration of 2.2 micrograms/g zopiclone. Further toxicological analysis revealed also the presence of diazepam in a concentration of 150 ng/g.

Adult↗
PubMed1996

Development of a stereospecific radioimmunoassay for the analysis of zopiclone and metabolites in urine.

A sensitive and specific radioimmunoassay has been developed, allowing the stereospecific detection of nanogram amounts of (+)- and (-)-enantiomers of zopiclone and its major metabolites in urine, without prior extraction or purification. Antisera were obtained from two series of four rabbits, immunized with optically pure (+)- and (-)-N-hemisuc-cinyldesmethylzopiclone, conjugated to bovine serum albumin according to the active ester method. The assay was stereospecific, allowing discrimination between the two enantiomers of N-desmethylzopiclone with mutual cross-reactivities below 2%. Substantial cross-reaction was observed with the parent compound, although lower than expected, and to a lesser extent with the N-oxide metabolite. A selection of hypnotics, anxiolytics, antidepressants and some other widely used drugs did not interfere with the assay (< 0.1%), when tested at a concentration level of 10 micrograms/ml. The sensitivity of the assay was 50 pg/ml and 10 pg/ml for the (+)- and (-)-enantiomers, respectively. The binding assay described here was used to evaluate the stereoselective excretion pattern of zopiclone. Analysis of cumulative excretion samples from a volunteer revealed a mean metabolic excretion ratio (+)/(-) of 2.2, ranging from 1.7 (7 h) to 4.4 (36 h) A mean excretion ratio (+)/(-) of 2.5 +/- 1 was calculated after analysis of urine samples from 20 patients receiving zopiclone as a hypnotic daily.

Animals↗