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PubMed1999

Monocyte stimulation by reactive oxygen species: role of superoxide and intracellular Ca2+.

OBJECTIVE: Production of reactive oxygen species has generally been linked to inflammatory processes. Whether the presence of superoxide and hydrogen peroxide affects mononuclear cell function was investigated with an in vitro model using isolated human mononuclear cells. MATERIALS AND METHODS: Human mononuclear cells were isolated from buffy coat. Toxicity was measured with Trypan blue exclusion, secreted TNF-alpha was measured with an ELISA, reactive oxygen species within mononuclear cells were quantified with dichlorodihydrofluorescein diacetate fluorescence, intracellular TNF-alpha was measured with flow cytometry and fluorescence microscopy. RESULTS: The enzymatic production of superoxide caused a dose-dependent increase in TNF-alpha synthesis, whereas hydrogen peroxide was ineffective. Flow cytometric measurements and immunofluorescence microscopy demonstrated that monocytes were the main TNF-alpha producing population. This proinflammatory reaction was further characterized by pharmacologically investigating Ca2+ signalling pathways. Superoxide stimulated TNF-alpha secretion was inhibited by intracellular Ca2+-buffers (MAPT-AM and BAPT-AM) or VOC operating antagonists (diltiazem and verapamil) and only to a small extent by pharmacological inhibitors of ligand-gated pathways (TMB-8 and SKF 96368). CONCLUSION: We propose that superoxide activates human mononuclear cells in a Ca2+-dependent manner.

Calcium↗

PubMed2002

Nasal midazolam as a novel anticonvulsive treatment against organophosphate-induced seizure activity in the guinea pig.

Seizures and status epilepticus, which may contribute to brain injury, are common consequences of exposure to organophosphorus (OP) cholinesterase inhibitors. Effective management of these seizures is critical. To investigate the efficacy of nasal midazolam as an anticonvulsive treatment for OP exposure, as compared to intramuscular midazolam, guinea pigs were connected to a recording swivel for electrocorticograph (ECoG) monitoring and clinical observation. The experimental paradigm consisted of pyridostigmine pretreatment (0.1 mg/kg i.m.) 20 min prior to sarin exposure (1.2x LD(50,) 56 micro g/kg i.m.). One minute post-exposure, atropine (3 mg/kg i.m.) and TMB-4 (1 mg/kg im) were administered. Within 3-8 min after sarin exposure all animals developed electrographic seizure activity (EGSA), with convulsive behavior. Treatment with midazolam (1 mg/kg i.m.) 10 min after the onset of EGSA abolished EGSA within 389+/-181 s. The same dose was not effective, in most cases, when given 30 min after onset. However, a higher dose (2 mg/kg) was found efficacious after 30 min (949+/-466 s). In contrast, nasal application of midazolam (1 mg/kg) was found most effective, with significant advantages, in amelioration of EGSA and convulsive behavior, when given 10 min (216+/-185 s) or 30 min (308+/-122 s) following the onset of EGSA ( P<0.001). Thus, nasal midazolam could be used as a novel, rapid and convenient route of application against seizure activity induced by nerve agent poisoning.

Administration, Intranasal↗

PubMedDate not supplied

Toxicokinetics of 1,2,4-trimethylbenzene in humans exposed to vapours of white spirit: comparison with exposure to 1,2,4-trimethylbenzene alone.

This study compares the toxicokinetics of inhaled 1,2,4-trimethylbenzene (124TMB) in men exposed to white spirit with that previously observed in the same individuals exposed to 124TMB alone. The appropriateness of using dimethylhippuric acid (DMHA) metabolites of 124-, 123- and 135TMB in urine as biomarkers of exposure is also addressed and the kinetics of n-decane, n-undecane and 123TMB is investigated. The toxicokinetics of 124TMB was studied in nine male, healthy volunteers exposed to solvent vapours in an exposure chamber for 2 h during a work load of 50 W. The subjects were exposed to 2 ppm (11 mg/m3) of 124TM B during exposure to 300 mg/m3 of white spirit. The 124TMB isomer was analysed in blood, urine and exhaled air by gas chromatography. The DMHA metabolites of all three TMB isomers were analysed in urine by high-performance liquid chromatography. The results were compared with previously published exposures to 2 and 25 ppm (120 mg/m3) of 124TMB vapour alone. In addition, the occurrence of acute effects was studied by means of a questionnaire. Irritation and central nervous system (CNS) symptoms were recorded by ratings on a 100 mm visual analogue scale. Blood levels of 124TMB and excretion rates of 3,4-DMHA in urine were markedly elevated both during and after exposure to white spirit compared to the same exposure level of 124TMB alone. No irritation or CNS effects were reported in the questionnaire at any exposure condition. It appears that components in white spirit interfere with the metabolic elimination of 124TMB. This should be considered in biological exposure monitoring as well as in risk assessment.

Adult↗

PubMed2000

The phosphoryl oxime-destroying activity of human plasma.

The potential of obidoxime and other pyridinium-4-aldoximes to reactivate dimethyl- and diethylphosphorylated cholinesterases is markedly restricted by the inevitable formation of rather stable phosphoryl oximes (POXs) with high anticholinesterase activity. This effect is hardly seen with very dilute enzyme preparations, but becomes significant at physiological enzyme concentrations. Human plasma with the butyrylcholinesterase irreversibly blocked by soman was able to stimulate obidoxime-induced reactivation of concentrated erythrocyte acetylcholinesterase (Ery-AChE) to the same extent as was observed with a dilute preparation, suggesting phosphoryl oxime-destroying capacity. The inactivating factor, which was tentatively termed POX-hydrolase, had (1) a molecular weight of >100 kDa; (2) required Ca2+ , which could not be substituted by Zn2+ or Mg2+; and (3) lost its catalytic activity reversibly in the presence of ethylenediamine-tetraacetic acid (EDTA). The enzyme activity varied widely (20-fold) among different subjects and did not follow the activity pattern of human serum paraoxonase (PON1). Rabbit plasma with its particularly high paraoxonase content showed only weak POX-hydrolase activity. These data suggest POX-hydrolase to be a different entity. POX-hydrolase was most active with the putative phosphoryl-obidoxime from paraoxon-ethyl, less with the product from paraoxon-methyl and least with that from diisopropylfluorophosphate. The analogue TMB-4 reacted similarly to obidoxime. The putative phosphonyl oximes arising by the reaction of obidoxime with nerve agents were apparently not cleaved. The variation in POX-hydrolase activity may additionally contribute to the variable response to oxime therapy in patients with organophosphate insecticide poisoning.

Animals↗

PubMed2005

Amperometric detection in TMB/HRP-based assays.

3,3',5,5'-Tetramethylbenzidine (TMB) is the most commonly used chromogen for horseradish peroxidase (HRP) and so its performance as an electrochemical substrate was evaluated. Measurements of HRP activity in solution were carried out by using an amperometric detector coupled to a flow injection analysis (FIA) system. The enzymatic product was easily detected at a potential of +0.1 V (vs. Ag-pseudoreference electrode) at a bare screen-printed electrode placed in a homemade electrochemical flow cell. A high flow rate (4.3 mL min(-1)) of 0.5 M H2SO4 was used to obtain repeatable signals and a short analysis time. The detection limit achieved after 15 min of incubation was 2x10(-14) M of HRP. The applicability of the amperometric detector to ELISAs was demonstrated by using a commercially available kit for the quantification of interleukin-6 (IL-6) without modifying the kit manufacturer's protocol or the reagents for this test.

Benzidines↗

PubMed2006

Investigation of several parameters influencing signal generation in flow-through membrane-based enzyme immunoassay.

Rapid-response analytical tests that can be performed at the point of sampling are based on a visual detection system. The influence of different factors on the signal generation in a membrane-based enzyme immunoassay was investigated. The research was applied to a flow-through immunoassay for the detection of ochratoxin A (OTA). This assay format is a very convenient, simple and fast qualitative screening tool. Conjugates of OTA with horseradish peroxidase (HRP) and alkaline phosphatase (AP) were used as enzyme tracers. A new conjugate OTA-AP has been synthesized in our laboratory and its performance in the assay was compared with that of OTA-HRP. Different substrate systems for HRP and AP were compared. Several reagents, including polymers and surfactants, were tested for their possible effect on signal generation with the use of OTA-HRP conjugate. Polymers such as poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG) 6000 exerted a favourable effect on signal amplification, whereas surfactants negatively affected assay performance. The highest signal amplification (30-70% compared to the standard assay procedure) was achieved using 0.5% PVA in tetramethylbenzidine (TMB) Colorburst substrate solution and phosphate-buffered saline (PBS) for the washing step. It allowed more reliable visual estimation of the results from OTA-HRP assay. Exclusion of the detergent (Tween 20) from the washing solution exerted a favourable effect on assay performance using both enzyme tracers. The assay using OTA-HRP was more susceptible to matrix interferences than the assay with OTA-AP. Signal development in the matrix was better for the OTA-AP assay and visual estimation of the results was easier to perform in this case. For the analysis of spiked wheat samples, OTA-AP conjugate gave a more sensitive, stable and reproducible assay with a cut-off level of 4 microg kg(-1) for OTA. The application of the new OTA-AP conjugate resulted in improved assay performance for the food samples.

Alkaline Phosphatase↗

PubMed2000

Determination of low-level mercury based on a renewable-drops sensing technique.

The design and characteristics of a novel drop-based fluorescence-detection technique for the determination of mercury(II) are described. The method, using a flow injection technique, is based on the renewable-drops of 3,3',5,5'-tetramethylbenzidine(TMB), which are formed at the bottom tip of a silica capillary tube connected to the end of the flow system. An excitation beam from a high-pressure Hg lamp directly illuminates the drops, the fluorescence emission is conducted to a photodiode (PD) to convert the photocurrent into a voltage signal (mV). Optimum analytical conditions for Hg(II) assays have been established. In NaAc/HAc buffer at pH 3.09 this assay has a wide linear range for Hg(II) from 8.0 x 10(-8) to 2.0 x 10(-5) mol/L with a detection limit of 2.0 x 10(-8) mol/L. The use of renewable drops allowing a fresh reaction surface for each sample is of particular value to solving the problems of irreversible reactions. Besides its high sensitivity, the method permits a simple, fast, and inexpensive measurement with only micro-quantities of reagent consumption. The technique described provides a simple and sensitive way to fabricate sensors of feasible prospects and commercial advantages.

Benzidines↗

PubMed2000

Sorption-catalytic determination of cadmium using bromobenzothiazo noncovalently bound to silica and paper.

Cadmium (along with Fe(II), Co(II), Zn(II), and Pb(II) ions) decreases the rate of oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) with KIO4 conducted either without or with Mn(II) as a catalyst. Cadmium(II) is preconcentrated from aqueous solutions on silica plates or paper filters physically modified with a reagent for selective determination of Cd(II), namely 1-[(6-bromo-2-benzothiazolyl)azo]-2-naphthol (bromobenzothiazo, or BBT). The modifier is strongly retained on the both supports at pH 6-10 and does not affect the inhibiting effect of Cd(II) in the indicator reaction. Cadmium is determined by its inhibiting action directly on the sorbents by measuring transmittance (BBT/paper) or reflectance (BBT/silica) with limits of detection of 2 x 10(-4) and 0.03 mg/L, respectively. The proposed hybrid combination of sorption with catalytic detection on the sorbent allows to increase the selectivity factors several times (up to 2 orders) relatively to the determination in solution. Tap water samples and soil extracts were analyzed.

Journal Article↗

PubMed2003

Genioglossal hypoglossal motoneurons contact substance P-like immunoreactive nerve terminals in the cat: a dual labeling electron microscopic study.

This study investigated the synaptic interactions between hypoglossal motoneurons that project to the genioglossus muscle and substance P (SP) containing immunoreactive nerve terminals. Cholera toxin B conjugated to horseradish peroxidase (CTB-HRP) was injected into the right half of the genioglossus muscle in four anesthetized cats. Two days later, the animals were perfused with acrolein fixative. Tetramethylbenzidine (TMB) was the chromogen used to detect retrogradely labeled cells containing CTB-HRP. The tissues were then processed for immunocytochemistry using an antiserum raised against SP with diaminobenzidine (DAB) as the chromogen. At the light microscopic level, labeled cells were observed primarily ipsilaterally in ventral and ventrolateral subdivisions of the hypoglossal nucleus. The majority of these labeled cells were observed at the level of the area postrema. At the electron microscopic level, SP-like immunoreactive nerve terminals formed synaptic contacts with retrogradely labeled dendrites and perikarya. Nineteen percent of the terminals that contacted retrogradely labeled cells contained SP. These are the first ultrastructural studies demonstrating synaptic interactions between protruder hypoglossal motoneurons and SP terminals. These studies demonstrate that hypoglossal motoneurons which innervate the major protruder muscle of the tongue, the genioglossus muscle, may be modulated by SP. Thus, SP may play a role in the control of protrusive movements of the tongue acting via neurokinin receptors.

Afferent Pathways↗

PubMed2025

Integrating necroptosis and immune landscapes: a multi-omics-derived NecropImmScore stratifies prognosis and therapy in ovarian cancer.

BACKGROUND: Ovarian cancer (OC) remains the deadliest gynecologic malignancy, largely due to its immunosuppressive tumor microenvironment (TME) and resistance to therapy. Necroptosis, a regulated lytic cell death pathway mediated by the RIPK1-RIPK3-MLKL axis, can trigger immunogenic cell death, but its specific role in shaping the OC immune landscape and its clinical translation potential are posorly understood. METHODS: We employed multi-omics analysis (transcriptomics, genomics, clinical data) from TCGA-OV (n = 380), ICGC OV-AU, and IMvigor210 cohorts, combined with rigorous in vitro functional validation using OC cell lines (SKOV3, HEY), macrophages (THP-1 derived), and T cells (Jurkat). Computational immunology approaches (ESTIMATE, CIBERSORT, ssGSEA) quantified immune infiltration. We identified MLKL-associated immune genes, performed survival analysis (Kaplan-Meier, Cox regression), and constructed a necroptosis-immune signature (NecropImmScore) using consensus clustering and PCA of 102 prognostic genes. Drug sensitivity was predicted via pRRophetic and CellMiner. RESULTS: MLKL emerged as a protective prognostic biomarker (p = 0.018), significantly correlated with enhanced immune infiltration (ImmuneScore, StromalScore, ESTIMATEScore; p < 2.22e-16), M1 macrophage polarization (p = 0.006), activated CD4 + T cells (p = 0.003), and elevated immune checkpoint expression (PD-L1, CTLA4, LAG3, TIGIT). In vitro, MLKL overexpression in OC cells promoted M1 polarization (p < 0.05), activated Jurkat T cells (upregulated CCR4/5/7/9, CD69, CD3D/E, GZMB; p < 0.05), and induced key chemokines (CXCL9/10/11/13) critical for immune cell recruitment. Integration of MLKL-related and immune-related DEGs (n = 632) revealed enrichment in T-cell activation, chemokine signaling, and antigen presentation pathways (FDR < 0.05). Consensus clustering based on 102 survival-associated genes defined three molecular subtypes (Clusters A-C) with divergent survival (p = 0.019), necroptosis activity, and immune infiltration (Cluster C: best prognosis, highest MLKL/ImmuneScore). The derived NecropImmScore robustly stratified patients: high-score correlated with superior overall survival (TCGA: p < 0.001; ICGC: p = 0.014), inflamed TME phenotype, elevated checkpoint expression, and improved response to anti-PD-L1 in IMvigor210. Critically, high NecropImmScore predicted higher BRCA1 mutation frequency (AUC = 0.802), synergy with BRCA1 status for prognosis, higher homologous recombination deficiency (HRD) score, sensitivity to cisplatin (p = 0.014), paclitaxel (p = 0.016), gemcitabine (p = 0.017), and provided superior prognostic stratification when combined with TMB and HRD score (p < 0.001). CONCLUSION: This study establishes MLKL as a master regulator of anti-tumor immunity in OC, driving chemokine-mediated immune cell recruitment and TME reprogramming. The novel NecropImmScore is a multifaceted biomarker that effectively predicts prognosis, immunotherapy response, BRCA1 deficiency, and chemosensitivity, offering significant potential for guiding precision therapeutic strategies in OC.

Humans↗

PubMed2001

Ca++ regulation of paracellular permeability in the middle intestine of the eel, Anguilla anguilla.

The role of Ca++ on the regulation of the paracellular pathway permeability of the middle intestine of Anguilla anguilla was studied by measuring the transepithelial resistance and the dilution potential, generated when one half of NaCl in the mucosal solution was substituted iso-osmotically with mannitol, in various experimental conditions altering extracellular and/or intracellular calcium levels. We found that removal of Ca++ in the presence of ethylene glycol-bis(beta-aminoethyl ether) (EGTA) from both the mucosal and the serosal side, but not from one side only, reduced both the transepithelial resistance and the magnitude of the dilution potential. The irreversibility of this effect suggests a destruction of the organization of the junction in the nominal absence of Ca++. However a modulatory role of extracellular Ca++ cannot be excluded. The decrease of the intracellular Ca++ activity, produced by using verapamil to block the Ca++ entry into the cell, or by adding 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (hydrochloride) (TMB-8), an inhibitor of Ca++ release from the intracellular stores, reduced both the transepithelial resistance and the magnitude of the dilution potential, indicating a role of cytosolic Ca++ in the modulation of the paracellular permeability. However the rise of calcium activity produced by the Ca++ ionophore calcimycin (A23187) evoked an identical effect, suggesting that any change in physiological intracellular Ca++ activity alters the paracellular permeability.

Anguilla↗

PubMed2026

Pediatric intracranial inflammatory myofibroblastic tumor harboring DCTN1::ALK fusion: a case report with radiologic-pathologic-molecular correlation.

Central nervous system inflammatory myofibroblastic tumors are rare; pediatric DCTN1::ALK fusion cases are exceptionally uncommon. Here, we present an eight-year-old boy who presented with headache, vomiting, and a rapidly enlarging right frontal scalp mass. An MRI showed a dural, extra-axial lesion with mass effect. Histology confirmed IMT, and ALK immunohistochemistry was positive; next-generation sequencing (NGS) identified DCTN1 (exon 1-27)-ALK (exon 20-29) fusion, and FISH confirmed ALK rearrangement (33/100 nuclei). Genomic metrics showed tumor mutational burden (TMB) of 0.94/Mb, microsatellite stability, and CNV burden of 2.1%. He underwent near total resection followed by alectinib; to our knowledge, this is the first reported young pediatric (<10 years old) CNS IMT with this fusion.

Humans↗

PubMed2002

Distribution of kerosene components in rats following dermal exposure.

The systemic distribution of kerosene components in blood and tissues was analysed in rats following dermal exposure. Four types of trimethylbenzenes (TMBs) and aliphatic hydrocarbons (AHCs) with carbon numbers 9-16 (C(9)-C(16)) were analysed as major kerosene components by capillary gas chromatography/mass spectrometry (GC/MS). The kerosene components were detected in blood and all tissues after a small piece of cotton soaked with kerosene was applied to the abdominal skin. The amounts of TMBs detected were higher than those of AHCs. Greater increases in TMB levels were found in adipose tissue in an exposure duration-dependent manner. The amounts of TMBs detected were only at trace levels following post-mortem dermal exposure to kerosene. These findings suggest that kerosene components were absorbed percutaneously and distributed to various organs via the blood circulation. Post-mortem or ante-mortem exposure to kerosene could be distinguished when the exposure duration was relatively long. Adipose tissue would seem to be the most useful for estimating the degree of kerosene exposure.

Animals↗

PubMedDate not supplied

Multi-colour brightfield in situ hybridisation on tissue sections.

We describe the brightfield microscopical detection of multiple DNA target sequences in cell and tissue preparations. For this purpose, chromosome-specific DNA probes labelled with biotin, digoxigenin or fluorescein were simultaneously hybridised and detected by enzyme cytochemistry using two horseradish peroxidase (PO) reactions and one alkaline phosphatase (APase) reaction. For triple-colour detection on single cell preparations, the combination of the enzyme precipitates PO/diaminobenzidine (DAB, brown colour), APase/fast red (FR, red colour) and PO/tetramethylbenzidine (TMB, green colour) resulted in an accurate detection of DNA targets. Embedding of the preparations in a thin cross-linked protein layer further stabilised the enzyme reaction products. For in situ hybridisation on tissue sections, however, this detection procedure showed some limitations with respect to both the stability of the APase/FR and PO/TMB precipitates, and the sequence of immunochemical layers in multiple-target procedures. For this reason, the APase/FR reaction was replaced by the APase/new fuchsin (NF, red colour) reaction and the washing steps after the PO/TMB reaction were restricted to the use of phosphate buffer pH 6.0. Furthermore, to improve the efficiency of the ISH reaction, APase/NF was applied in an avidin-biotin complex detection system and, to avoid target shielding in the triple-target ISH, the third primary antibody was applied prior to the second enzyme cytochemical reaction. These adaptations resulted in stable, well contrasting brown, red and green coloured precipitates. After quick haematoxylin counterstaining, the tissue preparations were directly mounted in phosphate buffer and, optionally, embedded in the cross-linked protein layer.

Color↗

PubMed1997

Determination of dimethylhippuric acid isomers in urine by high-performance liquid chromatography.

In order to analyse metabolites in urine after trimethylbenzene (TMB) exposure a method based on high-performance liquid chromatography (HPLC) for determination of the six dimethylhippuric acids (2,3-DMHA, 2,6-DMHA, 2,5-DMHA, 2,4-DMHA, 3,4-DMHA and 3,5-DMHA) in urine has been developed. In contrast to earlier published methods, the present method allows detection of all possible isomers of DMHA in a single analysis. The DMHAs were extracted from urine with dichloromethane. After evaporation, the residue was dissolved in mobile phase and analysed by a stepwise gradient HPLC system with ultraviolet (UV) detection at 225 nm. Mobile phase A (1.25% acetonitrile and 0.3% acetic acid in water) was used up to a retention time of 59.5 min and mobile phase B (5% acetonitrile in water containing 0.3% acetic acid) was used for completion of the analysis at approximately 90 min. The DMHA isomers were chromatographed on a reversed phase Radial-Pak C18 column (4 microns; 100 mm x 5 mm inner diameter). The detection limit for the six isomers was 1.5 micrograms/ml (range 0.5-3.4, 100 microliters injection volume). The precision of the method was 4.2% relative standard deviation (range 3.8-4.4; 100 micrograms/ml). Standard curves of the DMHAs were linear over the interval 10-500 micrograms/ml in human urine. Individual DMHAs or the sum of DMHA isomers may be used as biological indicators of occupational exposure to TMBs.

Benzene Derivatives↗

PubMed2002

Possible role of calcium ions, calcium channels and calmodulin in excystation and metacystic development of Entamoeba invadens.

The effect of calcium ions (Ca(2+)) and calmodulin (CaM) on the excystation and metacystic development of Entamoeba invadens was examined by transfer of cysts to a growth medium containing calcium antagonists and CaM inhibitors. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by the calcium chelators ethyleneglycol bis (beta-aminoethyl ether)- N,N'-tetraacetate (EGTA) and ethylene-diaminetetraacetate (EDTA), with EDTA being more potent than EGTA. The inhibitory effect of higher concentrations of these chelators on excystation was associated with reduced viability of cysts. Metacystic development, when determined by the number of nuclei in an amoeba, was delayed by EGTA, because the percentage of four-nucleate amoebae was higher than in controls at day 3 of incubation. EDTA made metacystic development unusual by producing a large number of metacystic amoebae with more than ten nuclei. The inhibition of excystation by these chelators was partially abrogated by their removal. A putative antagonist of intracellular calcium flux, 8-( N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8) also inhibited the excystation and metacystic development, but had little effect on cyst viability. The slow Na(+)-Ca(2+) channel blocker bepridil but not verapamil inhibited the excystation and metacystic development, associating with reduced cyst viability at higher concentrations. The inhibitory effect of bepridil on excystation was abrogated by removal of the drug. The CaM inhibitor trifIuoperazine (TFP) but not W-7 [ N-(6-aminohexyl)-chloro-1-naphtalene sulphonamide] inhibited the excystation and metacystic development. The inhibitory effect of TFP on excystation was also abrogated by removal of the drug. These results indicate that extracellular calcium ions, amoebic intracellular calcium flux, calcium channels, and a CaM-dependent process contribute to the excystation and metacystic development of E. invadens.

Animals↗

PubMed2001

Effect of calcium antagonists, calcium channel blockers and calmodulin inhibitors on the growth and encystation of Entamoeba histolytica and E. invadens.

The effects of calcium antagonists, calcium channel blockers, and calmodulin inhibitors on the growth of Entamoeba histolytica and the growth and encystation of Entamoeba invadens were examined. Calcium chelators ethyleneglycol bis (beta-aminoethyl ether)-N,N'-tetraacetate (EGTA) and ethylene-diaminetetraacetate (EDTA) inhibited the growth of both Entamoeba and also the encystation of E. invadens in a dose-dependent manner, with EDTA being more effective than EGTA. A putative antagonist of intracellular calcium flux, 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8) also inhibited both growth and encystation, with the E. histolytica being more sensitive than E. invadens, and with the growth of E. invadens being more sensitive than encystation. The slow Na+-Ca2+ channel blockers bepridil and verapamil inhibited both growth and encystation. Bepridil was more effective than verapamil. The calmodulin (CaM) inhibitors, W-7 (N-(6-aminohexyl)-chloro-1-naphtalene sulphonamide) and trifluoperazine (TFP), were also inhibitory for both the growth and encystation; TFP was more effective than W-7, and encystation was more sensitive than growth in E. invadens. These results indicate that extracellular calcium ions, amebic intracellular calcium flux, calcium channels, and a CaM-dependent process contribute to the growth and encystation of Entamoeba.

Animals↗

PubMed1999

Induction of epithelio-mesenchymal transformation of quail embryonic neural cells by inhibition of atypical protein kinase-C.

Epithelio-mesenchymal transition, which involves the re-organisation of cell-cell adhesion molecules and the actin cytoskeleton, can be induced in embryonic neural epithelium in vitro by protein kinase-C inhibitors. A non-inhibitory analogue, BIM V, and potent inhibitors of other kinases are not active. This suggests a central role for C-kinases, although the powerful specific C-kinase inhibitors BIM I and Ro 31-8220 show lower than expected activity. Co-inhibition by several kinases is unlikely to account for this, since no potentiation occurs when these are combined with potent inhibitors of other kinases. BIM I and Ro 31-8220 strongly inhibit only conventional calcium-regulated C-kinases; this and the lack of effect of TMB-8, which inhibits calcium release, suggests that novel and/or atypical isoforms are involved. Various potentiators and activators of conventional and novel C-kinases have no obvious effect alone and fail to reduce the effect of staurosporine, suggesting that atypical C-kinases are critical. The presence of C-kinase isoforms in the E2 embryonic neural tissues has been probed on Western blots, revealing immunoreactivity for the atypical isoforms iota (or lambda) and zeta and the alpha, gamma, epsilon and mu isoforms. Immunofluorecent localisation on sections of embryos has shown the widespread distribution of conventional and novel isoforms but only the atypical isoforms lambda and zeta are enriched at the apical margins of the neural and other epithelia; they overlap with the cell-cell adhesion molecule N-cadherin and with F-actin. Thus, epithelio-mesenchymal transition in the embryonic neural epithelium in vitro is induced by inhibiting protein kinase activity, probably via an atypical protein kinase-C; atypical protein kinase-C isoforms are present in the tissue at the appropriate developmental stage and subcellular site in cells capable of epithelio-mesenchymal transition.

Animals↗