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PubMedDate not supplied

[Evaluation of a spectrophotometric technique for plasmatic haemoglobin determination on Konelab 30].

INTRODUCTION: the measurement of plasma haemoglobin is utilized in the evaluation of various haemolytic phenomenons, especially at the time of sampling. Besides, the methods used to titrate plasmatic haemoglobin, an analysis which is of great interest to cardiac surgery, must be very accurate, because plasmatic levels are much lower than those found in whole blood. BACKGROUND: the purpose of this work is to present a new technique for direct determination of plasmatic haemoglobin. MATERIAL AND METHODS: this method was studied and adapted on a multiparametric analyzer Konelab 30 (Thermo Electron Corporation). The correlation was assessed with a Sigma Diagnostics spectrophotometric method, using the 3,3',5,5'-tetramethylbenzidine (TMB), a non carcinogenic reagent. The reaction of haemoglobin with TMB was studied with a SECOMAM S500P UV-Visible spectrometer. RESULTS AND DISCUSSION: the method demonstrates linearity, precision, and sensitivity suitable for clinical use and not affected by the presence of bilirubin, nor triglycerides. The method was precise (CV < 1,00%), accurate (CE < 5,00%) and showed excellent recovery of haemolysis (R > 0,95). The CV for the automated assay was less than 1%. Reference ranges were studied and lithium heparinate seems to be the best anticoagulant to use so as to minimize haemolysis. CONCLUSION: the studied technique is practical, specific, accurate, fast, reliable and inexpensive, and was adopted in emergency as in daily routine in our laboratory for plasmatic haemoglobin determination in various situations.

Hemoglobins↗
PubMed1990

[Catalytic properties and stability of catalase in reversed micelles of aerosol OT in octane].

The catalytic function of catalase and its peroxidatic activity during tetramethylbenzidine (TMB) oxidation by cumene hydroperoxide were studied in reversed micelles of Aerosol OT (AOT) in octane relative to the [H2O]/[AOT] ratio and the initial catalase concentration. The optimum conditions permitting to retain the catalytic activity of the enzyme and its ability to induce peroxidation of TMB, were found. The catalytic function of the enzyme was shown to be dependent on its concentration in AOT micelles. The catalase stability monitored by the catalytic reaction and the decrease of the Soret band were analyzed. Both processes have two phases differing by the rate constants of the pseudo-first order. The catalase inserted into AOT micelles is characterized by the high stability as compared to other hemoproteins (cytochrome P-450, myoglobin, hemoglobin, peroxidase) under identical conditions.

Animals↗
PubMedDate not supplied

Effect of calcium-modifying drugs on mouse in vitro fertilization and preimplantation development.

Calcium ions are required for normal in vitro fertilization and preimplantation development in the mouse. This study examined the effects of alterations in Ca2+ flux and distribution on sperm penetration of eggs and embryo cleavage. Compounds used included diltiazem, a Ca2+ channel blocker, and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an antagonist of intracellular calcium release. Incubation of sperm and eggs with diltiazem at 30 and 10 microM and TMB-8 at 30, 10, and 3 microM resulted in depressed fertilization compared with controls. Motility was not inhibited at these concentrations of either drug. Both drugs also depressed progression of mouse embryos from 2-cell to blastocyst at 30 and 3-microM concentrations. This study suggests that both Ca2+ flux and distribution to specific cellular sites are required for normal mouse in vitro fertilization and early preimplantation development.

Animals↗
PubMedDate not supplied

[Evaluation of the degree of muscle blockade: a comparative study using electromyography of the muscular relaxation induced by pancuronium, atracurium and vecuronium].

To compare the muscle relaxing effect of pancuronium, atracurium and vecuronium, 99 patients operated on under neuroleptanesthesia were divided in three groups depending on whether they had received, during induction, pancuronium 0.1 mg/kg, atracurium 0.5 mg/kg, or vecuronium 0.1 mg/kg. One-fourth of the initial dose was repeated if necessary. The electromyographic study of the muscle relaxing effect was carried out with stimulation of the cubital nerve with courses of supramaximal square wave electric stimuli in 'trains of four'. The time to maximal blockade (TMB), the time of clinical effectiveness (TCE), the total duration time (TDT), the time of duration of the maintenance dose (DM 25) and the recovery index (RI) were measured. TMB was 4.3 +/- 1 min for pancuronium, 3.5 +/- 0.8 min for atracurium, and 3.3 +/- 0.98 min for vecuronium. The differences between pancuronium and the other drugs were statistically significant, but they were not so between the latter two. TCE was 67.9 +/- 13.5 min for pancuronium, significantly longer than with vecuronium and atracurium (28.2 +/- 5.7 and 31.5 +/- 4.7, respectively). TDT was 126.2 +/- 19.9 min for pancuronium, 61.2 +/- 11.5 min for atracurium and 55.5 +/- 16.7 for vecuronium. The mean duration of the repeated dose was 52.7 +/- 8.4 min for pancuronium, 19.9 +/- 5 min for vecuronium and 10.9 +/- 5 min for atracurium. RI, which was similar for atracurium and vecuronium (12.7 +/- 1.7 min and 12.8 +/- 3.3 min), was longer for pancuronium (27.7 +/- 4.3 min).

Adolescent↗
PubMed1990

[Experimental studies on the mechanism of the action of staphylococcal epidermolytic toxin A utilizing recombinant toxin].

The pathogenic role of proteinases and Ca++ in the action of staphylococcal epidermolytic toxin A (ETA) was investigated using recombinant ETA (rETA). rETA was released from the periplasmic space of E. coli transformed with the plasmid carrying ETA gene and purified by high performance liquid chromatography. The epidermolytic activity of the purified rETA was 5,000 epidermolytic unit per mg of protein. Pieces of newborn mouse skin were cultured in minimum essential medium containing rETA. Various concentrations of alpha 2-macroglobulin, N-ethylmaleimide, leupeptin, L-transepoxysuccynyl-leucylamide (4-guanidino) butane, phenylmethylsulfonyl fluroide, pepstatin A, ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis (2-aminoethylether) tetraacetic acid (EGTA) and 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8) were added to the medium. Splitting in the upper epidermis occurred after 4 hr of incubation in the presence of 10 micrograms/ml rETA and was not inhibited by the proteinase inhibitors except EDTA and EGTA. EDTA, EGTA and TMB-8 inhibited the splitting completely at concentrations of 0.1-1 mM. The inhibitions caused by these agents were restored by the addition of Ca++ to the medium. These results strongly suggest that the action of ETA is mediated by the increase in cytoplasmic Ca++ concentration resulting from Ca++ influx and/or intracellular Ca++ mobilization.

Amino Acid Sequence↗
PubMed1990

Role of intracellular calcium concentration and protein kinase C activation in IFN-gamma stimulation of U937 cells.

IFN-gamma enhances many monocyte functions, including oxidative metabolism and Ag presentation. IFN-gamma has been reported to increase the intracellular concentration of calcium ([Ca2+]i) and modulate protein kinase C activity in murine macrophages, but the signal transduction pathways induced by IFN-gamma in human cells and their functional significance are poorly understood. Our study examined the hypothesis that an increases in [Ca2+]i and protein kinase C activation are required for functional responses to IFN-gamma. The U937 cell line was used as a model of an IFN-gamma responsive cell. IFN-gamma caused a rapid and concentration-dependent increase in [Ca2+]i, which was partly inhibited by calcium-free medium, diltiazem, and TMB-8. IFN-gamma induced a fourfold increase in the concentration of inositol 1,4,5-trisphosphate. Induction of HLA-DR, Fc gamma R, CR3, and Mo3e Ag expression by IFN-gamma was blocked by concentrations of TMB-8 that inhibited an increase in [Ca2+]i, but not by protein kinase C inhibition by H-7 or inhibition of calmodulin with W-7. Ionomycin did not enhance Ag expression and PMA induced the expression of only the Mo3e Ag. We conclude that IFN-gamma induces antigenic expression on human U937 cells by a mechanism dependent on, but not limited to, an increase in intracellular calcium, which is likely due to inositol 1,4,5-trisphosphate generation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Two distinct pathways of platelet-activating factor-induced hydrolysis of phosphoinositides in primary cultures of rat Kupffer cells.

Stimulation of rat Kupffer cells in primary culture with platelet-activating factor (PAF) caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate with a concomitant increase in the levels of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,4-bisphosphate. This phospholipase C-mediated hydrolysis of polyphosphoinositides was independent of extracellular Ca2+ but was inhibited by the intracellular Ca2+ antagonist TMB-8. A second slower response to PAF was characterized by deacylation of PI leading to the accumulation of glycerophosphoinositol (GPI). PAF-induced GPI synthesis was not inhibited by TMB-8. These effects of PAF were accompanied by initial transient mobilization of Ca2+ from intracellular stores followed by a rather slow influx of Ca2+ from the extracellular medium. PAF-stimulated deacylation and phosphodiesteric hydrolysis of inositol lipids were differentially affected by cholera toxin and pertussis toxin. Pretreatment of the Kupffer cells with either of these toxins caused inhibition of phospholipase C activity. Pertussis toxin also inhibited PAF-stimulated deacylation. However, cholera toxin itself stimulated GPI release and addition of PAF to the cholera toxin-treated cells caused a further increase in GPI release. Phorbol ester inhibited PAF-induced phosphodiesteric hydrolysis of phosphoinositides, but not deacylation. PAF-induced metabolism of phosphoinositides was inhibited by the PAF antagonist, U66985. These results suggest that PAF-induced phosphodiesteric hydrolysis and deacylation of inositol phospholipids are regulated via distinct mechanisms involving activation of separate G-proteins in rat Kupffer cells. Also the regulation of phosphoinositide metabolism by Ca2+ mobilization from two separate Ca2+ pools is indicated by this study.

Animals↗
PubMed1985

Diffusion of horseradish peroxidase at the injection site.

The sensitivity of diaminobenzidine (DAB), tetramethylbenzidine (TMB) and Hanker-Yates (H-Y) methods was compared in order to determine which procedure shows best the effective site of horseradish peroxidase (HRP) uptake. The lateral geniculate nucleus was chosen as the target nucleus for HRP injections, and the neuronal labelling in the superior colliculus and in zona incerta was used as a control. While TMB showed images that overestimated the effective site of uptake, the H-Y procedure showed more restricted HRP spots, similar to those obtained with DAB, that corresponded well with the neuronal labelling observed in the control areas. These results underline the usefulness of the H-Y procedure upon evaluation of the effective site of uptake. In addition, they do not support an underestimation of this effective site by this method.

Animals↗
PubMed1989

Characterization of platelet cytoplasmic Ca2+ mobilization in patients with congenital cyclo-oxygenase deficiency and with defective platelet aggregation to A23187.

Agonist-induced platelet cytoplasmic Ca2+ concentrations ([Ca2+]i) in patients with congenital cyclo-oxygenase deficiency (A) and with impaired aggregation to A23187 (B) were measured with aequorin in the presence or absence of extracellular Ca2+. The influence of TMB-8 or ONO3708 on agonist-induced [Ca2+]i in those platelets was also investigated. In Patient 1, there was a single aequorin luminescence peak in response to arachidonate, which was a thromboxane A2(TXA2) independent Ca2+ influx. The luminescence peak due to the formation of TXA2 was not detectable. The A23187-induced [Ca2+] i was decreased in the presence of extracellular Ca2+, but was within normal limits in the absence of extracellular Ca2+. A thrombin or STA2-induced elevation of [Ca2+] i was always within normal limits under any conditions. These results suggest that cyclo-oxygenase activity (CO activity) contributes to the A23187-induced Ca2+ influx, but does not contribute to the Ca2+ release from intracellular stores, and that the thrombin or STA2-induced Ca2+ influx and release do not depend on the CO activity. In Patient 2, the time lag from the addition of A23187 to the aequorin luminescence peak was found both in the presence and absence of extracellular Ca2+, which was more obvious in the latter. This A23187-induced elevation of [Ca2+] i disappeared after treatment of the platelets with TMB-8 in the absence of extracellular Ca2+, which is rarely seen in normal platelets. The most striking finding was that the thrombin-induced rise in [Ca2+] i in the absence of extracellular Ca2+ was not detectable. These findings might be closely related to abnormal platelet function in this patient.

Blood Platelet Disorders↗
PubMed1989

[Ammonium molybdate as stabilizing agent in tetramethyl benzidine reaction of CB-HRP neurohistochemistry and its application to electron microscopy and immunocytochemistry].

Ammonium molybdate (AHM) was used as a stabilizing agent in the tetramethyl benzidine (TMB) reaction of choleragen subunit B conjugated horseradish peroxidase (CB-HRP) neurohistochemistry. In comparison with Mesulam's TMB method using sodium nitroprusside (SNP) as stabilizing agent, this new stabilizer offers similar sensitivity in regard to visualization of CB-HRP labelled neurons and their extranuclear Golgi-phobic dendrites. The nearly physiological pH (6-8) of the reaction medium demonstrates better preservation of tissue and cell structures of reacted sections and avoids non-specific needle-like crystal formation. Under the electron microscope, the histochemical reaction products can be clearly distinguished, without the usual shrinkage or distention of cellular and subcellular structures. This method was also applied with success to immunocytochemistry through small modifications.

Animals↗
PubMed1989

Occult intracellular calcium pools: relevance to neutrophil oxidant production.

Human neutrophils use divalent cations for the stimulus-response coupling leading to the respiratory burst. We have studied the relevance of the extracellular versus intracellular calcium stores necessary for superoxide generation in neutrophils and neutrophil cytoplasts stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol ester and our findings are as follows. (1) FMLP triggered superoxide anion (O2-) generation only in the presence of extracellular calcium, but phorbol myristate acetate (PMA) activation was independent of extracellular calcium. (2) inhibition of calcium transport channels with verapamil produced a dose-related inhibition of FMLP- and PMA-stimulated O2- production. Verapamil inhibition of FMLP-stimulated but not PMA-stimulated O2- was reversed simply by increasing extracellular calcium concentration. Cytosolic calcium loading with substimulatory concentrations of A23187 (0.3 to 0.6 mumol/L) (only in calcium-replete medium) could reverse the verapamil inhibition of PMA-triggered O2- generation. An inhibitor of intracellular calcium availability, 3,4,5-trimethoxybenzoic acid (TMB-8), similarly inhibited FMLP-stimulated and PMA-stimulated O2- production; this inhibition was reversible with A23187 calcium loading. (3) Verapamil, although it inhibited the functional oxidant response, had no inhibitory effect on fura 2-monitored cytosolic calcium rise induced by FMLP stimulation. (4) No cytosolic calcium increment was observed with PMA. O2- generation from enucleated polymorphonuclear leukocyte (PMN) cytoplasts was sensitive to inhibition by verapamil but was less so than O2- generation from intact PMNs. Cytoplasts, nearly depleted of cytoplasmic organelles and cytoskeleton, the potential sites of intracellular calcium stores, were markedly insensitive to the inhibitory effects of TMB-8.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcimycin↗
PubMed1988

Atrial natriuretic peptide inhibits angiotensin II-induced contraction of isolated glomeruli and cultured glomerular mesangial cells of rats: the role of calcium.

We examined the contractile effect of angiotensin II on isolated rat glomeruli and cultured glomerular mesangial cells, as well as the possible involvement of calcium in this contraction. In addition, the effect of rat atrial natriuretic peptide (ANP) on angiotensin II-induced contraction was studied. Computer-assisted image analysis was used to measure glomerular cross-sectional area and planar mesangial cell surface area in several experimental conditions. Angiotensin II induced a decrease in glomerular cross-sectional area and planar mesangial cell surface area that was both time and dose dependent. This effect was partially blocked by preincubation with verapamil or TMB-8, the latter compound being more effective. Incubation with ANP prevented angiotensin II-induced reduction of glomerular cross-sectional area and planar mesangial cell surface area and reversed contraction in mesangial cells. Incubation with TMB-8 impaired the effect of ANP. These data suggest that angiotensin II could exert part of its physiologic effects in the kidney through calcium-dependent mesangial and glomerular contraction. ANP inhibited this contraction, possibly explaining some of its renal effects.

Angiotensin II↗
PubMed1985

[Peroxidase activity of catalase with respect to aromatic amines].

The catalase dissociation into subunits has been studied at pH less than 3.5 and greater than 11.0. This process is characterized by pseudo-first order rate constants, depending on the initial concentrations of the enzyme and H+. At pH 2.85, the steady-state kinetics of five aromatic amines oxidation by catalase monomers has been studied for orthodianisidine (o-DA), 3,5,3',5'-tetramethylbenzidine (TMB), ortho- and para-phenylene diamine (p-PDA) and 5-aminosalycilic acid. The optimal substrates for catalase in acidic solutions are o-DA, TMB and p-PDA. A comparison has been carried out for the catalase peroxidative activity, and the catalytic characteristics of horseradish peroxidase in the oxidation of the same substrate. The mechanisms of peroxidatic amines oxidation by catalase and horseradish peroxidase are discussed.

Amines↗
PubMed1988

Release of prostacyclin and EDRF from endothelial cells is differentially controlled by extra- and intracellular calcium.

The aim of this study was to define the roles of extra- and intracellular Ca++ in the release of PGI2 and EDRF from cultured bovine endothelial cells stimulated with receptor-mediated and receptor-independent substances. The receptor-mediated stimulant bradykinin (10 nM) elicited transient releases of PGI2 (assayed with radioimmunoassay of 6-keto PGF1 alpha) and EDRF (assayed by its stimulatory effect on purified soluble guanylate cyclase). Bradykinin also elicited dose-dependent increases in intracellular free calcium [( Cai++], measured with the fluorescent probe indo-1). In the absence of extracellular Ca++ (nominally Ca+(+)-free, EGTA 0.1 mM) or in the presence of the intracellular calcium antagonist TMB-8 (0.1 mM), PGI2 release was significantly attenuated. Bradykinin-induced EDRF release was not significantly affected by TMB-8 but was completely abolished in Ca+(+)-free medium. When endothelial cells were stimulated with thimerosal (an inhibitor of the enzyme acyl-CoA-lysolecithin-acyl-transferase; 5 microM), a long-lasting release of EDRF and PGI2 was induced, associated with only a slight increase in [Cai++]. Removal of extracellular Ca++ had little effect on [Cai++], completely abolished EDRF release, and did not change PGI2 release. It is concluded that there is a close association between PGI2 release and [Cai++] in bradykinin-stimulated endothelial cells. In contrast to PGI2 synthesis, EDRF production is directly dependent on extracellular Ca++ and independent of [Cai++].

Animals↗
PubMed1985

Calcium requirements for increased complement receptor expression during neutrophil activation.

It has recently been shown that human neutrophils rapidly increase surface expression of membrane receptors for C3b (CR1) and C3bi (CR3) in response to chemoattractants and other stimuli. In the present studies, we used monoclonal antibodies and flow cytometry to assess the role of Ca2+ in this process. Stimulation with ionophore A23187 in the presence of 1.2 mM Ca2+ increased CR1 330% and CR3 650% compared with unstimulated cells at 37 degrees C. Because this indicated that increasing the cytosolic free Ca2+ caused increased receptor expression, we examined the role of Ca2+ in the response to other stimuli as well. Adding 1.2 mM Ca2+ or 5 mM EDTA to the media in which the polymorphonuclear leukocytes were suspended had no effect on the CR1 response to f-MLP or LTB4, whereas Ca2+ slightly enhanced and EDTA markedly inhibited the CR3 response to these stimuli. The effects of Mg2+-EGTA and EDTA were identical. TMB-8 (200 microM), which inhibits the release of Ca2+ from intracellular stores, completely blocked the increased expression of both receptors induced by fMLP or LTB4. Increased expression of both receptors was also prevented by the calmodulin antagonists chlorpromazine (50 microM) and trifluoperazine (10 microM), but not by chlorpromazine sulfoxide. Phorbol myristate acetate (0.1 ng/ml) increased CR1 230% and CR3 265%. Again Ca2+ and EDTA did not alter the CR1 response, whereas Ca2+ increased CR3 to 287% and EDTA reduced CR3 to 187% of control. TMB-8 (250 microM) completely blocked both CR1 and CR3 responses to this stimulus as well. Thus, release of intracellular Ca2+ is necessary and sufficient for increased CR1 expression in response to diverse stimuli, but maximal increases in CR3 expression require an additional influx of extracellular Ca2+. These results indicate that the mechanisms by which the surface expression of the two different complement receptors increase are different in their requirements for extracellular Ca2+. In comparison with the work of others, we suggest that the processes of increased complement receptor expression and secretion of granular enzymes may also differ.

Calcimycin↗
PubMed1988

Androgen regulates synaptic input to motoneurons of the adult rat spinal cord.

Adult male rats (Sprague-Dawley) were castrated and implanted subcutaneously with Silastic capsules containing testosterone or nothing. Sham-castrated males served as controls. Four weeks following castration, cholera toxin-horseradish peroxidase (CT-HRP) was injected bilaterally into the bulbocavernosus muscles and animals were sacrificed 2 d later. The spinal cords containing the spinal nucleus of the bulbocavernosus (SNB) were dissected, processed with a modified tetramethylbenzidine (TMB) method for visualization of retrogradely transported CT-HRP, and examined at the ultrastructural level. Neuronal structures apposing the membranes of 150 TMB-labeled SNB neurons were analyzed by measuring the percentage of somatic and proximal dendritic membranes covered by synaptic contacts, synaptoid contacts, and neuron-neuron contacts. Most of the neuronal structures in the control and experimental SNB motoneurons consisted of synaptic contacts. The mean percentage of somatic and proximal dendritic membranes covered by synapses 4 weeks after castration was reduced to approximately 30% of those in control animals. However, treatment with testosterone for 4 weeks after castration prevented this decline. Castration and testosterone treatment also influenced the size and number of synaptic contacts per unit length of somatic and proximal dendritic membranes, and the incidence of neuron-neuron contacts and double synapses onto SNB motoneurons. These results indicate that androgen is critical for maintaining the organization of synaptic inputs to these spinal motoneurons in adult male rats.

Androgens↗
PubMed1986

Effects of calcium antagonists and calcium-buffered salines on wound healing in Xenopus early embryos.

The role of calcium in the process of wound closure in Xenopus early embryos was studied. Embryos were wounded in the presence of the calcium antagonists D-600 and TMB-8 or in calcium-buffered salines, and the effects on wound healing were observed by scanning electron microscopy. D-600 and TMB-8 inhibit wound closure and these antagonists appear to act synergistically since their combined effect is greater than their individual effects. Experiments with calcium-buffered salines suggest that wound closure can proceed in the presence of low extracellular calcium. In all conditions there is a correlation between the degree of wound closure and the shapes of the cells at the wound margin; closing wounds are accompanied by cells elongated radial to the wound, gaping (non-closing) wounds are accompanied by cells stretched tangential to the wound. Thus the results suggest that calcium influx may not be a requirement for the changes in cell shape which accompany, and probably effect, wound closure in Xenopus early embryos.

Animals↗
PubMed1985

Involvement of intracellular Ca2+ in chemotaxis and metabolic burst by neutrophils: the use of antagonists of intracellular Ca2+.

The effect of TMB-8, quin2, chlortetracycline, chlorpromazine and prenylamine on two neutrophil functions - chemotaxis towards fMLP and metabolic burst activated by PMA or fMLP - was compared. All drugs inhibited both functions in the absence of extracellular Ca2+. The inhibitory effect of all drugs on chemotaxis, and the effect of TMB-8, quin2 and chlortetracycline on the metabolic burst was antagonized by extracellular Ca2+. There were differences between inhibition of chemotaxis and the metabolic burst, and between inhibition of PMA activated and fMLP-activated metabolic burst. The results are consistent with the view that intracellular Ca2+ is required in chemotaxis, and in PMA or fMLP activated metabolic burst, but that the role of Ca2+, or the location where Ca2+ is derived from, is different and depends on function and the type of activator.

Animals↗