Duration Of Preincubation Research Articles

The effects of vitamin d on insulin release from isolated islets of rats.

Background:Vitamin D (vit D) affects glucose metabolism. Receptors of vitamin D have been identified in β cells and studies show that vitamin D deficiency reduces glucose-stimulated insulin secretion (GSIS).Objectives:The aim of this study was to examine the effect of vitamin D on insulin release from isolated islets of rats.Materials and Methods:Islets were isolated from male Wistar rats, weighing 200-250 grams, using the collagenase digestion method. Insulin release was assessed following 24 and 48 hours coincubation of islets with vitamin D (0.1, 1 and 10 nM) and glucose (5.6, 11.1 and 16.7 mM). In addition, islets were preincubated with vitamin D for 24 and 48 hours and GSIS was assessed for one hour in the presence of 5.6 and 16.7 mM glucose.Results:Coincubation of islets with vitamin D (10 nM) and 11.1 mM glucose increased islet insulin release (37.27 ± 3.75 vs. 24.64 ± 2.83 ng/islet/24 hours; P < 0.05), while vitamin D (1 and 10 nM) decreased insulin release in the presence of 16.7 mM glucose (21.14 ± 3.58 and 18.65 ± 3.84 vs. 37.71 ± 4.63 ng/ islet/24 hours; P < 0.05). Islets preincubation with vitamin D (1 and 10 nM) increased GSIS in the presence of 16.7 mM glucose (4.39 ± 0.73 and 4.39 ± 0.63 vs. 2.07 ± 0.43 ng/islet/1 hour; P < 0.05).Conclusions:Preincubation of islets with vitamin D increased GSIS but decreased insulin release in coincubation with high levels of glucose. Insulin secretion from β cells in the presence of glucose seems to be related to the dosage of vitamin D and duration of preincubation.

Effects of gold-chloroquine complexes on respiratory burst of polymorphonuclear leukocytes.

The effects of gold-chloroquine derivatives with the formula [Au(PR3)(CQ)]PF6 (where R = Ph (1), Et or Me) on the superoxide anion production by human neutrophils (PMNs, polymorphonuclear cells) were investigated. When these complexes (0.1-3 mumol/l) were added to PMNs prior to the activators formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), they inhibited isoluminol-horseradish peroxidase-dependent chemiluminescence (CLisol). The inhibition was a direct result of effects on PMNs since chemiluminescence in the cell-free system (horseradish peroxidase-hydrogen peroxidase-isoluminol) was not affected. The above mentioned concentrations of the complexes did not show in vitro toxicity on the cells. On the other hand, when 1 mumol/l of complex 1 was added to cells after stimulation, the chemiluminescence of PMNs stimulated by PMA was inhibited, but not the chemiluminescence stimulated by fMLP. The gold-chloroquine binding was essential for the referred activity as chemiluminescence was not influenced by the precursors chloroquine (CAS 54-05-7) and AuCl(PPh3). Furthermore, the extent of inhibition of chemiluminescence in PMNs activated by PMA did not increase with the duration of preincubation in presence of 1 mumol/l of 1. Extensive washing of cells after preincubation with 1 mumol/l of 1 reversed the aforementioned inhibition. All these results show that the gold-chloroquine binding can lead to compounds with specific properties that could make them useful in the treatment of some inflammatory diseases.

Respiratory burst function of ovine neutrophils

BackgroundRespiratory burst function resulting in the release of reactive oxygen species such as superoxide anion (O2-) from neutrophils is one of the key mechanisms of the innate immune system, and maladaptive control of this mechanism is thought to play a pivotal role in the development of pathologies such as acute lung injury and sepsis. Ovine models of these pathologies are limited by the poor understanding of ovine neutrophil respiratory burst function.ResultsAspects of ovine neutrophil respiratory burst function to be characterised were: i) the maximum rate of O2- generated (Vmax); ii) the time taken to reach Vmax; iii) the total amount of O2- generated during the reaction; and iv) the duration of the reaction. As well as for unstimulated neutrophils, these aspects were also characterised after incubation with a priming agonist (platelet activating factor [PAF], tumour necrosis factor alpha [TNF-α] and lipopolysaccharides [LPS]) activating agonists (N-formylmethionyl-leucyl-phenylalanine [fMLP] and phorbol 12-myristate 13-acetate [PMA]) or a combination of a priming and an activating agonist. In the absence of priming or activating agonists, ovine neutrophils displayed a low level of respiratory burst function which was not enhanced by either PAF, TNF-α, LPS or fMLP, but was significantly enhanced by PMA. The PMA-induced respiratory burst function was further enhanced by pre-incubation with PAF, but not with TNF-α or LPS. By varying the length of pre-incubation with PAF it was demonstrated that this effect decreased as the duration of pre-incubation with PAF increased, and that PAF was enhancing PMA's effects rather than PMA enhancing PAF's effects.ConclusionThis study successfully adapted a commonly used method of measuring human neutrophil respiratory burst function to characterise different aspects of ovine neutrophil respiratory burst function. This improved understanding of ovine neutrophils will facilitate the validitation of ovine biomedical models of human pathologies in which neutrophils have been implicated.

Neuroprotective Effects of the Novel Glutamate Transporter Inhibitor (–)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]-isoxazole-4-carboxylic Acid, Which Preferentially Inhibits Reverse Transport (Glutamate Release) Compared with Glutamate Reuptake

(+/-)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo [3,4 -d]-isoxazole-4-carboxylic acid (HIP-A) and (+/-)-3-hydroxy-4,5,6, 6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B) are selective inhibitors of excitatory amino acid transporters (EAATs), as potent as DL-threo-beta-benzyloxyaspartic acid (TBOA). We report here that the active isomers are (-)-HIP-A and (+)-HIP-B, being approximately 150- and 10-fold more potent than the corresponding enantiomers as inhibitors of [3H]aspartate uptake in rat brain synaptosomes and hEAAT1-3-expressing cells. Comparable IC(50) values were found on the three hEAAT subtypes. (-)-HIP-A maintained the remarkable property, previously reported with the racemates, of inhibiting synaptosomal glutamate-induced [3H]D-aspartate release (reverse transport) at concentrations significantly lower than those inhibiting [3H]L-glutamate uptake. New data suggest that the noncompetitive-like interaction described previously is probably the consequence of an insurmountable, long-lasting impairment of EAAT's function. Some minutes of preincubation are required to induce this impairment, the duration of preincubation having more effect on inhibition of glutamate-induced release than of glutamate uptake. In organotypic rat hippocampal slices and mixed mouse brain cortical cultures, TBOA, but not (-)-HIP-A, had toxic effects. Under ischemic conditions, a neuroprotective effect was found with 10 to 30 microM (-)-HIP-A, but not with 10 to 30 microM TBOA or 100 microM (-)-HIP-A. The effect of (-)-HIP-A suggests that, under ischemia, EAATs mediate both release (reverse transport) and uptake of glutamate. The neuroprotection with the lower (-)-HIP-A concentrations may indicate a selective inhibition of the reverse transport confirming the data obtained in synaptosomes. The selective interference with glutamate-induced glutamate release might offer a new strategy for neuroprotective action.

Stepwise binding of nickel to horseradish peroxidase and inhibition of the enzymatic activity

The incubation of horseradish peroxidase C (HRPC) with millimolar concentrations of nickel, at room temperature and at pH 4.0, induced the progressive formation of a metal-enzyme complex characterized by alterations of the enzyme Soret absorption band that were time- as well as nickel concentration- dependent. For any given incubation period between 1 and 60 min, 2 values for the apparent dissociation constant (K(d)) were found, suggesting the presence of binding sites with different affinities for nickel. The value of each K(d) dropped as the incubation time increased, indicating a progressive stabilization of the metal-enzyme complex. Hill plots suggested a cooperative binding of up to four Ni2+ ions per molecule of HRPC. The inhibition of the enzymatic activity by nickel was studied by following the H2O2-mediated oxidation of o-dianisidine by HRPC under steady-state kinetic conditions. Ni2+ was found to be either a noncompetitive or a mixed inhibitor of HRPC depending both on the duration of preincubation with the enzyme and on Ni2+ concentration. The enzyme remained active only over a limited metal concentration range and data indicated that binding of one Ni2+ affected the substrate binding site, binding of a second Ni2+ affected both substrate and peroxide binding sites, and binding of more than 2 Ni2+ per HRPC molecule led to complete loss of enzymatic activity. Results pointed to the damaging effects of prolonged exposure to heavy metals and also to the existence of a critical metal concentration beyond which immediate abolishing of enzymatic activity was observed.

Effect of preincubation of cryopreserved porcine epididymal sperm

Preincubation of spermatozoa is important for capacitation and successful fertilization in vitro. The effects of preincubation time on frozen-thawed boar epididymal spermatozoa as measured by sperm motility, acrosomal integrity and fertilization ability in vitro were examined. Epididymal spermatozoa were collected from three Large White boars and frozen. The thawed spermatozoa were preincubated for 0, 15, 30, 60 and 120 min. Their motility was evaluated by a sperm motility analyzer and then the sperm motility indexes (SMIs) were calculated. The status of their acrosomal integrity was evaluated by triple-staining. Then, their fertilization ability was examined by in vitro fertilization (IVF) using porcine oocytes matured in vitro. SMIs of spermatozoa and the incidences of acrosome-intact live spermatozoa from the three boars were high (21–39 for SMI and 50–61% for acrosome-intact live spermatozoa) just after thawing, but both decreased as the duration of preincubation was prolonged (2–10 and 23–40%, respectively). The incidences of sperm penetration were high (61–89% of inseminated oocytes) when the sperm were preincubated for 0–60 min. However, sperm penetration decreased as the preincubation period was prolonged to 120 min. The degree of this decrease differed depending upon the boar from which the spermatozoa were obtained (10–72%). When the two parameters, sperm motility and acrosomal integrity, were analyzed statistically, the latter parameter rather than the former one showed a significant effect on penetration ability in vitro after each duration of preincubation. These results suggest that preincubation of frozen-thawed boar epididymal spermatozoa is not required for IVF and also that the maintenance of acrosomal integrity in unreacted status, rather than the maintenance of sperm motility, is important for fertilization ability after thawing and during preincubation of boar epididymal spermatozoa.

A model describing the effect of temperature history on lag time for Listeria monocytogenes

A model was built to describe the influence of the temperature and the duration of pre-incubation on the lag time for regrowth of Listeria monocytogenes at low temperature. This model is consistent with the usual procedure used to calculate lag times of cultures growing under fluctuating temperatures. It also describes the effect of prolonged starvation conditions on the regrowth lag time and takes into account the influence of the physiological state of inocula in predictive models.

Determination of porphobilinogen deaminase activity in human erythrocytes: pertinent factors in obtaining optimal conditions for measurements

Determination of porphobilinogen deaminase (PBGD; EC 4.3.1.8) activity in erythrocytes can contribute to the identification of patients suspected of acute intermittent porphyria. PBGD catalyses the polymerization of four molecules of porphobilinogen (PBG) to the highly unstable 1-hydroxymethylbilane. The 1-hydroxymethylbilane is transformed into uroporphyrinogen III by uroporphyrinogen III synthase. When this enzyme is inactivated, 1-hydroxymethylbilane cyclizes non-enzymatically to uroporphyrinogen I, which can be oxidized to uroporphyrin I. PBGD activity can be measured by quantitation of uroporphyrin I formed from PBG under conditions where this is the only end product. The purpose of the present study was to define the optimal conditions for quantitating PBGD activity in human erythrocytes. The preanalytical factors examined were: anticoagulants and methods for disruption of the erythrocytes. The analytical factors examined were: duration of preincubation, reaction time, reaction temperature, pH, ionic strength and conditions for the oxidation of uroporphyrinogen I to uroporphyrin I. Based on the results, we propose an optimized method for determination of PBGD activity in erythrocytes.

Dextran-70 inhibits equine platelet aggregation induced by PAF but not by other agonists.

Dextrans of mean molecular weight 70 kDa (dextran-70) have had clinical use as anti-thrombotics in man. A major part of the anti-thrombotic action is mediated via inhibition of platelet function. Greatorex (1975, 1977) treated thromboembolic colic in horses with infusions of dextran-70 and reported a 90% recovery rate, but this treatment is nonetheless rarely used. We have used an in vitro method to examine the effect of dextran-70 on equine platelet suspensions, in the hope that understanding the mechanism of action of dextran-70 might lead to the development of alternative therapeutic agents. The effects of dextran-70 on equine platelets occurred immediately in vitro with an initial activation and shape change. Subsequent assessment of aggregation revealed a dose-dependent specific inhibition of platelet-activating factor (PAF)-induced aggregation, significant in rate of aggregation at dextran-70 concentrations >40 g/l (P<0.05) and in extent of aggregation at dextran-70 concentrations >50 g/l (P<0.05). Pre-incubation with 60 g/l dextran-70 significantly inhibited the rate and extent of aggregation in response to PAF (1 nmol/l) (P<0.001 and P = 0.003, respectively) but this was not dependent on the duration of pre-incubation (from 0 to 150 min). No effects were seen when the agonist was adenosine 5'-diphosphate (200 nmol/l), collagen (10 mg/l), 5-hydroxytryptamine (100 micromol/l) or U44069 (600 nmol/l) (all P>0.1). Analysis of PAF concentration-aggregation curves after pre-incubation with 60 g/l dextran-70 indicated significant noncompetitive inhibition by dextran-70 (P<0.001 for rate and extent of aggregation). The ability of dextran-70 to inhibit responses of equine platelets to PAF is probably an important component of its beneficial effect as an anti-thrombotic in colic cases.

Dopamine D2-type agonists protect mesencephalic neurons from glutamate neurotoxicity: mechanisms of neuroprotective treatment against oxidative stress.

Oxidative stress, a process in which neurotoxic oxygen free radicals cause dopaminergic neuronal degeneration, has been implicated in the degenerative process in Parkinson's disease. Glutamate-induced neurotoxicity is a model of oxidative stress. We demonstrated that preincubation with D2-type dopamine agonists bromocriptine and quinpirole provides neuroprotection against glutamate-induced neurotoxicity in cultured rat mesencephalic neurons. Simultaneous administration of D2 agonists, however, did not provide neuroprotection. The protective effects were dependent on the duration of preincubation and were blocked by a D2 antagonist and a protein synthesis inhibitor. Furthermore, preincubation with D2 agonists provided neuroprotection against toxicity induced by calcium overload and exposure to superoxide anions. Confocal microscopic analysis, using 2,7-dichlorofluorescin diacetate, revealed that bromocriptine preincubation suppressed the action of radicals on neurons. These findings indicate that dopamine D2 agonists provide protection mediated not only by the inhibition of dopamine turnover but also via D2-type dopamine receptor stimulation and the subsequent synthesis of proteins that scavenge free radicals.

Cellular and Molecular Mechanisms of Nitric Oxide–Induced Heart Muscle Relaxation

1. The nitric oxide (NO) donor S-nitro-N-acetyl-penicillamine (SNAP) inhibits Helix aspersa heart activity and relaxes muscles. 2. K-free saline and ouabain both depress SNAP-induced relaxation in most experiments, but in a few preparations they either had no effect or potentiated SNAP-induced relaxation. 3. Na-K pump reactivation following preincubation in K-free saline leads to the pronounced transient relaxation of heart muscle, the magnitude of which depends on the duration of preincubation. 4. 0.1 mM SNAP inhibited the ouabain sensitive part of 86Rb uptake, which reflects Na-K pump activity. This inhibition is potentiated by phospholipase C. 5. SNAP increased cGMP levels in the heart. 6. These results indicate that SNAP-induced relaxation depends on Na and Ca gradients across the membrane, which suggests that Na:Ca exchange is involved in the mechanisms of SNAP-induced relaxation. It is postulated that SNAP elicits its inhibitory effect on the heart through a cGMP-dependent Na:Ca exchange.

A rapid assay of endotoxin in whole blood using autologous neutrophil dependent chemiluminescence

A rapid (30 min) whole blood assay for the detection of lipopolysaccharide (LPS) is described. This chemiluminescent (CL) assay utilizes the CR 1 and CR 3 receptor-induced oxidant production of polymorphonuclear leucocytes as a detection platform. The differential priming of neutrophils in whole blood by LPS-antibody complexes allows the specificity of the assay to be achieved. Oxidant released in response to complement opsonized zymosan results in luminol oxidation and subsequent light emission. This is dependent on heat labile putative complement proteins in the plasma. The assay consists of a control which measures baseline whole blood neutrophil oxidant production. The test assay contains murine monoclonal IgM antibody against the Lipid A epitope of LPS and measures the enhanced chemiluminescent response of the neutrophils in the presence of LPS-antibody complexes. Maximal sensitivity of the CL assay is dependent upon optimal antigen–antibody equivalence and duration of pre-incubation with the whole blood sample. The quantification of LPS is possible by inclusion of a positive control containing a maximally reactive LPS dose (800 pg/ml Escherichia coli 055:B5 LPS at an antibody concentration of 0.8 μg/assay). The CL assay is insensitive to variations in patient neutrophil concentration over a minimum range of 0.5 to 20×10 9 cells/l. The CL assay is widely reactive with the LPS of many strains of gram negative bacteria but not with the cell wall products of gram positive bacteria or Candida and Aspergillus. In comparison to acid extraction chromogenic LAL, the CL assay demonstrates superior recovery precision and accuracy in in vitro studies. This was reproducible over a wide range of LPS concentrations (0.017–1.6 EU/ml or 20–2000 pg/ml). This assay may be a clinically useful tool for the diagnosis of infection or endotoxin in patients.

Increasing effect of ethanol on 5-HT3 receptor-mediated 14C-guanidinium influx in N1E-115 neuroblastoma cells.

N1E-115 mouse neuroblastoma cells were used to study the influence of ethanol on the 5-HT- and veratridine-induced influx of 14C-guanidinium via the 5-HT3 receptor channel and the fast sodium channel, respectively. Ethanol (10-100 mM) concentration-dependently increased the 5-HT-induced 14C-guanidinium influx, leaving the basal and veratridine (100 microM)-induced influx unaffected. The increasing effect of ethanol (100 mM) was observed at all 5-HT concentrations investigated; accordingly, ethanol increased the maximum response to 5-HT. Whereas in the absence of ethanol the concentration-response curve for 5-HT was bell-shaped, this was no longer the case when ethanol (100 mM) was present in the incubation buffer; the descending branch of the concentration-response curve for 5-HT at concentrations above 300 microM was virtually no longer observed. When, in the presence of substance P (10 microM) the 5-HT-induced 14C-guanidinium influx was already enhanced, the ability of ethanol (100 mM) to increase the 5-HT-induced influx was considerably diminished (by 72%). Preincubation of N1E-115 cells with 5-HT caused a decay of the subsequent 5-HT response ("desensitization") which was dependent on the duration of preincubation; ethanol (100 mM) did not affect the rate of this decay of the 5-HT response. The 5-HT (30 microM)-induced 14C-guanidinium influx was also increased by methanol (100 mM) and n-propanol (100 mM). The rank order of the increasing effect of the n-alkanols (at 100 mM) was: methanol < ethanol < n-propanol; i.e. the degree of enhancement increased with the lipophilicity of the alcohols.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiplatelet effects of a novel antianginal agent, nicorandil.

Nicorandil (nicotinamidoethyl nitrate) is a novel vasodilator. Its vasodilator properties are related both to the nicotinamide and nitrate moieties. Classic nitrates such as nitroglycerin (NTG) and isosorbide dinitrate demonstrate in vitro inhibition of ADP-induced platelet aggregation. Such effects have been shown to occur in a dose-related manner, are potentiated by reduced thiols and by increasing preincubation time, and are associated with increases in intracellular cyclic GMP. We explored the effect of nicorandil on ADP-induced human platelet aggregation and the role of reduced thiol N-acetylcysteine (NAC) in modulating this response. Nicorandil significantly inhibited aggregation to ADP dose dependently (IC50 3.0 mM). These effects were associated with inhibition of fibrinogen binding to the platelet surface (IC50 2 mM). Addition of nicorandil after maximal ADP-induced aggregation was achieved resulted in disaggregation. Addition of a source of reduced thiol (NAC) potentiated the antiaggregatory effects of nicorandil threefold (p < 0.05). Platelet inhibition by nicorandil was also augmented by increase in duration of preincubation, with maximal effects observed at 180 min. Preincubation of platelets with 10 mM nicorandil resulted in attenuated inhibition of platelet aggregation on gel filtration and subsequent exposure to additional nicorandil, indicative of tolerance induction. Methylene blue (MB), an inhibitor of guanylate cyclase, significantly reversed nicorandil-induced inhibition of platelet aggregation. Moreover, in accordance with this mechanism, nicorandil increased intracellular platelet cyclic GMP levels. Although the antiplatelet effect of nicotinamide was partially reversed by the K+ channel inhibitor iberotoxin, preincubation with iberotoxin had no impact on inhibition of platelet aggregation by nicorandil.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of bovine serum albumin concentration and source on sperm capacitation in the golden hamster.

Since virtually all experiments on sperm capacitation routinely include serum albumin in the culture media, the relative effect of BSA concentration on sperm capacitation was investigated. The fertile life of hamster sperm, as measured by the ability to penetrate the zona pellucida, decreased from 5 to 3 h as the concentration of BSA was increased from 3 to 18 mg/ml. To determine the extent of sperm capacitation in sperm populations that resulted in 100% penetration, the rate and timing of sperm penetration through the zona pellucida was determined. For each insemination, the rate of sperm penetration (%/min), the time when 50% of the eggs were zona-penetrated (T50), and the sperm capacitation index (T50/rate) were calculated. The extent of sperm capacitation as measured by all three parameters increased as the duration of preincubation or the BSA concentration was increased. The data on the duration of preincubation and the BSA concentration matrix indicated two properties of the effect of BSA on sperm capacitation: 1) the extent of sperm capacitation after the first hour of preincubation was independent of BSA concentration; and 2) when sperm were preincubated for two or more hours, the capacitating effectiveness of BSA was saturable; the saturating concentration of BSA decreased as the duration of preincubation increased. To determine whether the first hour of preincubation is independent of BSA, sperm were preincubated for 1 h without BSA and then 2 h with BSA. The extent of sperm capacitation of these sperm was equal to that of the 3-h control sperm; thus the first hour of capacitation is independent of BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetcyclacis and Abscisic Acid-Sensitive Reduction of Extracellular Ferricyanide by Mesophyll Cells ofValerianella locustaandLemna gibba

Electron transport across the plasma membrane of Valerianella locusta mesophyll cells and intact fronds of Lemna gibba, induced by 10 -3 M ferricyanide, was inhibited by tetcyclacis, an inhibitor regarded to be specific for cytochrome P-450 mono-oxygenases. The effect was dependent on the concentration of tetcyclacis and the duration of preincubation. The apparent rate of transmembrane electron transport increased in the presence of catalase, indicating tetcyclacis-induced H 2 O 2 -production or additional tetcyclacis-independent H 2 O 2 release

Effect of aphidicolin on DNA synthesis, PLD-recovery and DNA repair of human diploid fibroblasts.

The effect of aphidicolin, a specific inhibitor of DNA polymerases alpha and delta, was studied on DNA synthesis, PLD-recovery and DNA double-strand break rejoining in X-irradiated human fibroblasts. In unirradiated, exponentially growing cells, aphidicolin (0.5-5 micrograms ml) inhibited DNA synthesis almost completely. This effect depended not only on aphidicolin concentration but also on the duration of pre-incubation. The action of aphidicolin was found to be reversible. When aphidicolin had been removed, colony forming ability was not affected in aphidicolin pretreated cells. Aphidicolin pretreated and irradiated cells showed a reduction in PLD-recovery, dependent on aphidicolin concentration and duration of pretreatment. The initial number of DNA double-strand breaks (calibrated by 125I decay) was not affected by aphidicolin. However, after incubation for 90 min in the presence of aphidicolin there was a large reduction in double-strand break rejoining. With long incubation periods in aphidicolin rejoining was almost completely inhibited.

Dynamics of human sperm acrosome reaction: relation with in vitro fertilization

Acrosomal status has been studied on human sperm prepared for in vitro fertilization (IVF) and related to the rate of fertilization. A group of 41 men with normal classical semen parameters, included in the IVF program of University of Nice for feminine tubal obstruction (n = 37) or unexplained infertility (n = 4), were evaluated in a prospective study and compared with a control group of 10 fertile donors. Evaluation of acrosome status (spontaneous and A23187-induced acrosome loss) after 6 hours incubation in Ménézo's B2 medium was made by flow cytometry on suspended cells with a new immunofluorescence test recently reported by the authors based on a monoclonal antibody GB24. Spontaneous acrosome loss remained low even after 6 hours capacitation (mean + 1 SD, 6.5% + 4.9%). Response to A23187 increased with the duration of preincubation with a marked response after 6 hours (29.5% + 8.9%). Low spontaneous acrosome loss (less than mean + 1 SD) and high response to A23187 (greater than mean - 1 SD) were observed in 25 out of 26 cases of group A with a high fertilization rate (greater than 50% fertilized oocytes). A high level of spontaneous acrosome loss and/or a lack of response to A23187 was observed in 2 of 7 cases of group B (fertilization rate less than 50%) and 6 of 8 cases of group C (unexplained unsuccessful fertilization). Impaired acrosomal status can be associated with unexplained unsuccessful fertilization.

Reversible elimination of myofibrillar Ca2+ sensitivity by diamide and other sulfhydryl reagents: comparison with reversible contracture produced in intact cells

Cardiac contractile activity is usually controlled by intracellular Ca2+, but it can also be modified by oxidizing agents. Incubation of guinea pig heart myofibrils with diamide (3 mM, 1 h) increased basal (no Ca2+) ATPase activity by 580% and abolished Ca2+ dependence. The effect was proportional to diamide concentration (0.01-1 mM) and duration of preincubation (up to 2 h). Dithiothreitol (5 mM, 1 h) reversed most of the basal ATPase activation and restored Ca2+ sensitivity. Other sulfhydryl reagents produced a similar effect but also produced inhibition of total ATPase. In intact cell preparations, diamide produced a slow tonic contraction, consistent with myofibril activation. In the perfused rat heart, 1 mM diamide slowly increased diastolic ventricular pressure; this increase was partially reversed by dithioerythritol. In isolated rat heart myocytes, 1 mM diamide produced a slow tonic contraction, increased contractility in response to stimulation. Cardiocytes superfused for 1 h with buffer containing EGTA to deplete Ca2+ did not contract in response to stimulation but showed a slow tonic contraction with diamide. This contraction could be slowly and only partially reversed by dithioerythritol. Response to stimulation was restored by addition of Ca2+. The results show that diamide can produce contraction in viable cells. This contraction does not require extracellular Ca2+ and is unlikely to involve intracellular Ca2+. The direct activation of myofibrillar ATPase may contribute to the increased myocardial stiffness seen in ischemia and to ischemic contracture.

Effect of duration of pre-incubation on the specificity of the fructosamine assay.

Effect of duration of pre-incubation on the specificity of the fructosamine assay.