Improvement of rheological properties of firm acid gels by skim milk heating is conserved after stirring.

The enhancement of the strength of set acid gels by heating milk was related to rheological parameters (water retention capacity, storage modulus) of corresponding stirred gels. To obtain accurate rheological data from stirred gel it was necessary to maintain a constant granulometry of gel particles and to recognize time after stirring as a contributing factor. Two hours after stirring, the gel exhibited a higher storage modulus when milk was heated above 80 degrees C. A measurement of viscosity of just-stirred yoghurt was sufficient to predict correctly the quality of a stirred gel analysed by viscoelastic measurements. Increased resistance to syneresis of just-stirred gels was related to higher viscosity. The quantity of beta-lactoglobulin (beta-Ig) bound to casein micelles explains the improvement of these gel qualities. We have considered that the structure of the initial firm gel (mesostructure level) was conserved in fragments within the stirred gel. Consequently, the explanation given by various authors for the effect of heating milk on the properties of set gels can also be applied to stirred gels. The same mechanism, described in literature for structure formation of set gels from acidified milk is purposed to explain the role of heating milk on the recovery of gel structure after stirring. The beta-Ig association with casein micelles during heating favoured micelle connections during the acidification. It also favoured the association of gel fragments after stirring during the recovery in gel structure.

Regulation of InsP3-mediated Ca2+ release by CaMKII in Xenopus oocytes.

Inhibition of calmodulin (CaM) sensitizes Ca2+ release mediated by D-myo-inositol (1,4,5)-trisphosphate (InsP3) in Xenoplus oocytes, which results in spontaneous Ca2+ -dependent Cl- current oscillations or in a shift of the concentration threshold for lysophosphatidic acid (LPA) by a tenfold factor. The oscillatory currents appear at a low initial Ca2+ concentration and without any significant increase in the inositol phosphate (InsPs) concentrations. These data led us to rule out the direct involvement of CaM, as well as the implied involvement of InsP3 3-kinase. The response to intracellular injection of the non-metabolizable InsP3 analog 3-deoxy-3-fluoro InsP3 (InsP3-F) is obviously affected by previous treatment with CaM inhibitory peptide. Furthermore, these effects have been consistently obtained with specific CaMKII inhibitors such as KN-93 and AIP. CaM plays a key role in the Ca2+-dependent inactivation of type I InsP3 receptors. The experiments presented hereby allow us to postulate that CaM could also exert its inhibitory effect through CaMKII in a way that does not involve InsP3 metabolism regulation. It is concluded that CaMKII could participate in Ca2+-evoked inhibition of InsP3-mediated Ca2+ release by inhibiting the InsP3 receptor.

Simple rapid procedure for preparation of large quantities of ovalbumin.

A simple rapid procedure for preparation of large quantities of highly purified homogeneous ovalbumin from egg white by using an anion exchanger is described. It is based on the principle of frontal chromatography. The volume of "mucin-free" egg white loaded onto the column was determined in order to exceed resin capacity. Thus, competition between proteins for resin sites was created. Owing to its high negative charge density, ovalbumin drives other egg white proteins from the column progressively. Two hundred and fifty milliliters of Q-Sepharose FF gel eluted isocratically with 0. 5 M NaCl extracted 9.55 g of ovalbumin with a purity rate of 83%. A 6.75 g amount of ovalbumin, with a purity rate of 94%, was recovered with an isocratic elution program using 0.14 M NaCl. Purified ovalbumins were compared by electrophoresis and analytical chromatography with other ovalbumin preparations.

The carcinogen Cd(2+) activates InsP(3)-mediated Ca(2+) release through a specific metal ion receptor in Xenopus oocyte.

The effects of the carcinogen Cd(2+) on Xenopus oocyte were evaluated by Inositol (1,4,5)-trisphosphate (InsP(3)) assays and electrophysiological experiments. The stimulation of the Ca(2+)-dependent Cl(-) current by Cd(2+) is clearly linked to InsP(3) formation since the effects of the metal are antagonized by neomycin, heparin and caffeine. A similar inhibition of the Cd(2+) effects is observed when the oocytes are pretreated with thapsigargin. Moreover, the use of sulfhydryl groups reductors such as 2-mercaptoethanol or N-ethylmaleimide strongly suggests that the Cd(2+) response is mediated by an extracellular receptor. Finally, measurements of InsP(3) production demonstrate that Cd(2+) superfusion actually leads to a PIP(2) breakdown. We conclude that extracellular Cd(2+) evokes an increase in [Ca(2+)](i) by stimulating the emptying of the InsP(3)-sensitive Ca(2+) stores, and that it may do so by interacting with a specific cell-surface ion receptor. This putative ion receptor may be important in allowing oocytes to respond to heavy metals.

Caffeine exerts a dual effect on capacitative calcium entry in Xenopus oocytes.

Caffeine increases the amplitude of the Cl- currents evoked by capacitative Ca2+ entry (CCE) on thapsigargin-treated Xenopus oocytes. The caffeine-induced potentiation of the CCE process appears to rest on two distinct and additive components. The first component involves the cAMP second messenger system since it can be mimicked by either IBMX perfusion or cAMP microinjection into the oocyte and inhibited by the PKA inhibitory peptide i-PKA. The second component, although activatory, is dynamically related to the caffeine-evoked inhibition of InsP3-mediated Ca+ release and may arise from an interaction between caffeine and the InsP3 receptor in the context of a conformational coupling between the InsP, receptor and the channels responsible for CCE.

High intensity pulsed electric fields applied to egg white: effect on Salmonella Enteritidis inactivation and protein denaturation.

High-intensity electric fields have been successfully applied to the destruction of Salmonella Enteritidis in diaultrafiltered egg white. The effects of electric field strength (from 20 to 35 kV x cm(-1)), pulse frequency (from 100 to 900 Hz), pulse number (from 2 to 8), temperature (from 4 to 30 degrees C), pH (from 7 to 9), and inoculum size (from 10(3) to 10(7) CFU x ml(-1)) were tested through a multifactorial experimental design. Experimental results indicate that, for Salmonella inactivation, the electric field intensity is the dominant factor with a strongly positive effect, strengthened by its positive interaction with pulse number. Pulse number, temperature, and pH have also significant positive effects but to a lesser extent. In the most efficient conditions, the pulsed electric field (PEF) treatment is capable of 3.5 log10 reduction in viable salmonellae. Simultaneously, the measure of surface hydrophobicity does not indicate any increase after PEF treatment. These results suggest that no protein denaturation occurs, unlike what is observed after comparable heat treatment in terms of Salmonella inactivation (55 degrees C for 15 min).

Rapid growth of Salmonella enteritidis in egg white reconstituted from industrial egg white powder.

The aim of this study was to evaluate the consequences of the egg white-drying process on egg white ability to limit Salmonella Enteritidis growth in addition to the elucidation of the factors involved. We observed rapid growth of Salmonella Enteritidis inoculated in egg white reconstituted from industrial powder in comparison with that observed in liquid egg white collected in the laboratory: Salmonella cell counts rose from 10(3) to 10(8) cells/ml of egg white from powder during 24 h incubation at 30 degrees C. This rapid growth was observed in powder from all egg-breaking factories investigated, and it was comparable to that observed in optimum medium (tryptone soy broth). In view of the mechanism of egg white resistance and the major role played by iron availability and by ovotransferrin, we investigated several hypotheses to explain this rapid growth: iron provided during the drying process and/or denaturation of protein (especially ovotransferrin). The rapid growth observed in egg white reconstituted from powder was in relation to egg white protein denaturation and especially ovotransferrin denaturation during powder pasteurization that enhanced the availability of iron necessary for Salmonella growth. The major role played by ovotransferrin and iron deficiency on Salmonella growth in egg white was illustrated in this study.

The inositol (1,4,5)-trisphosphate 3-kinase of Xenopus oocyte is activated by CaMKII and involved in the regulation of InsP3-mediated Ca2+ release.

The effect of Ca2+ on inositol (1,4,5)-trisphosphate 3-kinase (3-kinase) activity was measured on Xenopus oocyte cytosolic extracts. The Ca2+-evoked elevation in 3-kinase activity appeared to be mediated by calmodulin (CaM) and the calmodulin-dependent protein kinase II (CaMKII). The results observed in vitro were totally retrieved in intact oocytes and tend to demonstrate the involvement of a CaMKII-mediated phosphorylation in the regulation of 3-kinase activity. Finally, electrophysiological recordings of InsP3-elicited chloride current transients in the presence of CaM/CaMKII inhibitors allowed to postulate an involvement of 3-kinase activity in the regulation of InsP3-mediated Ca2+ release.

Involvement of the Ca2+/calmodulin-dependent protein kinase II pathway in the Ca2+-mediated regulation of the capacitative Ca2+ entry in Xenopus oocytes.

Activation of the phosphoinositide transduction pathway induces capacitative Ca2+ entry in Xenopus oocytes. This can also be evoked by intracellular injection of Ins(1,4.5)P3, external application of thapsigargin and/or incubation in a Ca2+-free medium. Readmission of Ca2+ to voltage-clamped, thapsigargin-treated Xenopus oocytes triggers Ca2+-dependent Cl- current variations that reflect capacitative Ca2+ entry. Inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) by specific peptides markedly increased the amplitude of the transients, suggesting an involvement of the CaMKII pathway in the regulation of capacitative Ca2+ entry. Biochemical studies provide evidence for the activation of CaMKII in response to the development of capacitative Ca2+ entry. In effect, a CaMKII assay in vivo allows us to postulate that readmission of Ca2+ to thapsigargin-treated oocytes can induce a burst of CaMKII activity. Finally, analysis of the Cl- transient kinetics at high resolution of time suggests that CaMKII inhibition blocks the onset of the inactivation process without affecting the activation rate. We therefore postulate that CaMKII might participate in a negative feedback regulation of store-depletion-evoked Ca2+ entry in Xenopus oocytes.

Novel monoacetoneglucose derivatives with calcium antagonist activity.

Ethers and thioethers of monosaccharides have been synthesised which show potent toxicity to mouse (LD50 > or = 4 g.kg-1 O.W. and 0.2 to 1.5 g.kg-1 I.P.W.). A study of calcium antagonist activity for the full series of compounds indicated that the activity was similar for both O- and S- ethers and maximum activities were observed for monoacetoneglucose ethers possessing carbon chain close to 8 carbons.

Sensitization of InsP3-dependent calcium signalling through structural modification of voltage-dependent calcium channel: a physiological relevance of the calcium channel beta subunit.

The expression in Xenopus oocytes of the human voltage-dependent Ca2+ channel (VDCC) beta 2 subunit subtype (h beta 2) enhances the endogenous Ca2+ channel activity. By using the native Ca(2+)-dependent chloride conductance to monitor fast intracellular Ca2+ variations, we point out that the beta-enhanced Ca2+ entry (T1 component) is currently associated with a second delayed elevation of internal Ca2+ (T2 component). Further experiments show that this additional component absolutely requires Ca2+ entry through the beta-modulated channels although it directly derives from a Ca2+ release from intracellular inositol (1,4,5)-trisphosphate (InsP3)-sensitive stores. Finally, our study demonstrates that InsP3-evoked response in oocytes is dramatically modified since it gains a new shape of voltage dependency directly derived from the beta-modified Ca2+ influx. The main conclusion is that the spatiotemporal pattern of InsP3-dependent Ca2+ release may be closely influenced by the intrinsic characteristics of working VDCCs.

[The inhibitory action of a saccharide derivative on a calcium channel in the oocyte of Xenopus]. / Mise en évidence de l'action inhibitrice d'un dérivé saccharidique sur le canal calcique de l'ovocyte de Xénope.

Endogenous calcium channels of Xenopus oocyte membrane do not fit with pharmacological classification of calcium channels. The present study demonstrates that the saccharidic derivate, OC8-MAGlu-MAGlu, has potent inhibitory effect on this channel activity.

Two-step chromatographic procedure for the purification of hen egg white ovomucin, lysozyme, ovotransferrin and ovalbumin and characterization of purified proteins.

An improved procedure is described involving gel permeation and anion-exchange chromatography for the purification of four major hen egg white proteins. The procedure involves a first-step purification of ovomucin and lysozyme by gel permeation on a Superose 6 Prep Grade column. In the second step, anion-exchange chromatography on Q Sepharose Fast Flow led to the isolation of ovotransferrin and ovalbumin from a gel permeation chromatographic peak. The purities were estimated as ca. 80, 100, 80 and 100% for ovomucin, lysozyme, ovotransferrin and ovalbumin, respectively. The purification yield was over 60% for each protein. Further characterization of purified lysozyme revealed that it was fully active and homogeneous in relation to the electrospray ionization mass spectrum. The electrospray ionization mass spectrum showed different ovotransferrin species. The amino acid composition of purified ovomucin was compared to those published previously.

Interaction between Ca2+ release from inositol trisphosphate sensitive stores and Ca2+ entry through neuronal Ca2+ channels expressed in Xenopus oocyte.

Rat cerebellar RNA injected into Xenopus oocytes leads to the expression of putative P-type voltage-dependent Ca2+ channels (VDCCs). The monitoring of intracellular Ca2+ variations by recording the Ca2+ dependent chloride current in voltage clamped oocytes indicates that activation of these Ca2+ channels by depolarization gives rise to two distinct components of cytosolic Ca2+ elevation. If the early component (T1) can be directly attributed to the Ca2+ entry through VDCCs, the second delayed one (T2) is related to a Ca2+ release from InsP3 sensitive stores activated following Ca2+ entry. Modifications of cytosolic Ca2+ by direct injection of Ca2+ into oocytes or by increasing the Ca2+ influx through VDCCs suggest that the Ca2+ release from intracellular InsP3 sensitive stores can be modulated in a differential manner. Namely, discrete elevations of cytosolic Ca2+ switch on the Ca2+ release whereas higher Ca2+ concentrations dampen the release. These results suggest a functional coupling between P-type VDCCs and InsP3 receptors.

[Effects of the monosaccharide derivative 8RN-DAGal on the putative P-type calcium channel expressed in Xenopus oocytes]. / Effets du dérivé monosaccharidique 8RN-DAGal sur un courant calcique apparenté au type P exprimé dans l'ovocyte de Xénope.

P-type calcium channels are expressed in Xenopus oocytes after injection of rat cerebellar mRNA. The FTX and omega-Aga-IVa toxins extracted from Agelenopsis aperta venom are known to inhibit the activity of this channel. The present results demonstrate that 8RN-DAGal is also a antagonist of P-type calcium channels. The inhibition of the current, obtained with Ba2+, as charge carrier, is voltage dependent.

Positive regulation by protein kinase C of slow Na current in Xenopus oocytes.

The slow inward Na current observed during sustained depolarization of the Xenopus oocyte membrane is due to a complex mechanism described as the induction of the channels. The present work investigates the role of protein phosphorylation in Na channel function. Injection of alkaline phosphatase in the oocytes decreased inward current. Therefore, the possible involvement of protein kinase in Na channel induction was explored. Treatment of oocytes with two activators of protein kinase C (PKC) resulted in enhanced Na current amplitude, whereas the treatment of oocytes with two potent PKC inhibitors decreased the inward current. These results imply that PKC phosphorylation is a fundamental step of Na channel induction. The possibility that the depolarization of the oocyte membrane may be the factor involved in PKC activation is discussed.

[Structure-activity relationship of a new family of calcium antagonist molecules derived from monosaccharides]. / Relations structure-activité d'une nouvelle famille de molécules antagonistes calciques derivées de monosaccharides.

The Ca++ antagonist effects of new drugs derived from monosaccharides were tested in the rat duodenal muscle preparation in vitro. The structure-activity relationship shows that: 1. The monoacetonide products (nR-O-MAG and nR-S-MAG) with alkyl chain from 8 to 9 C atoms induce a maximal reduction of muscular tonus and contraction. 2. The inhibitory effect rapidly decreases when the alkyl chain has a number of C atoms smaller than 7 or larger than 9. 3. The diacetonide (DAG) and acetonide (G) products induce an inhibition of less extent. 4. The Ca++ antagonist effect is very slightly changed by the type of heteroatom (O or S) linking the alkyl chain to the monosaccharide. 5. The type of monosaccharide affects the Ca++ antagonist activity.

Regulation by protein kinase-C of putative P-type Ca channels expressed in Xenopus oocytes from cerebellar mRNA.

Xenopus oocytes injected with rat cerebellar mRNA expressed functional voltage-dependent Ca channels detected as an inward Ba current (IBa). The pharmacological resistance to dihydropyridines and omega-conotoxin together with the blockade obtained with Agelenopsis aperta venom suggest that these channels could be somehow assimilated to P-type Ca channels. The precise nature of the transplanted Ca channels was assessed by hybrid-arrest experiments using a specific oligonucleotide antisense-derivated from the recently cloned alpha 1-subunit of P channels (BI-1 clone). In addition, we demonstrate that exogenous Ca channel activity was enhanced by two different PKC activators (a phorbol ester and a structural analog to diacylglycerol). The general electrophysiological and pharmacological properties of the stimulated Ca channels remain unchanged. This potentiation induced by PKC activators is antagonized by a PKC inhibitor (staurosporine) and by a monoclonal antibody directed against PKC. It is concluded that P-type Ca channels are potentially regulated by PKC phosphorylation and the functional relevance of this intracellular pathway is discussed.

Expression of calcium channels in Xenopus oocyte injected with crab skeletal muscle fibre mRNA.

Using the whole cell voltage-clamp technique and a C1 free and Na free Ba methane sulfonate solution, stage V and VI Xenopus oocytes demonstrated a Ba current (endogenous component) with a peak amplitude average of 6 nA (6 +/- 2 nA). When oocytes were injected with crustacean skeletal muscle mRNA, an additional component of IBa could be detected (exogenous IBa). The latter current could be distinguished from the native one by several electrophysiological means: a peak amplitude average of 90 nA (90 +/- 4 nA), activation potential threshold, steady state inactivation properties and sensitivity to Ca blockers. As shown by Jdaïâa and Guilbault in crustacean skeletal muscle fibres, exogenous IBa could be divided into two components: a "fast component" and a "slow component" probably passing through two types of Ca channels (fast and slow) since the peak Ba current voltage relationship was biphasic and the fast component of exogenous IBa was less sensitive than the slow to nifedipine. The features of the newly synthesized channels incorporated in the Xenopus oocyte membrane suggest that they may be associated with fast and slow channels, previously described in many preparations, particularly in crustacean skeletal muscle fibres.

Expression of Ba currents in Xenopus oocyte injected with pregnant rat myometrium mRNA.

Using the single electrode voltage-clamp technique, stage V and VI Xenopus oocytes showed a Ba inward current (endogenous IBa) with a peak amplitude of -15 +/- 2 nA in a Cl and Na-free Ba methane sulphonate medium. When oocytes were injected with pregnant rat (18 days gestation) myometrium mRNA, an additional component of Ba current could be detected (exogenous IBa). This inward current could be distinguished from the native IBa by several means: i) peak amplitude (-75 +/- 3 nA); ii) activation voltage threshold; iii) steady-state inactivation parameter; iv) sensitivity to dihydropyridines. The features of the exogenous IBa were compared to those of the inward Ca channel current recorded with the tight-seal patch-clamp technique in single myometrial cells maintained in primary culture.