Increasing Preincubation Time Research Articles

PSX-B-1 Effect of exposure time and dose of effective microorganisms (EM®) on feed in vitro rumen fermentation and digestibility

Abstract The objective was to evaluate the effect of feed inoculation with effective microorganisms (EM®) (mainly containing Lactobacillus spp.,Rhodopseudomona palustrisand Saccharomyces cerevisiae) on rumen fermentation using in vitrogas production technique. We hypothesized that increasing doses and allowing exposure of EM® for up to 48 hours, would improve digestibility and rumen fermentation. The experimental design was a 4×4 completely randomized block design including 4 EM® levels [(0(EM0), 0.5(EM0.5), 1.0 (EM1) and 1.5 (EM1.5) mL EM® / kg DM] and 4 preincubation times [0 (T0), 12(T12), 24(T24), 48 (T48) h], with four repetitions per treatment. Treatments were evaluated using 100ml glass bottles with 0.5g of the diet (20% corn stover, 20% oat hay, 48.8% ground corn, 7% molasses, 1.2% urea, 1% soybean meal, 0.9% mineral premix,1.1% salt, dry matter basis) incubated with sheep ruminal fluid in 3 different occasions. Data were analyzed with PROC MIXED of SAS and orthogonal contrasts to determine the linear and quadratic effects of EM dose and exposure time. Interaction (P < 0.05) of EM x T was observed for in vitrodry matter digestibility (IVDMD), maximum gas volume (Vmax), total volatile fatty acids (VFA), acetate (ACE), propionate (PROP), butyrate (BUT) and ammonia-nitrogen (NH3), IVDMD was higher (P < 0.01, 4.8 and 3.72%) for T48EM1.5 than T12EM0 and T0EM0, PROP was higher (P < 0.05) for T48EM0, T48EM1 and 1.5 than T12EM0. The ACE:PROP ratio was higher (P < 0.05, 17.2%) for T12EM0 than T48EM1.5. IVDMD, PROP and NH3 linearly increased (P < 0.01) with increasing exposure time. EM levels have a quadratic effect (P < 0.01) with maximum response at EM0.5. It was concluded that the addition of 0.5 to 1.5 mL/kg DM of EM® to a sheep diet and increasing preincubation time, up to 48h, improve feed fermentation and digestibility.Project was supported by UNAM, DGAPA, PAPIIT (IT202120).

Differential Reversible and Irreversible Interactions between Benzbromarone and Human Cytochrome P450s 3A4 and 3A5.

Mounting evidence has revealed that despite the high degree of sequence homology between cytochrome P450 3A isoforms (i.e., CYP3A4 and CYP3A5), they have the propensities to exhibit vastly different irreversible and reversible interactions with a single substrate. We have previously established that benzbromarone (BBR), a potent uricosuric agent used in the management of gout, irreversibly inhibits CYP3A4 via mechanism-based inactivation (MBI). However, it remains unelucidated if CYP3A5-its highly homologous counterpart-is susceptible to inactivation by BBR. Using three structurally distinct probe substrates, we consistently demonstrated that MBI was not elicited in CYP3A5 by BBR. Our in silico covalent docking models and molecular dynamics simulations suggested that disparities in the susceptibilities toward MBI could be attributed to the specific effects of BBR covalent adducts on the F-F' loop. Serendipitously, we also discovered that BBR reversibly activated CYP3A5-mediated rivaroxaban hydroxylation wherein apparent V max increased and K m decreased with increasing BBR concentration. Fitting data to the two-site model yielded interaction factors α and β of 0.44 and 5.88, respectively, thereby confirming heterotropic activation of CYP3A5 by BBR. Furthermore, heteroactivation was suppressed by the CYP3A inhibitor ketoconazole in a concentration-dependent manner and decreased with increasing preincubation time, implying that activation was incited via binding of parent BBR molecule within the enzymatic active site. Finally, noncovalent docking revealed that CYP3A5 can more favorably accommodate both BBR and rivaroxaban in concert as compared with CYP3A4, which further substantiated our experimental observations. SIGNIFICANCE STATEMENT: Although it has been previously demonstrated that benzbromarone (BBR) inactivates CYP3A4, it remains uninterrogated whether it also elicits mechanism-based inactivation in CYP3A5, which shares ∼85% sequence similarity with CYP3A4. This study reported that BBR exhibited differential irreversible and reversible interactions with both CYP3A isoforms and further unraveled the molecular determinants underpinning their diverging interactions. These data offer important insight into differential kinetic behavior of CYP3A4 and CYP3A5, which potentially contributes to interindividual variabilities in drug disposition.

The Scaffold-Articular Cartilage Interface: A Combined In Vitro and In Silico Analysis Under Controlled Loading Conditions.

The optimal method to integrate scaffolds with articular cartilage has not yet been identified, in part because of our lack of understanding about the mechanobiological conditions at the interface. Our objective was to quantify the effect of mechanical loading on integration between a scaffold and articular cartilage. We hypothesized that increased number of loading cycles would have a detrimental effect on interface integrity. The following models were developed: (i) an in vitro scaffold-cartilage explant system in which compressive sinusoidal loading cycles were applied for 14 days at 1 Hz, 5 days per week, for either 900, 1800, 3600, or 7200 cycles per day and (ii) an in silico inhomogeneous, biphasic finite element model (bFEM) of the scaffold-cartilage construct that was used to characterize interface micromotion, stress, and fluid flow under the prescribed loading conditions. In accordance with our hypothesis, mechanical loading significantly decreased scaffold-cartilage interface strength compared to unloaded controls regardless of the number of loading cycles. The decrease in interfacial strength can be attributed to abrupt changes in vertical displacement, fluid pressure, and compressive stresses along the interface, which reach steady-state after only 150 cycles of loading. The interfacial mechanical conditions are further complicated by the mismatch between the homogeneous properties of the scaffold and the depth-dependent properties of the articular cartilage. Finally, we suggest that mechanical conditions at the interface can be more readily modulated by increasing pre-incubation time before the load is applied, as opposed to varying the number of loading cycles.

Ethacrynic acid as a lead structure for the development of potent urease inhibitors

Abstract Ethacrynic acid and a series of its analogues were synthesized and subsequently evaluated for their inhibitory effect on jack bean urease. Ethacrynic acid showed, even at low concentrations, very potent inhibitory activity against the enzyme. For ethacrynic acid, the inhibition potential increased with increasing preincubation time of ethacrynic acid and enzyme, whereas for some other compounds a higher preincubation time lead to a significant reduction of their activity. We could demonstrate that the α,β-unsaturated carbonyl unit of our compounds is mandatory to inhibit the enzyme, possibly due to its ability to bind to cysteine residues in the active site of the jack bean urease.

In Vitro Cytotoxicity Assessment of an Orthodontic Composite Containing Titanium-dioxide Nano-particles.

Background and aims. Incorporation of nano-particles to orthodontic bonding systems has been considered to prevent enamel demineralization around appliances. This study investigated cytotoxicity of Transbond XT adhesive containing 1 wt% titanium dioxide (TiO2) nano-particles. Materials and methods. Ten composite disks were prepared from each of the conventional and TiO2-containg composites and aged for 1, 3, 5, 7 and 14 days in Dulbecco’s Modified Eagle’s Medium (DMEM). The extracts were obtained and exposed to culture media of human gingival fibroblasts (HGF) and mouse L929 fibroblasts. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results. Both adhesives were moderately toxic for HGF cells on the first day of the experiment, but the TiO2-containing adhesive produced significantly lower toxicity than the pure adhesive (P<0.05). No significant differences were found in cell viability percentages between the two groups on the other days (P>0.05). There was a significant reduction in cell toxicity with increasing pre-incubation time (P<0.001). L929 cells showed similar toxicity trends, but lower sensitivity to detect cytotoxicity of dental composites. Conclusion. The orthodontic adhesive containing TiO2 nano-particles indicated comparable or even lower toxicity than its nano-particle-free counterpart, indicating that incorporation of 1 wt% TiO2 nano-particles to the composite structure does not result in additional health hazards compared to that occurring with the pure adhesive.

Mechanism-based CYP2D6 inactivation by acridone alkaloids of Indonesian medicinal plant Lunasia amara

Fourteen acridone alkaloids isolated from Lunasia amara Blanco were tested for their mechanism-based inhibition on human liver microsomal dextromethorphan O-demethylation activity, a prototype marker for cytochrome P450 2D6 (CYP2D6). Among the 14 compounds, 5-hydroxygraveroline (1), 8-methoxyifflaiamine (2), lunamarine (3), and lunine (12) increased their inhibitory activity with increasing preincubation time. Then, we further examined the possibility of mechanism-based inhibition on 5-hydroxygraveroline (1) and lunamarine (3), which showed the potent inhibition. Further investigations on 1 and 3 showed that the characteristic time- and concentration-dependent inhibition, which required a catalytic step with NADPH, was not protected by nucleophiles, and was decreased by the presence of a competitive inhibitor. Thus, 1 and 3 were concluded as mechanism-based inactivators of CYP2D6.

Corydaline Inhibits Multiple Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

Corydaline is a bioactive alkaloid with various antiacetylcholinesterase, antiallergic, and antinociceptive activities found in the medicinal herb Corydalis Tubers. The inhibitory potential of corydaline on the activities of seven major human cytochrome P450 and four UDP-glucuronosyltransferase enzymes in human liver microsomes was investigated using LC-tandem MS. Corydaline was found to inhibit CYP2C19-catalyzed S-mephenytoin-4’-hydroxylatoin and CYP2C9-catalyzed diclofenac 4-hydroxylation, with Ki values of 1.7 and 7.0 μM, respectively. Corydaline also demonstrated moderate inhibition of UGT1A1-mediated 17β-estradiol 3-glucuronidation and UGT1A9-mediated propofol glucuronidation with Ki values of 57.6 and 37.3 μM, respectively. In the presence of corydaline, CYP3A-mediated midazolam hydroxylation showed a decrease with increasing preincubation time in a dose-dependent manner with Ki values of 30.0 μM. These in vitro results suggest that corydaline should be evaluated for potential pharmacokinetic drug interactions in vivo due to potent inhibition of CYP2C19 and CYP2C9.

Mechanism-Based Inhibition of Human Liver Microsomal Cytochrome P450 2D6 (CYP2D6) by Alkamides ofPiper nigrum

Nineteen alkamides isolated from Piper nigrum L. were tested for their mechanism-based inhibition on human liver microsomal dextromethorphan O-demethylation activity, a prototype marker for cytochrome P450 2D6 (CYP2D6). All compounds increased their inhibitory activity with increasing preincubation time. Among them, 15 and 17 showed more than 50 % decrease of the CYP2D6 residual activity after 20 min preincubation. Further investigations on 15 and 17 showed that the characteristic time- and concentration-dependent inhibition, which required a catalytic step with NADPH, was not protected by nucleophiles, and was decreased by the presence of a competitive inhibitor. The kinetic parameters for inactivation (kinact and KI) were 0.028 min-1 and 0.23 microM for 15 and 0.064 min-1 and 0.71 microM for 17, respectively, which were stronger than the known mechanism-based inhibitor, paroxetine (a positive control). Thus, 15 and 17 are potent mechanism-based inhibitors of CYP2D6.

Cytotoxicity of a calcium aluminate cement in comparison with other dental cements and resin-based materials

The objective of this study was to compare the cytotoxic effects of a calcium aluminate cement with several currently used direct restorative materials. Specimens of three composites (QuiXfil, Tetric Ceram, Filtek Supreme), one zinc phosphate cement (Harvard Cement), one glass ionomer cement (Ketac Molar), and one calcium aluminate cement (DoxaDent), were used fresh or after 7-days’ preincubation in cell culture medium at 37°C, pH 7.2. PVC strips for ISO 10993-5 cytotoxicity test were used as positive control and glass specimens as negative control. L-929 fibroblasts (5-ml aliquots, containing 3×104 cells/ml), cultivated in DMEM with 10% FCS, 1% glutamine, and 1% penicillin/streptomycin at 37°C/5% CO2 and trypsinized, were exposed to the specimens for 72 h. The cells were harvested, centrifuged, and resuspended in 500 µl DMEM and then counted in 500 µl DMEM for 30 s with a flow cytometer at 488 nm. The analysis of variance comparing the six materials showed different influences on L-929 fibroblast cytotoxicity (p<0.0001). The cytotoxicity of all specimens diminished with increasing preincubation time (p<0.0001). Fresh DoxaDent exhibited the lowest cytotoxicity, followed by QuiXfil. Ketac Molar showed the highest cytotoxicity. After 7 days of preincubation, Harvard Cement and Filtek Supreme demonstrated more cytotoxicity than the other materials (p<0.005).

Cytotoxic effects of packable and nonpackable dental composites

Objectives. Advanced dental packable and nonpackable composite materials have been recently introduced onto the market as ‘amalgam alternatives’ for the restoration of posterior teeth. Most established composites are applied in increments of no more than 2 mm and light cured to ensure complete polymerization, whereas new formulations have been claimed to be suitable for application in increments of 4–5 mm. The aims of the present study were to analyze the cytotoxicity of these new composites in comparison to an established nonpackable composite using standardized cell culture systems and to examine the influence of thickness of the light cured increments on the cytotoxicity of these materials. Methods. Specimens were prepared in polyethylene tubes covered with mylar. All materials were light cured in 2.5 or 5 mm increments with a Demetron curing light (light intensity: 550 mW/cm 2). Specimens were added to the cultures immediately after production or after preincubation for 1, 2, 7 days or 6 weeks under cell-culture conditions. Specimens were incubated with L-929 fibroblasts for 72 h and cell numbers determined by flow cytometry. In a different series of experiments, dopaminergic cells were incubated with composite supernatants. Results. Results with L-929 fibroblasts demonstrated that all freshly prepared composite materials reduced cell numbers ( p<0.05) in comparison to controls. The cytotoxicity of all substances diminished after increasing preincubation times ( p<0.0001). In a rank order of cytotoxicity, the established composite and two of the advanced composite materials exhibited least cytotoxicity, whereas the other advanced composites showed moderate or severe cytotoxic effects. The cytotoxicity of each material was much higher when polymerized in 5 mm increments than in 2.5 mm composite increments ( p<0.0001). Significance. Our data demonstrate that the advanced composites tested show similar or more severe cytotoxicity than an established nonpackable composite and that cytotoxicity of all materials investigated increases when applied in a 5 mm bulk increment.

Relationship between the crosslinking of caseins by transglutaminase and the gel strength of yoghurt

After pre-incubation of skim milk with microbial transglutaminase (10.5 U/100 ml skim milk or 3 U/g milk protein) at 40 °C for 150 min and subsequent heat inactivation, yoghurt was prepared. The breaking strength of this yoghurt increased in the given system from 550 cN to 920 cN with increasing pre-incubation time, reaching a plateau after 60 min. This increase in breaking strength correlates with a rather small amount of casein oligomerization, ranging from 10.9% to 25.8% using gel-permeation chromatography under reducing and denaturing conditions (sum of predominantly formed dimers and trimers in the total casein fraction). After enzymatic hydrolysis, Ne-(γ-glutamyl)-l-lysine was quantified by amino acid analysis. The isopeptide was formed exclusively intermolecularly, indicating that even a very small amount of casein crosslinking is capable of inducing significant changes in the functional properties of milk proteins.

Transient low-affinity agonist binding to Torpedo postsynaptic membranes resolved by using sequential mixing stopped-flow fluorescence spectroscopy.

We have detected the binding of the fluorescent agonist Dns-C6-Cho to both low- and high-affinity states of the nicotinic acetylcholine receptor (nAcChoR) using sequential mixing stopped-flow fluorescence spectroscopy. Our approach to resolving low- and high-affinity binding was to first preincubate receptor membranes with the fluorescent partial agonist Dns-C6-Cho for 15 ms to 1000 s and then to follow the fluorescence decay upon chemical dilution into excess acetylcholine. The fast and slow decays, reflecting Dns-C6-Cho dissociation from low- and high-affinity receptors, had rates of 140 +/- 27 s-1 and 0.1 +/- 0.02 s-1, respectively. With increasing preincubation times, the number of low-affinity receptors decreased while the number of high-affinity receptors increased in a Dns-C6-Cho concentration-dependent manner consistent with current models for agonist-induced affinity state conversion. At receptor-activating concentrations of Dns-C6-Cho, the apparent rates with which high-affinity receptors formed approximated those of ion flux desensitization, implying that the fast desensitized state has an agonist dissociation rate that is indistinguishable from the equilibrium slow desensitized state. The KD for the low-affinity binding site was determined to be 1.1 microM from the increase in the amplitude of the fast decay with Dns-C6-Cho concentration with preincubation times that were sufficiently brief to minimize affinity state conversion. Assuming a bimolecular association rate of 10(8) M-1 s-1, a second estimate of 1.4 microM was made for low-affinity binding. We also detected a fluorescence enhancement consistent with a conformational isomerization of Dns-C6-Cho-inhibited nAcChoRs.

Inhibition of H + efflux from Saccharomyces cerevisiae by insoluble metal phosphates and protection by calcium and magnesium: inhibitory effects a result of soluble metal cations?

Insoluble metal phosphates were capable of inhibitory effects on plasma membrane H + -ATPase-mediated glucose-dependent H + efflux from Saccharomyces cerevisiae . The relative toxicities of the different compounds used was Zn > Co which was the same order as that obtained for soluble chlorides of these metals, although the toxicity of the latter was manifest at lower concentrations. Inhibitory effects of the metal phosphates were dependent on the amount in suspension and increased with increasing preincubation time. The inhibitory effect of toxic metal compounds on glucose-dependent H + efflux could be reduced or prevented by the addition of chlorides of calcium and magnesium, calcium being more effective than magnesium. The relative protective effects of Ca and Mg were similar for both soluble and insoluble compounds, and Ca could neutralize the influence of both cobalt and zinc phosphates. Although toxic effects could result from the solubilization of the insoluble metal phosphate compounds as a result of H + efflux releasing potentially toxic metal cations, there was no difference in the amount of metal cations released whether glucose was present or absent from the treatments. In addition, it was found that significant concentrations of soluble Co 2+ and Zn 2+ were released into metal phosphate suspensions in the absence or presence of cells with equilibrium concentrations in cell free suspensions (attained after 10–20 min) being about 60 m for Zn 3 (PO 4 ) 2 and about 65 m for Co 3 (PO 4 ) 2 . In the presence of cells, a lo equilibrium concentration was attained in the presence of Zn, probably due to uptake by glucose-dependent and -independent processes. Such uptake was not observed for released Co 2+ and could account for the higher toxicity of zinc phosphate compared to cobalt phosphate. Although some direct interactions with the insoluble phosphates must have been involved in overall inhibitory effects, it is concluded that a significant proportion of toxic effects resulted from free metal cations in solution, with protective effects of Ca and Mg resulting from competitive and stabilizing interactions at the cell surface.

Effect of acid predissolution on fibril size and fibril flexibility of synthetic beta-amyloid peptide

beta-amyloid peptide (A beta) is the major protein component of senile plaques and cerebrovascular amyloid deposits in Alzheimer's patients. Several researchers have demonstrated that A beta is neurotoxic in in vitro and in vivo systems. Peptide aggregation state and/or conformation might play a significant role in determining the toxicity of the peptide. The size and flexibility of fibrils formed from the synthetic peptide beta (1-39), corresponding to the first 39 residues of A beta, were determined. Samples were prepared either directly from lyophilized peptide or diluted from a 10 mg/ml stock solution in 0.1% trifluoroacetic acid (TFA). All samples had a final peptide concentration of 0.5 mg/ml, a final pH of 7.4, and a final NaCl concentration of 0.14 M. The molecular weight and linear density of the fibrils increased with increasing pre-incubation time in TFA, based on static light scattering measurements. Analysis of the angular dependence of the intensity of scattered light indicated that the fibrils were semi-flexible chains and that the fibril flexibility decreased with increasing pre-incubation time in TFA. There was a concomitant change in phase behavior from precipitation to gelation with the decrease in fibril flexibility.

Inhibition of the Plastidic Pyruvate Dehydrogenase Complex in Isolated Plastids of Oat

The activity of the plastidic pyruvate dehydrogenase complex (pPDHC) is one source of acetyl-CoA in plastids of higher plants needed for de novo fatty acid biosynthesis. This plastidic enzyme reaction is specifically inhibited by acetylmethylphosphinate (AMPI), a com ­ pound which had hitherto been known only as an inhibitor of the mitochondrial pyruvate dehydrogenase complex (mPDHC). In the test system of isolated intact oat plastids (Avena sativa) [2-14C]pyruvate was used for de novo fatty acid biosynthesis. The incorporation of label from [2-14C]pyruvate in fatty acids was inhibited by AMPI in a dose-dependent manner. The inhibition rose with increasing preincubation time of plastids with the inhibitor. I50 values for the inhibition of de novo fatty acid biosynthesis from [2-14C]pyruvate by AMPI for iso­lated etioplasts and chloroplasts were 4.5 and 80 μm , respectively. The activity of the pPDHC decreased during greening of oat seedlings, as is seen from the decreasing incorporation of [2-14C]pyruvate into fatty acids during the light-induced transformation of etioplasts into chloroplasts. In contrast to the decreasing pPDHC activity, the activity of the plastidic acetyl-C oA synthetase (ACS), which transfers acetate to acetyl-CoA, rose parallel to the transfor­mation of etioplasts into chloroplasts. During the assay time of 20 min we could not detect an incorporation of radiolabel from pyruvate or acetate into β-carotene or any other carotenoid

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)

A cryptic promoter in the O(R) region of bacteriophage lambda

A cryptic promoter, designated P alpha, initiates transcription within the O(R) region of bacteriophage lambda. Transcription from P alpha proceeds in the direction of the cI repressor gene from sites 46 and 48 bp preceding the PRM transcription start site. P alpha is likely to compete with both PR and PRM for formation of open complexes, since it is only active when PR is mutated and can be suppressed by mutations that increase PRM activity. In addition, transcription initiation at P alpha is blocked by lambda repressor. Kinetic analysis of relative abundance of the products of in vitro transcription indicated that P alpha was approximately 1/3 as strong as PRM. However, a P alpha mutation had little effect on KBkf (the association rate constant) for PRM. These observations can be explained by the finding that open complexes formed at P alpha are relatively unstable (half-life = 20 to 25 min). Dissociation of RNA polymerase from P alpha allows additional open complexes to form at PR or PRM, and thus the apparent strength of P alpha decreases with increasing preincubation times.

Functional cholecystokinin receptors are distinguished kinetically by biotinyl-Tyr-Gly-(Thr28,Nle31)CCK(25–33) in rat pancreatic acini

Biotinyl-tyrosine-glycine(Thr28,Nle31)CCK(25-33) (BTG-TN-CCK-9) promoted amylase secretion and phosphatidylinositol (PI) metabolism with the same potency and efficacy as TN-CCK-9 in dispersed rat pancreatic acini. A 1 min preincubation of the ligand with a 20-fold excess of streptavidin completely suppressed this biological activity. On the other hand, amylase secretion and PI metabolism prestimulated with BTG-TN-CCK-9 were blocked within 1-5 min after streptavidin addition. [125I]BTG-TN-CCK-9 bound to high (Kd 0.17 nM) and low (Kd 13 nM) affinity receptors. Its dissociation, in the presence of either streptavidin or TN-CCK-9, showed a rapid component and a slow component. The proportion of tracer dissociating slowly increased with increasing preincubation time as did the proportion of tracer that could not be washed away quickly by acidic treatment, in parallel experiments. This phenomenon occurred less readily at 4 degrees C or in the presence of 1 mM CCCP. In acini preincubated for 30 min with 0.3 nM [125I]BTG-TN-CCK-9 and various concentrations of unlabelled BTG-TN-CCK-9, then washed at neutral pH (in order to eliminate rapidly dissociating ligand preferentially), the tracer displacement curve was shifted leftward, suggesting that rapidly dissociating receptors corresponded to low affinity receptors. When acini were preincubated for 1 min with BTG-TN-CCK-9, then washed at neutral pH with buffer only, we observed residual stimulated secretion over the next 30 min period, that correlated with the BTG-TN-CCK-9 concentration offered during the short preincubation period. This phenomenon was inhibited by streptavidin suggesting that intracellularly accumulated intact BTG-TN-CCK-9 (as shown, by radio-HPLC) promoted residual secretion when free to bind again to cell surface receptors in the absence of streptavidin. Taken collectively, these data suggest the coexistence of at least 2 types (or states) of CCK receptors.

Phosphatidic acid and polyphosphoinositide formation in a broken cell preparation from rat brain: Effects of different incubation conditions

The formation of phosphatidic acid (PA) and polyphosphoinositides in rat brains in vitro was studied under hypotonic conditions by incubating a lysed crude synaptosomal fraction with [?-(32)P]ATP for 10 s. Only phosphatidic acid, phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP(2)) were labelled. The formation of PIP(2) decreased with increasing preincubation times. PIP and PA formation, however, were not affected. The rate of phosphorylation of PIP, PIP(2) and PA was linear with incubation time for at least 10 s, indicating that the ATP/enzyme ratio was sufficiently high. The maximal incorporation of phosphate into the three phospholipids was found at 30 degrees C. Ten millimolar Mg(2+) and pH 6.5 were the best compromise for measuring the incorporation of phosphate into PIP, PIP(2) and PA simultaneously. A sodium acetate-magnesium acetate buffer system gave optimal kinase activities. The results obtained show that, with minor adjustments of the incubation conditions, it is possible to measure simultaneously the formation of PIP, PIP(2) and PA.

Action of botulinum neurotoxin on acetylcholine release from rat brain synaptosomes: putative internalization of the toxin into synaptosomes.

The action of botulinum neurotoxin type C1 on the release of acetylcholine from rat brain synaptosomes was studied by using anti-toxin heavy chain Fab and anti-toxin light chain Fab. The toxin was bound to synaptosomes at 0 degrees C for 10 min, in which [14C]acetylcholine had been accumulated previously. The toxin-binding synaptosomes were pre-incubated at 37 degrees C, and the release of acetylcholine was determined after the synaptosomes had been incubated in 25 mM KCl-incubation medium for 20 min at 37 degrees C. Inhibition of [14C]acetylcholine release from the synaptosomes was observed with increasing pre-incubation time and toxin concentration, and the maximum inhibition was seen after pre-incubation for at least 15 min, which was called the "lag time." The toxin-binding synaptosomes were reacted with anti-toxin heavy chain and anti-toxin light chain Fabs at 0 degrees C for 1.5 min before pre-incubation of the synaptosomes at 37 degrees C. Both Fabs reversed the acetylcholine release inhibition by the toxin. However, when the Fabs were added during the pre-incubation time at 37 degrees C, they showed less restoration with increasing pre-incubation time. The restoration was completely abolished if the Fabs were added to the synaptosomes after the first half of the "lag time." On the other hand, when 125I-labeled toxin-binding synaptosomes were reacted with the Fabs at 0 degrees C for 1.5 min before pre-incubation of the synaptosomes at 37 degrees C, anti-heavy chain Fab removed 125I-toxin from the synaptosomes, but anti-light chain Fab did not. However, if the Fabs were added to toxin-binding synaptosomes during the pre-incubation time at 37 degrees C, the Fabs could not remove 125I-toxin from the synaptosomes, and the synaptosomes retained more labeled toxin with increasing pre-incubation time. These results suggest that there are three distinct steps in the inhibition of acetylcholine release from synaptosomes by botulinum neurotoxin. The first is binding, which is reversible, temperature-independent, and mediated by the heavy chain of the toxin. The second is temperature-dependent internalization, that takes place in the first half of the "lag time," in which both the chains are internalized into synaptosomes. The third is the development of toxicity, which requires the latter half of the "lag time."