Quantitative determination of the soluble O antigen of Salmonella serogroup B and of specific antibodies in different classes by using immunoenzyme analysis

The results of measurements of S. typhimurium O-antigen and specific IgA-, IgG- and IgM-antibodies in 115 serum samples from patients with salmonellosis induced by group B salmonellae are analyzed. As determined in this study, the concentrations of IgA-antibodies ranged 0-15 micrograms/ml, the minimal diagnostically significant value being 3.6 micrograms/ml; the concentrations of IgG-antibodies ranged 0-13 micrograms/ml, the minimal diagnostically significant value being 1.3 micrograms/ml; and the concentrations of IgM-antibodies ranged 0-50 microgram/ml, the minimal diagnostically significant value being 5.0 micrograms/ml. The minimal diagnostically significant concentration of O-antigen, determined in selected serum samples obtained from healthy donors, was 0.1 microgram/ml. There is a significant correlation between the concentrations of IgA-, IgG- and IgM-antibodies, but the concentrations of these antibodies do not correlate with the concentration of soluble O-antigen. The study showed that the simultaneous determination of S. typhimurium O-antigen and specific antibodies in the material under test significantly enhances the effectiveness of the serodiagnosis of the disease.     

Quantitative determination of the soluble antigens of intestinal bacteria using a method of immunoenzyme analysis. I. Processing of the parameters of the method

The parameters of the assay based on the quantitative evaluation of the neutralization of specific antibodies by the antigen under study and the subsequent detection of free antibodies on the fixed reference antigen with the aim of the quantitative determination of the specific O-antigens of Salmonella, groups D, B, C1, as well as those of Shigella sonnei and Shigella flexneri, have been developed. The proposed method makes it possible to detect the O-antigen of the causative agent at concentrations of 0.001 micrograms/ml to 100 micrograms/ml.     

The determination of the antibodies and the somatic O-antigen of Salmonella group B by using the complement-bound lysis of liposomes]

A simple and sensitive method for the determination of group B Salmonella O-antigen and specific antibodies to group B Salmonella by means of the complement-dependent lysis of liposomes sensitized with S. typhimurium lipopolysaccharide (LPS) is proposed. The factors affecting the sensitivity of the method during the determination of antibodies and free antigen have been studied. The method permits the determination of soluble LPS antigen in concentrations of 0.5-200 micrograms/ml.     

Effect of dimethyl sulphoxide and some antibiotics on cultured human T-lymphoma cells as measured by microcalorimetry

The influence of dimethyl sulphoxide (I), penicillin/streptomycin (II), gentamicin (III), and amphotericin B (IV) on growing human T-lymphoma cells was measured by microcalorimetry. There was a dose-dependent decrease in the heat production rate of the cells after 24 h of incubation with I in concentrations ranging from 0-2% (v/v). At 3.6%, about half of the cells died. II and III had no effect on the cells after incubation for 6 days, at concentrations from 1 to 10 times that of the normal (50-500 IU/ml; 50-500 micrograms/ml). IV was used in combination with II (50 IU/ml; 50 micrograms/ml) and III (50 micrograms/ml), respectively, at concentrations between 0.25 and 7.5 micrograms/ml. After 6 days of incubation, the results were similar to those obtained with II and III separately.     

Immune response and immune tolerance to the O-antigen of Shigella flexneri VI

The method for the determination of the number of cells synthetizing antibodies to S. flexneri VI O-antigen in the spleen of mice has been developed. Primary immune response to this antigen has been studied with the use of the new method. Immune response to the optimum immunogenic dose of O-antigen has a manifest variable character. The intensity of primary immune response has been shown to rise with the increase of the dose of O-antigen from 0.004 to 50 micrograms. The preliminary injection of 200 micrograms of O-antigen, followed by the injection of cyclophosphamide 2 days later, leads to the development of specific immunological tolerance to O-antigen in experimental animals.     

Sex difference in the saturable binding of low-density lipoprotein by liver membranes in ageing rats

It is well documented that women of child-bearing age tend to have lower serum low-density lipoprotein (LDL) concentrations than men. In order to explore the metabolic basis of this sex difference, we have compared the saturable binding of 125I-labeled LDL (d 1.02-1.05 g/ml) at 37 degrees C by liver membranes from healthy male and female Wistar rats of different ages (15-213 days). Woolf plots of saturable binding curves over the concentration range 15-65 micrograms LDL protein/ml were linear and compatible with a single class of binding sites. Maximum binding capacity (Bmax) was not significantly different in male and female animals of 15-19 days of age (respectively, 0.331 +/- 0.018 vs. 0.427 +/- 0.044 micrograms LDL protein/mg membrane protein, mean +/- S.E.). Thereafter, Bmax increased in females, reaching a peak of 0.635 +/- 0.042 micrograms LDL protein/mg membrane protein at 60 days. As no increase in Bmax occurred in males, values were significantly higher (P less than 0.02) in females than in males (by a mean of 61-117%) at all ages after 30 days. During ageing, serum cholesterol concentration changed reciprocally with Bmax in females (Pearson's correlation coefficient, r = -0.761, P less than 0.01) and remained essentially constant in males. The equilibrium dissociation constant for 125I-labelled LDL binding to the hepatic membranes was unaffected by both age and sex. These results provide evidence that the sex difference in the plasma total and LDL cholesterol concentrations is related, at least in part, to a greater mean LDL receptor density in the livers of females.     

Induction of heat shock proteins in Chinese hamster ovary cells and development of thermotolerance by intermediate concentrations of puromycin

During 4 hr after puromycin (PUR: 20 micrograms/ml) treatment, the synthesis of three major heat shock protein families (HSPs: Mr = 110,000, 87,000, and 70,000) was enhanced 1.5-fold relative to that of untreated cells, as studied by one-dimensional gel electrophoresis. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed at 10(-3) isosurvival as a thermotolerance ratio (TTR) of either 2 or greater than 5 after heating at either 45.5 degrees C or 43 degrees C, respectively. However, thermotolerance was induced by only intermediate concentrations (3-30 micrograms/ml) of puromycin that inhibited protein synthesis by 15-80%; a high concentration of PUR (100 micrograms/ml) that inhibited protein synthesis by 95% did not induce either HSPs or thermotolerance. Also, thermotolerance was never induced by any concentration (0.01-10 micrograms/ml) of cycloheximide that inhibited protein synthesis by 5-94%. Furthermore, after PUR (20 micrograms/ml) treatment, the addition of cycloheximide (CHM: 10 micrograms/ml), at a concentration that reduces protein synthesis by 94%, inhibited both thermotolerance and synthesis of HSP families. Thus, thermotolerance induced by intermediate concentrations of PUR correlated with an increase in newly synthesized HSP families. This thermotolerance phenomenon was compared with another phenomenon termed heat resistance and observed when cells were heated at 43 degrees C in the presence of CHM or PUR immediately after a 2-hr pretreatment with CHM or PUR. Heat protection increased with inhibition of synthesis of both total protein and HSP families. Moreover, this heat protection decayed rapidly as the interval between pretreatment and heating increased to 1-2 hr, and did not have any obvious relationship to the synthesis of HSP families. Therefore, there are two distinctly different pathways for developing thermal resistance. The first is thermotolerance after intermediate concentrations of PUR treatment, and it requires incubation after treatment and apparently the synthesis of HSP families. The second is resistance to heat after CHM or PUR treatment immediately before and during heating at 43 degrees C, and it apparently does not require synthesis of HSP families. This second pathway not requiring the synthesis of HSP families also was observed by the increase in thermotolerance at 45.5 degrees C caused by heating at 43 degrees C after cells were incubated for 2-4 hr following pretreatment with an intermediate concentration of PUR.     

Activation of purified cytoplasmic 17 beta-hydroxysteroid dehydrogenase from human placenta by human albumin and globulin

The cytoplasmic 17 beta-hydroxysteroid dehydrogenase of human placenta, purified more than 2500-fold, was activated by small amounts of human albumin and globulin. This activation was dependent on substrate concentration. At 20 microM estradiol (10 X KM) and two different concentrations of enzyme (0.01 and 2 micrograms/ml), the activation was greatest at albumin or globulin concentrations between 0 and 30 micrograms/ml. At "low" concentrations of estradiol (20 nM = 10(-2) X KM) and enzyme (0.01 microgram/ml), maximal activity occurred at approximately 10 micrograms/ml. Higher concentrations of albumin and globulin led to a decline in activity.     

Indian red scorpion venom modulates spontaneous activity of rat right atria through the involvement of cholinergic and adrenergic systems.

The effect of Indian red scorpion (Mesobuthus tamulus concanesis, Pocock; MBT) venom was investigated on isolated rat right atrial preparations. MBT venom (0.001-3.0 micrograms/ml) exhibited a peculiar concentration-response pattern with respect to rate. The venom concentrations between 0.001-0.01 microgram/ml increased the atrial rate (phase I), followed by a relative decrease with 0.03-0.3 microgram/ml (phase II), and then an abrupt increase with 0.6-3.0 micrograms/ml (phase III). On the other hand, the force was unaltered by venom at phases I and II, while an increase was seen at phase III (3.0 micrograms/ml). Propranolol (0.1 microM) completely blocked the cardiostimulant action of venom at phase III. Further, this stimulant action of venom was absent in atria obtained from reserpinized animals. Pretreatment with atropine (0.3 microM), produced tachycardia at concentrations 0.1-0.3 microgram/ml of venom. But, hexamethonium (30 microM) had no influence on the venom (0.1 microgram/ml)-induced alterations in rate. However, MBT venom increased the acetylcholinesterase (AChE) activity (2-3 fold) in a concentration-dependent manner. Tetrodotoxin (2 microM), did not block the increase in rate produced by 0.01 microgram/ml of venom. Results suggest that, MBT venom-induced alterations of cardiac rhythmicity are mediated through cholinergic as well as adrenergic mechanisms depending upon the concentrations. The modulation of atrial rate at very low concentrations may be due to the direct action of venom on the atrium.     

Induction of luteal regression in the marmoset monkey (Callithrix jacchus) by a gonadotrophin-releasing hormone antagonist and the effects on subsequent follicular development

Doses of 100 or 200 micrograms of a novel GnRH antagonist ([N-acetyl-D beta Na11-D-pCl-Phe2-D-Phe3-D-Arg6-Phe7-Arg8-D-Ala10]NH2 GnRH) (4 animals/dose) were administered on Days 10/11 of the luteal phase and induced a marked suppression of circulating bioactive LH and progesterone concentrations within 1 day of treatment (P less than 0.01). Thereafter, progesterone concentrations remained low or undetectable until after the next ovulation. Similar results were obtained when 200 micrograms antagonist were given on Days 5/6 of the luteal phase (N = 4). The interval from injection of antagonist (200 micrograms but not 100 micrograms) to ovulation (based on a rise in progesterone above 10 ng/ml) was significantly longer than that from prostaglandin-induced luteal regression to ovulation in control cycles (N = 4/treatment) (range, 13-15 days after antagonist vs 8-10 days after prostaglandin, P less than 0.01). This delay of 4-5 days was equivalent to the duration for which LH concentrations were significantly suppressed by 200 micrograms antagonist when administered to ovariectomized animals (N = 3). Corpus luteum function during the cycle after GnRH antagonist treatment appeared normal according to the pattern of circulating progesterone. These results show that corpus luteum function and preovulatory follicular development in the marmoset monkey are dependent on pituitary gonadotrophin secretion.     

Secondary immune response to flagellin conjugates with polyacrylic acid

The conditions of the activation of immunological memory by means of flagellin and its conjugate with polyacrylic acid (PAA) have been studied. The flagellin-PAA conjugate has proved to be capable of inducing the appearance of memory cells when introduced in doses, considerably lower (0.01 and 0.001 micrograms) than those of native protein (0.1, 1 and 10 micrograms). At the same time no manifest differences in the reactivation of flagellin-induced and conjugate-induced memory cells by the antigen have been established. Immunization with protein, as well as with its conjugate, has been found to induce the formation of mainly anti-Hi : 1,2 IgG in secondary immune response.     

Interaction of vasopressin and prostaglandins in the nonpregnant human uterus

The interaction of vasopressin (VP) and prostaglandins (PG) on the nonpregnant human uterus was studied in vitro and in vivo. In organ baths arginine (A)- and lysine (L)-VP in concentrations of 0.6 to 100 ng/ml stimulated small human myometrial strips and uterine artery preparations to a similar degree. When these VPs were given in the presence of indomethacin or naproxen in concentrations of 1 microgram/ml and 5 micrograms/ml, respectively, the myometrial and arterial responses were not significantly influenced. PGF2 alpha in concentrations of 0.01-100 ng/ml stimulated the myometrial preparations but caused a slight relaxation of the arteries, with PGE2 the myometrial effects were insignificant and the relaxation of the arteries greater. When AVP was given together with either of the PGs to the bath the result was generally a summation of the individual effects of both types of substances.--In vivo during intrauterine pressure recordings in nonpregnant women 1-2 days before onset of menstruation LVP in single intravenous injection of 1.2 micrograms markedly stimulated uterine contractions. The response remained practically unaltered after pretreatment with 500 micrograms of naproxen given orally. The responses to LVP were also closely similar before, during and after intravenous infusion of PGF2 alpha at a rate of 5 micrograms/min.--It is concluded that the effect of VP on myometrium and uterine arteries is not to any great extent mediated by local synthesis of PG and that PGs do not cause potentiation or inhibition of the VP effects on the nonpregnant uterus.     

Development of preovulatory follicles expected to form short-lived corpora lutea in beef cows

Oestrus, expected to be followed by a short luteal phase, was induced in post-partum cows by weaning their calves at 35 days after parturition. Ovaries containing the first preovulatory follicles (Type F) formed after parturition were collected 3 h after the onset of oestrus. For comparison, preovulatory follicles (Type C) were collected 3 h after the onset of oestrus in normally cycling cows. The number of granulosa cells was determined and the concentrations of receptors for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in granulosa cells and for LH in theca cells were measured. Concentrations of oestradiol-17 beta, testosterone, androstenedione and progesterone in follicular fluid were also measured. Type F follicles contained about twice the number of granulosa cells (based on DNA) as did Type C follicles (45.8 +/- 11.3 and 24.5 +/- 3.9 micrograms DNA/follicle, respectively; P less than 0.05) but these cells had fewer receptors for LH (0.13 +/- 0.02 vs 0.29 +/- 0.03 fmol/micrograms DNA; P less than 0.01) and FSH (0.61 +/- 0.08 vs 1.3 +/- 0.29 fmol/micrograms DNA; P less than 0.08) than did those from Type C follicles. Additionally, there were fewer receptors for LH in theca tissue from Type F than from Type C follicles (28.3 +/- 5.2 vs 51.3 +/- 6.1 fmol/follicle; P less than 0.01). Concentrations of oestradiol-17 beta (475.8 +/- 85.6 vs 112.9 +/- 40.0 ng/ml; P less than 0.01) and androstenedione (214.1 +/- 48.7 vs 24.7 +/- 7.7 ng/ml; P less than 0.01) in follicular fluid were higher in Type C than in Type F follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

An enzyme-linked immunosorbent assay (ELISA) of denatured cartilage link protein

Antiserum (MB007) was raised in rabbits to SDS-denatured cartilage link protein in order to develop an enzyme-linked immunosorbent assay (ELISA) to quantify link protein in cartilage extracts. The antibodies were characterized by using native and denatured link protein, either as the immobilised or the inhibiting antigen in the assay, and shown to bind more effectively to denatured link protein. At low concentrations, neither hyaluronate (0-30 micrograms/ml), proteoglycan (0-50 micrograms/ml) nor hyaluronate-binding region (0-3 micrograms/ml) competitively inhibited the link protein assay. However, at higher concentrations of proteoglycan (50 micrograms/ml-4 mg/ml) and hyaluronate-binding region (3-40 micrograms/ml) inhibition was observed. A more highly purified proteoglycan and a further purified hyaluronate-binding region preparation showed identical behaviour. The inhibition produced by proteoglycan and hyaluronate-binding region occurred at approximately equivalent molar concentrations (assuming Mr of 10(6) and 7 X 10(4), respectively). These results suggest that a significant proportion of these polyclonal antibodies recognize an epitope common to link protein and hyaluronate-binding region. However, the possibility that these effects are due to contamination with covalently bound link protein cannot be excluded. Trypsinated aggregates (0-10 micrograms/ml) produced no inhibition in the link protein ELISA, but as higher concentrations the inhibition was approximately 2000-fold lower than might have been expected from the link protein concentration present. Thus, the accessibility and/or binding of the antibodies to link protein was substantially decreased, illustrating masking of the link protein antigenic sites, as found by A. Ratcliffe and T.E. Hardingham (Biochem. J. 213 (1983) 371-378). These studies indicate that link protein in tissue extracts may be quantified in the concentration range 30-200 ng/ml and in the presence of hyaluronate, proteoglycan and hyaluronate-binding region, provided that both the immobilised and extracted link proteins are denatured.     

Impaired secretion and increased insolubilization of IgE-receptor complexes in mycophenolic acid-treated (guanine nucleotide-depleted) RBL-2H3 mast cells.

In RBL-2H3 rat leukemic mast cells, cross-linking anti-DNP IgE-receptor complexes with multivalent antigen (DNP-BSA) activates a signal transduction pathway leading to Ca2+ influx and secretion. Cross-linking IgE-receptor complexes also stimulates a pathway that inactivates (desensitizes) receptors; this pathway becomes important at high concentrations of cross-linking antigen. Recent evidence that antigen-induced secretion is impaired by mycophenolic acid (MPA), an inhibitor of guanine nucleotide synthesis de novo, has implicated a GTP-binding protein (G protein) in the signaling pathway. Other recent studies have indicated that the conversion of cross-linked receptors to a detergent-insoluble (cytoskeleton-associated) form at high antigen concentrations is correlated with the loss of signaling activity. Here we show that secretion elicited by an optimal concentration of antigen (0.05 micrograms/ml DNP-BSA) is only inhibited by about 25% in guanine nucleotide-depleted cells, whereas secretion elicited by 5 micrograms/ml DNP-BSA, a concentration in the range that causes the high-dose inhibition of secretion, is inhibited by more than 60%. We also show that IgE-receptor complexes are insolubilized in response to 5 but not 0.05 micrograms/ml DNP-BSA in both control and guanine nucleotide-depleted cells. Importantly, the extent of insolubilization elicited by 5 micrograms/ml DNP-BSA is increased by more than 60% in the guanine nucleotide-depleted samples. These results raise the possibility that guanine nucleotide depletion reduces the secretory response to high antigen concentrations in two ways: by inhibiting the G protein-coupled signaling pathway and by increasing the availability of receptors to the pathway leading to receptor insolubilization and inactivation.     

Effects of synthetic ovine corticotropin-releasing factor (CRF) on plasma ACTH and cortisol in 31 normal human males

Responses of plasma ACTH and cortisol to corticotropin-releasing factor (CRF) were evaluated in 31 normal human males. 1.0 micrograms/ks of sterilized synthetic ovine CRF was administered to the subjects, aged 19 to 53 yr and weighing 50 to 78 kg, at between 9:30 a.m. and 10:30 a.m. as an intravenous bolus injection after an overnight fast. Blood specimens were drawn before and 15, 30, 60, 90 and 120 min after injection for later determination of plasma ACTH and cortisol concentrations by radioimmunoassays. Plasma ACTH and cortisol levels for all subjects rose significantly (p less than 0.001) from the basal level (mean +/- SEM, 26.8 +/- 4.5 pg/ml and 12.6 +/- 0.9 micrograms/dl) to peak levels (58.4 +/- 5.5 pg/ml and 22.9 +/- 1.0 micrograms/dl) at 30 min and at 60 min, respectively. Although the plasma concentrations of ACTH and cortisol thereafter declined gradually, the levels at 120 min (43.4 +/- 5.2 pg/ml and 18.9 +/- 0.9 micrograms/ml, respectively) were still significantly higher than the basal levels (p less than 0.001). Significant inverse correlations were observed between the basal levels of each hormone and the ratio of the peak level to the basal level (p less than 0.01), and the increases in plasma ACTH and cortisol concentrations were either not significant or much smaller for the individuals in whom the basal levels were higher than 65 pg/ml and 17.0 micrograms/dl, respectively. No serious subjective symptom was observed during the experimental period in any of the subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

The inter- and intra-subject variabilities of plasma GH response to human growth hormone-releasing hormone (1-44) NH2 in men

The effects of intravenously given human growth hormone-releasing hormone (1-44) NH2 (hGRH-44) on growth hormone (GH) secretion were studied in normal men. A wide variability of intersubject GH response to hGRH-44 was observed. The peak plasma GH levels in response to 50, 100 and 200 micrograms hGRH-44 in 7 normal men were 9.1 +/- 3.2 ng/ml (Mean + SEM), 19.3 +/- 3.3 ng/ml and 22.4 +/- 4.0 ng/ml, respectively. Both the mean peak values for plasma GH response to 100 and 200 micrograms were significantly greater than that for 50 micrograms hGRH-44 injection (p less than 0.01), although there was no significant difference of the mean peak plasma GH values and mean concentrations at each time point, except for those at 120 min, when 100 or 200 micrograms hGRH-44 was administered. A significant difference in the mean amount of plasma GH secreted in response to hGRH-44 was observed only between 50 and 200 micrograms hGRH-44 injection (p less than 0.01). Furthermore, a dose-related plasma GH increase in response to hGRH-44 was not always observed in each subject. In contrast to the wide intersubject variability, the difference among responses of plasma GH to 100 micrograms or 200 micrograms of hGRH-44 given at multiple times separated by intervals of at least 1 week in each individual was relatively small.(ABSTRACT TRUNCATED AT 400 WORDS)     

A suppression of gonadotropin secretion by cortisol in castrated male rhesus monkeys (Macaca mulatta) mediated by the interruption of hypothalamic gonadotropin-releasing hormone release

Four orchidectomized rhesus monkeys (3-3.5 yr of age) were treated for 62 days with daily i.m. injections of hydrocortisone acetate (HCA) at a dose of 10-20 mg/(kg BW X day), and blood samples were obtained daily or every other day before, during, and after treatment. Hydrocortisone acetate injections resulted in a progressive rise in mean plasma cortisol from basal concentrations of 17-35 micrograms/100 ml prior to initiation of steroid treatment to approximately 150 micrograms/100 ml 5 wk later. When serum cortisol concentrations reached 100 micrograms/100 ml, 3-4 wk after the initiation of HCA treatment, circulating luteinizing hormone (LH) and follicle-stimulating hormone (FSH) began to decline, reaching nondetectable concentrations 35 days later. Withdrawal of HCA resulted in a return in plasma cortisol concentrations to pretreatment control levels, which was associated with a complete restoration of gonadotropin secretion. In 2 animals, administration of an intermittent i.v. infusion of gonadotropin-releasing hormone (GnRH) (0.1 micrograms/min for 3 min once every hour), which appears to stimulate the gonadotropes in a physiologic manner, reversed the cortisol-induced inhibition of gonadotropin secretion, restoring circulating LH and FSH concentrations to within 80-100% of control. These results suggest that, in the rhesus monkey, the major site of the inhibitory action of cortisol on gonadotropin release resides at a suprapituitary level and is mediated by interruption of hypothalamic GnRH release.     

Bryostatin enhancement of memory in Hermissenda

Bryostatin, a potent agonist of protein kinase C (PKC), when administered to Hermissenda was found to affect acquisition of an associative learning paradigm. Low bryostatin concentrations (0.1 to 0.5 ng/ml) enhanced memory acquisition, while concentrations higher than 1.0 ng/ml down-regulated the pathway and no recall of the associative training was exhibited. The extent of enhancement depended upon the conditioning regime used and the memory stage normally fostered by that regime. The effects of two training events (TEs) with paired conditioned and unconditioned stimuli, which standardly evoked only short-term memory (STM) lasting 7 min, were--when bryostatin was added concurrently--enhanced to a long-term memory (LTM) that lasted about 20 h. The effects of both 4- and 6-paired TEs (which by themselves did not generate LTM), were also enhanced by bryostatin to induce a consolidated memory (CM) that lasted at least 5 days. The standard positive 9-TE regime typically produced a CM lasting at least 6 days. Low concentrations of bryostatin (<0.5 ng/ml) elicited no demonstrable enhancement of CM from 9-TEs. However, animals exposed to bryostatin concentrations higher than 1.0 ng/ml exhibited no behavioral learning. Sharp-electrode intracellular recordings of type-B photoreceptors in the eyes from animals conditioned in vivo with bryostatin revealed changes in input resistance and an enhanced long-lasting depolarization (LLD) in response to light. Likewise, quantitative immunocytochemical measurements using an antibody specific for the PKC-activated Ca2+/GTP-binding protein calexcitin showed enhanced antibody labeling with bryostatin. Animals exposed to the PKC inhibitor bisindolylmaleimide-XI (Ro-32-0432) administered by immersion prior to 9-TE conditioning showed no training-induced changes with or without bryostatin exposure. However, if animals received bryostatin before Ro-32, the enhanced acquisition and demonstrated recall still occurred. Therefore, pathways responsible for the enhancement effects induced by bryostatin were putatively mediated by PKC. Overall, the data indicated that PKC activation occurred and calexcitin levels were raised during the acquisition phases of associative conditioning and memory initiation, and subsequently returned to baseline levels within 24 and 48 h, respectively. Therefore, the protracted recall measured by the testing regime used was probably due to bryostatin-induced changes during the acquisition and facilitated storage of memory, and not necessarily to enhanced recall of the stored memory when tested many days after training.