Absence Of Cytochalasin B Research Articles

Developmental competence of ovine oocyte following vitrification: effect of oocyte developmental stage, cumulus cells, cytoskeleton stabiliser, FBS concentration, and equilibration time

The aim of the present study was to examine the effects of fetal bovine serum (FBS) concentration, equilibration time, and oocyte pre-treatment with cytochalasin B (CCB) on subsequent development of vitrified-warmed ovine immature (GVCOCs) and matured (MII) oocytes with (MIICOCs) or without cumulus cells (MIIDOs). In Experiment 1, the effects of FBS concentrations (10 and 20%) during the vitrification-warming procedure were examined. Survival rates after warming were not different between GVCOCs, MIICOCs and MIIDOs oocytes. After in vitro fertilization, rate of cleaved embryos in MIICOCs group at the presence of 20%FBS was higher than MIIDOs and GVCOCs groups. In Experiment 2, the effects of equilibration times (5, 7, and 10 min) were examined. There was no difference in survival rate of vitrified-warmed oocytes equilibrated at different times. Although, the rate of cleavage in MIICOCs and MIIDOs oocytes equilibrated for 10 and 7 min, respectively, was higher than 5 min equilibrated MIIDOs and 7 and 10 min equilibrated GVCOCs oocytes. In Experiment 3, the effects of oocyte pre-treatment with CCB were examined. Despite the insignificant difference in survival rate of vitrified-warmed ovine immature and matured oocytes, the rates of cleavage in CCB pretreated groups were significantly lower than untreated groups. Moreover, the blastocysts were only derived from those cumulus enclosed vitrified-warmed germinal vesicle (GV) and MII oocytes that had been exposed to 10% FBS in the absence of CCB. In conclusion, the presence of cumulus cells, 10% FBS, and the omission of CCB were beneficial for post-warming development of vitrified ovine oocytes.

240 EFFECTS OF TIMING AND DIPLOIDIZATION ON ACTIVATION AND DEVELOPMENT OF RAT OOCYTES

The objectives of these studies were (1) to compare the effects of Cytochalasin B (CB) or 6-dimethyl amino purine (DMAP) in combination with SrCl2 on activation and development of rat oocytes and (2) to determine the optimal timing for collection and activation of rat oocytes following superovulation. In Experiment 1, rat oocytes collected at 16–18 h after hCG injection were immediately cultured in 10 mm SrCl2 alone, 10 mm SrCl2 + 2 mm DMAP for 4 h and 10 mm SrCl2 + 5 μg mL–1 CB for 5–6 h, respectively, they were then cultured in KSOM for 20 h and finally cultured in R1ECM for an additional 4 days, all at 37°C, 5% CO2 in air. There were no significant differences in pronuclear formation (SrCl2 alone: 51/53; DMAP: 139/140; CB: 139/140) and cleavage rate (SrCl2 alone: 52/53; DMAP: 129/140; CB: 130/140) observed among three groups (χ2-test, P > 0.05). On Day 5 the frequency of development to blastocyst was not statistically different (P > 0.05) between DMAP (55.71%, 78/140) and CB (54.23%, 76/140) treated groups; however, no blastocysts were produced in 10 mm SrCl2 alone. In Experiment 2, oocytes were collected at 14, 16, 18, 20 and 22 hours after hCG injections, respectively, and they were immediately cultured in 10 mm SrCl2 + 2 mm DMAP for 4 h and then cultured in the same conditions described for Experiment 1. Although development to blastocyst was slightly lower in the 14-h group (43.50%, 47/108), no significant differences in the frequencies of pronuclear formation, cleavage on Day 1 or development to blastocyst on Day 5 were observed between groups (χ2-test, P > 0.05) (Table 1). All experiments were repeated at least three times. In conclusion, our results showed that SrCl2 combined with either CB or DMAP could equally, effectively activate rat oocytes. In contrast rat haploid oocytes activated in the absence of CB or DMAP were not able to develop to the blastocyst stage. Furthermore, rat oocytes collected between 14 and 22 h after hCG injection had an equal potential to develop to the blastocyst stage after activation by 10 mm SrCl2 + 2 mm DMAP. Table 1.Development of rat oocytes activated at different time points following

Parthenogenetic activation of mouse oocytes by strontium chloride: A search for the best conditions

Strontium has been successfully used to induce activation of mouse oocytes in nuclear transfer and other experiments, but the optimum treatment conditions have not been studied systematically. When cumulus-free oocytes were treated with 10 mM SrCl 2 for 0.5–5 h, activation rates (88.4 ± 4.1 to 91.2 ± 2.7%) did not differ (mean ± S.E.; P > 0.2), but rate of blastulation (57.3 ± 3.5%) and cell number per blastocyst (45.0 ± 2.4) were the highest after treatment for 2.5 h. When treated with 1–20 mM SrCl 2 for 2.5 h, the activation rate and cell number per blastocyst were higher ( P < 0.02) after 10 mM SrCl 2 treatment than other treatments. The best activation and development were obtained with Ca 2+–free Sr 2+ medium, but the activation rate was low (37.7 ± 1.6%) in Ca 2+-containing medium. Activation rates were the same, regardless of the presence or absence of cytochalasin B (CB) in the activating medium, but the blastulation rate was higher ( P < 0.001) in the presence of CB. Only 70% of the cumulus-enclosed oocytes were activated and 10% blastulated after a 10 min exposure to 1.6 mM SrCl 2, and many lysed, with increased intensity of Sr 2+ treatment. The presence of CB in SrCl 2 medium markedly reduced lysis of cumulus-enclosed oocytes. Media M16 and CZB did not differ when used as activating media. Only 10.5% of the oocytes collected 13 h post hCG were activated by Sr 2+ treatment alone, with 34% blastulating, but rates of activation and blastulation increased ( P < 0.001) to 94 and 60%, respectively, when they were further treated with 6-dimethylaminopurine (6-DMAP). The total and ICM cell numbers were less ( P < 0.001) in parthenotes than in the in vivo fertilized embryos. In conclusion, the concentration and duration of SrCl 2 treatment and the presence or absence of CB in activating medium and cumulus cells had marked effects on mouse oocyte activation and development. To obtain the best activation and development, cumulus-free oocytes collected 18 h post hCG should be treated for 2.5 h with 10 mM SrCl 2 in Ca 2+-free medium supplemented with 5 μg/mL of CB.

Monosodium urate-crystal-stimulated phospholipase D in human neutrophils

Protein kinase Calpha (PKCalpha) and small GTPases of the Rho and ADP-ribosylation factor (Arf) family are implicated in the regulation of phospholipase D1 (PLD1) activity. Although they are involved in fMet-Leu-Phe (fMLP)-mediated PLD activation, their role in monosodium urate (MSU)-stimulated PLD1 activity in human neutrophils is not clear. The translocation of PKCalpha, RhoA and Arf from the cytosol to the membranes was monitored. fMLP induced a cytochalasin B (CB)-dependent recruitment of Arf, RhoA and PKCalpha to neutrophil membranes. CB also increased the activation of PLD 10-fold. In contrast with fMLP, MSU stimulated a sustained and time-dependent relocalization of Arf and PKCalpha, but not of RhoA, to the membrane fraction. MSU-stimulated PLD was activated with a time course preceding membrane recruitment of Arf and PKCalpha in the absence of CB. Furthermore, MSU-induced PLD activation and the membrane recruitment of PKCalpha, but not that of Arf, were inhibited by CB. An anti-FcgammaRIIIB antibody, VIFcRIII, prevented the membrane relocalization of Arf and PKCalpha and the stimulation of the levels of tyrosine phosphorylation and of PLD activity induced by MSU. Erbstatin and ST-638, two inhibitors of tyrosine kinases, inhibited the MSU-induced translocation of Arf and PKCalpha but not MSU-induced tyrosine phosphorylation and PLD activation. Furthermore MSU crystals did not cause the tyrosine phosphorylation of PLD1. The present study indicates that soluble and particulate agonists show selectivity in inducing the translocation of RhoA in neutrophils and that the ability of MSU to increase PLD activation was independent of the membrane relocalization of Arf and PKCalpha.

Effect of culture media on the development of microinjected murine embryos

Strontium has been successfully used to induce activation of mouse oocytes in nuclear transfer and other experiments, but the optimum treatment conditions have not been studied systematically. When cumulus-free oocytes were treated with 10 mM SrCl2 for 0.5–5 h, activation rates (88.4 ± 4.1 to 91.2 ± 2.7%) did not differ (mean ± S.E.; P > 0.2), but rate of blastulation (57.3 ± 3.5%) and cell number per blastocyst (45.0 ± 2.4) were the highest after treatment for 2.5 h. When treated with 1–20 mM SrCl2 for 2.5 h, the activation rate and cell number per blastocyst were higher (P < 0.02) after 10 mM SrCl2 treatment than other treatments. The best activation and development were obtained with Ca2+–free Sr2+ medium, but the activation rate was low (37.7 ± 1.6%) in Ca2+-containing medium. Activation rates were the same, regardless of the presence or absence of cytochalasin B (CB) in the activating medium, but the blastulation rate was higher (P < 0.001) in the presence of CB. Only 70% of the cumulus-enclosed oocytes were activated and 10% blastulated after a 10 min exposure to 1.6 mM SrCl2, and many lysed, with increased intensity of Sr2+ treatment. The presence of CB in SrCl2 medium markedly reduced lysis of cumulus-enclosed oocytes. Media M16 and CZB did not differ when used as activating media. Only 10.5% of the oocytes collected 13 h post hCG were activated by Sr2+ treatment alone, with 34% blastulating, but rates of activation and blastulation increased (P < 0.001) to 94 and 60%, respectively, when they were further treated with 6-dimethylaminopurine (6-DMAP). The total and ICM cell numbers were less (P < 0.001) in parthenotes than in the in vivo fertilized embryos. In conclusion, the concentration and duration of SrCl2 treatment and the presence or absence of CB in activating medium and cumulus cells had marked effects on mouse oocyte activation and development. To obtain the best activation and development, cumulus-free oocytes collected 18 h post hCG should be treated for 2.5 h with 10 mM SrCl2 in Ca2+-free medium supplemented with 5 μg/mL of CB.

An Inherited Defect of Neutrophil Motility and Microfilamentous Cytoskeleton Associated With Abnormalities in 47-Kd and 89-Kd Proteins

AbstractA 2-month-old male Tongan infant presented with fever, severe skin and mucosal infections, hepatosplenomegaly, thrombocytopenia, and normal neutrophil counts. While polymorphonuclear neutrophil (PMN) morphology was normal, several neutrophil motile functions were found to be altered in the patient. Furthermore, two siblings had died in infancy with a similar clinical picture, raising the possibility of an inherited neutrophil defect. Random migration and chemotaxis, assessed by the under agarose method, were profoundly impaired. Actin polymerization, as measured by flow cytometry of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallaci- din (NBD-phallacidin)-stained PMNs, showed lower basal F-actin and a 1.75-fold increase in response to 10'7 mol/L formyl-methionyl-leucyl-phenylalanine (FMLP) compared with a 4.51-fold increase in control. Microscopic examination of NBD-phallacidin-stained PMN spread on glass showed decreased area of spreading and F-actin-rich filamentous projections distinct from control. The early phase of FMLP- induced right angle light scattering was absent, similar to the effect caused by cytochalasin-B (CB), an inhibitor of actin polymerization. Accordingly, FMLP induced secretion of elastase without the addition of CB. Staphylococcus aureus killing was 50% of control whereas superoxide production in response to FMLP and surface expression of CD11b were greater than twice normal. Partial defects in actin polymerization and scatter were seen in the parents and release of elastase, in the absence of CB, was also increased in both parents. Sodium dodecyl sulfate-polyacrylamide electrophoresis of whole cell proteins from the patient showed a marked decrease in an 89-Kd protein (8% of control) and a marked increase in a 47-Kd protein (4.2-fold). Both mother and father had decreased 89-Kd (77% and 42% of control) and increased 47-Kd proteins (2- and 3.4-fold), although neither had recurrent infections or chemotactic defects. These studies describe a new inherited actin dysfunction syndrome associated with severe propensity to fungal infection and draw attention to the proteins of apparent molecular weights of 89 Kd and 47 Kd, which may be of great importance in the regulation of actin polymerization in human PMNs.

An inherited defect of neutrophil motility and microfilamentous cytoskeleton associated with abnormalities in 47-Kd and 89-Kd proteins

A 2-month-old male Tongan infant presented with fever, severe skin and mucosal infections, hepatosplenomegaly, thrombocytopenia, and normal neutrophil counts. While polymorphonuclear neutrophil (PMN) morphology was normal, several neutrophil motile functions were found to be altered in the patient. Furthermore, two siblings had died in infancy with a similar clinical picture, raising the possibility of an inherited neutrophil defect. Random migration and chemotaxis, assessed by the under agarose method, were profoundly impaired. Actin polymerization, as measured by flow cytometry of N-(7-nitrobenz-2-oxa- 1,3-diazol-4-yl)phallacidin (NBD-phallacidin)-stained PMNs, showed lower basal F-actin and a 1.75-fold increase in response to 10(-7) mol/L formyl-methionyl-leucyl-phenylalanine (FMLP) compared with a 4.51- fold increase in control. Microscopic examination of NBD-phallacidin- stained PMN spread on glass showed decreased area of spreading and F- actin-rich filamentous projections distinct from control. The early phase of FMLP-induced right angle light scattering was absent, similar to the effect caused by cytochalasin-B (CB), an inhibitor of actin polymerization. Accordingly, FMLP induced secretion of elastase without the addition of CB. Staphylococcus aureus killing was 50% of control whereas superoxide production response to FMLP and surface expression of CD11b were greater than twice normal. Partial defects in actin polymerization and scatter were seen in the parents and release of elastase, in the absence of CB, was also increased in both parents. Sodium dodecyl sulfate-polyacrylamide electrophoresis of whole cell proteins from the patient showed a marked decrease in an 89-Kd protein (8% of control) and a marked increase in a 47-Kd protein (4.2-fold). Both mother and father had decreased 89-Kd (77% and 42% of control) and increased 47-Kd proteins (2- and 3.4-fold), although neither had recurrent infections or chemotactic defects. These studies describe a new inherited actin dysfunction syndrome associated with severe propensity to fungal infection and draw attention to the proteins of apparent molecular weights of 89 Kd and 47 Kd, which may be of great importance in the regulation of actin polymerization in human PMNs.

Lack of effect of opioid peptides, morphine and naloxone on superoxide formation in human neutrophils and HL-60 leukemic cells

There are controversial reports in the literature concerning the effects of opioids on superoxide (O2-) formation in phagocytes, these agents being either inhibitory or stimulatory. We re-examined this issue and compared the effects of the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), phorbol myristate acetate (PMA), ATP, platelet activating factor (PAF), cytochalasin B (CB) and prostaglandin E1 (PGE1) with those of various opioids on O2- formation in human neutrophils and HL-60 leukemic cells under defined experimental conditions. In the presence of CB, fMet-Leu-Phe and PAF concentration-dependently activated O2- formation in neutrophils with EC50 values of 20 nM and 100 nM, respectively. In the absence of CB, fMet-Leu-Phe and PAF were much less effective. PAF synergistically enhanced O2- formation induced by fMet-Leu-Phe. ATP at a concentration of 100 microM and the opioids, methionine enkephalin, beta-endorphin, dynorphin, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin, [D-Ala2-D-Leu5]-enkephalin and morphine at concentrations between 10 pM to 1 microM did not activate O2- formation. ATP but not beta-endorphin potentiated fMet-Leu-Phe-induced O2- formation. O2- formation induced by a maximally stimulatory concentration of PMA (100 ng/ml) was enhanced by fMet-Leu-Phe but was unaffected by methionine enkephalin or PGE1. PMA at a non-stimulatory concentration (2 ng/ml) potentiated the effect of fMet-Leu-Phe but did not induce responsiveness to PAF, ATP or beta-endorphin. PGE1 strongly inhibited fMet-Leu-Phe-induced O2- formation, whereas morphine, methionine enkephalin and the opioid antagonist, naloxone, were without effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulus-dependent differences in superoxide anion generation by normal human eosinophils and neutrophils

The role of eosinophils in allergic and hypersensitivity diseases has yet to be fully established and remains limited by techniques to isolate the eosinophil in high purity. Consequently, most studies that evaluate and characterize eosinophil function are conducted with isolates from patients with hypereosinophilia. There is, however, evidence to suggest that isolates from such patients do not represent normal function. Now, with new techniques to isolate and purify eosinophils from normal subjects without eosinophilia, metabolic function of the normal eosinophil can be assessed. To accomplish this, granulocytes from healthy volunteers were separated by continuous density Percoll gradients into populations of purified eosinophils (90.3 ± 1.9%) and neutrophils (98.2 ± 0.4%). Superoxide (O 2 −) generation was measured with a microassay of superoxide dismutase-inhibitable cytochrome c reduction in response to several soluble and particulate agonists. Normal eosinophils generated significantly more O 2 − in response to either phorbol myristate acetate or calcium ionophore A23187 than their matched neutrophil fractions. In contrast, differences in granulocyte response to zymosan and chemotactic peptide, N-formyl-methionyl-leucyl-phenyalanine, were dependent on the presence of cytochalasin B (CB) in the reaction. N-formyl-methionyl-leucyl-phenyalanine-stimulated eosinophils generated less O 2 − in the absence of CB but similar amounts in the presence of CB, compared to neutrophils. Activation by zymosan in the presence of 10% autologous serum generated similar amounts of O 2 − in all the cell populations when CB was present; however, in the absence of CB, neutrophils produced less O 2 − when they were compared to eosinophils. Therefore, normal eosinophils respond differently to some activators, compared to neutrophils, and these differences may prove significant as the contribution of eosinophils to inflammation becomes established.

Cytochalasin B slows but does not prevent monomer addition at the barbed end of the actin filament.

We used Limulus sperm acrosomal actin bundles to examine the effect of 2 microM cytochalasin B (CB) on elongation from both the barbed and pointed ends of the actin filament. In this paper we report that 2 microM CB does not prevent monomer addition onto the barbed ends of the acrosomal actin filaments. Barbed end assembly occurred over a range of actin monomer concentrations (0.2-6 microM) in solutions containing 75 mM KCl, 5 mM MgCl2, 10 mM Imidazole, pH 7.2, and 2 microM CB. However, the elongation rates were reduced such that the rates at the barbed end were approximately the same as those at the pointed end. The association and dissociation rate constants were 8- to 10-fold smaller at the barbed end in the presence of CB along with an accompanying twofold increase in critical concentration at that end. Over the time course of experimentation there was little evidence for potentiation by CB of the nucleation step of assembly. CB did not sever actin filaments; instead its presence increased the susceptibility of actin filaments to breakage from the gentle shear forces incurred during sample preparation. Under these experimental conditions, the assembly rate constants and critical concentration at the pointed end were the same in both the presence and the absence of CB.

Accessibility of sulfhydryl residues induced by cytochalasin B binding and conformational dynamics in the human erythrocyte glucose transporter

Studies with intact cells have implicated essential sulfhydryl groups in the carrier-mediated glucose transport of human erythrocytes. In an attempt to identify and characterize such essential sulfhydryl residues we have studied the interaction of p-chloromercuribenzoate (PCMB) with a purified glucose transporter preparation (band 4.5) from human erythrocytes, in the presence and absence of its ligands, and the effects of this interaction on the binding of cytochalasin B (CB) to the transporter. At least 3 mol of PCMB reacted per mol of this preparation. A portion of the reaction was significantly enhanced in the presence of cytochalasin B. This enhancement was a saturable function of CB concentration, and was half-maximal at a CB concentration equal to the dissociation constant for the CB binding to the preparation. This CB-sensitive, PCMB reaction product comigrated with the band 4.5 on lithium dodecyl sulfate-polyacrylamide gel electrophoresis. An excess of d-glucose did not affect the PCMB reaction by itself in the absence of CB, but totally abolished the CB-induced enhancement of the PCMB reaction. PCMB inhibited the CB binding activity of the transporter preparation, and this inhibition was also enhanced in the presence of CB. These results suggest that CB binding perturbs the conformational dynamics of the glucose transporter resulting in an exposure of at least two sulfhydryl residues to PCMB reaction, and that some of these CB-sensitive sulfhydryl groups are essential for CB binding to the transporter.

Effect of Cytochalasin B on Human Lymphocyte Responses to Mitogens: Time and Concentration Dependence

Human peripheral blood mononuclear cells were exposed to various concentrations of concanavalin A (Con A) and cytochalasin B (CB). Low concentrations of CB (0.5 to 2.0 microng/ml) augmented, whereas high concentrations of CB (greater than 2.0 microng/ml) inhibited the mitogenic response to Con A when Con A and CB were added at the initiation of culture. Augmented responses could also be found by adding normally inhibitory high concentrations of CB after 72 to 96 hr incubation with Con A. Preincubation of the cells with CB 5 min to 4 hr before adding Con A also resulted in potentiation of the mitogenic response. CB did not increase spontaneous transformation and worked as well in various solvents (dimethyl sulfoxide, acetone, and ethanol). The effect was totally reversible by removing CB before adding Con A. CB had no effect on 3H-Con A binding to cells. The potentiating effect of CB was still evident after removal of CB, Con A, or both from cultrues that had been simultaneously exposed to Con A and CB for at least 2 days. CB augmented the response of human lymphocytes to Con A-adsorbed, x-rayed autologous lymphocytes. In the presence of optimal concentrations of CB, x-ray-damaged cells respond better to Con A; the response to suboptimal and supraoptimal concentrations of Con A is better, and the response with delayed addition of alpha-methyl-D-mannoside is better. CB also potentiates the response of human lymphocytes to pokeweed mitogen and phytohemagglutinin. Direct observation of cells in culture demonstrated the development of huge clusters of cells during mitogenic responses to Con A in the absence of CB. Circumstances which inhibited the mitogenic response to Con A totally prevented the development of cellular aggregates (prolonged incubation with high concentrations of CB). Each of the conditions which augmented the mitogenic response to Con A was accompanied by the formation of multiple small, relatively uniform cellular aggregates. These observations suggest that CB affects cell-cell interaction possibly by affecting distribution of Con A on the cell surface, by affecting cell sruface net negative charge, or by increasing the response to chemotactic stimuli. CB may be acting in some manner to enable certain nonresponsive cells to respond to a mitogenic stimulus, although an increase in the response of individual cells cannot be ruled out.

Cytochalasin B, the blood platelet release reaction and cyclic GMP

CYTOCHALASIN B (CB) affects a wide range of cellular processes that appear to be contractile in nature1. The molecular mechanisms responsible for these effects are uncertain, though it is known that CB interferes with microfilament function1 and can block transport of glucose across cell membranes2,3. Secretion by exocytosis has been shown to be inhibited by CB in some cell types4–8 but to be potentiated by CB in others9–12. We describe here the effects of CB on the release reaction of blood platelets induced by collagen fibres, a process in which the contents of both amine storage granules (5-HT, ADP, ATP) and lysosomes are secreted13 and which leads to platelet aggregation mediated by the ADP released14. Although previous studies have shown that CB inhibits various platelet activities including clot retraction15–17 and platelet aggregation16,17, we now find that lower concentrations of CB markedly potentiate both the secretory activities of the platelet and release-dependent aggregation. Because collagen-induced platelet aggregation is associated with an increase in intracellular cyclic GMP18, a compound which potentiates secretion by some cells19,20, and because it has been suggested that CB may stimulate leukotaxis by increasing cyclic GMP21, we have studied the relationship between the platelet release reaction and platelet cyclic GMP levels in the presence and absence of CB. The results show that CB does not potentiate the release reaction through any direct effect on cyclic GMP levels.

Modification of zymosan-induced release of lysosomal enzymes from human polymorphonuclear leukocytes by cytochalasin B.

During the process of phagocytosis, polymorphonuclear leukocytes (PMN) release lysosomal enzymes into the extracellular medium. When the antibiotic cytochalasin B (CB) is present in the incubation medium along with phagocytable particles, enhanced recovery of enzyme activities from the incubation medium has been observed. These findings have led to the interpretation that CB enhances lysosomal enzyme release. Our results contradict this interpretation. The lysosomal enzymes acid phosphatase and beta-galactosidase are unstable after they are released from cells. During the first 5-15 min of phagocytosis, significant amounts of both acid phosphatase and beta-galactosidase can be recovered from the extracellular medium. After this, the recovery of enzyme from the medium declines, presumably because the rate of loss of lysosomal enzyme activity exceeds the rate of release at later time periods. In the presence of CB, the appearance of lysosomal enzymes in the extracellular medium of cells exposed to zymosan is retarded for 5-10 min, after which it begins and then continues for approximately 20 min. At the end of a 30-min incubation period, therefore, in the absence of CB, extracellular levels of lysosomal enzymes (especially those which are unstable) are declining toward low levels while, in the presence of CB, extracellular enzyme levels are continuing to rise. We also measured the lysosomal enzyme remaining within cells after exposure to zymosan. CB retarded the disappearance of enzyme from cells and resulted in significantly less total cell enzyme loss. Thus, in the presence of CB, a greater proportion of the lysosomal enzyme lost from cells is recovered in the extracellular medium. In contrast to the previous conclusions that CB enhances lysosomal enzyme release, our results indicate that CB delays and decreases the zymosan-stimulated release of lysosomal enzymes from PMN. Since CB inhibits phagocytosis by PMN, our results indicate that the antibiotic modifies the mechanism of release of lysosomal enzymes, resulting in zymosan stimulation of their release independently of phagocytosis.

Two actions of cyclic AMP on melanosome movement in frog skin. Dissection by cytochalasin B.

Photomicrography and reflectance microphotometry were used to monitor melanosome movement in frog skin melanocytes in vitro in response to hormonal stimulation and cytochalasin B (CB). Melanocyte-stimulating hormone (MSH), theophylline, and dibutyryl cyclic AMP (DiBcAMP) induced melanosome dispersion (darkening) which was promptly arrested by cytochalasin B in concentrations of 5-20 microg/ml. Melanosome aggregation (skin lightening) occurred only after removal of the darkening agent (MSH, theophylline, or DiBcAMP) and proceeded in the presence or absence of CB. When CB was added to darkened skins, they did not lighten and melanosomes remained in the dispersed state. Use of CB has permitted the dissection of cyclic AMP-mediated melanosome dispersion into two distinct events. The first, induction of melanosome dispersion, is CB sensitive. The second action of intracellular cyclic AMP involves an uncoupling of the centripetal motive force, and is CB insensitive. In the latter process, production of cyclic AMP appears to produce the same result as application of microtubule-disrupting agents.

Preimplantation development of microsurgically obtained haploid and homozygous diploid mouse embryos and effects of pretreatment with Cytochalasin B on enucleated eggs

Haploid embryos were obtained by microsurgical removal of one pronucleus, followed by doubling of the haploid chromosome set with Cytochalasin B (CB), either at the first or second mitosis. This procedure provides a source of fully homozygous diploid embryos which were grown in vitro or in vivo. The effect of CB treatment before and during operation on the course of enucleation and further development of embryos was studied. Out of 81 eggs made diploid at 2-cell stage and transplanted into the oviducts of immature or pseudopregnant recipients 27 morulae and blastocysts were recovered, but not a single case of implantation occurred by the eighth or ninth day of development. After 72-80 h of in vitro culture, most of the homozygous embryos were morulae but after an additional 24 h the majority of them transformed into blastocysts. The rate of development of homozygotes was markedly better than that of haploids, which progressed beyond morula stage. The immediate survival rate of operated eggs was dependent on whether or not the eggs were pre-incubated and the enucleation was performed in the presence of CB. In the former case the immediate survival rate was nearly twice as high as in the absence of CB, but more of the treated eggs underwent fragmentation and early developmental arrest.