The effect of cytochalasin B and colchicine on concanavalin A induced vacuolation in mouse peritoneal macrophages

Macrophages bind concanavalin A (ConA) and interiorize the ConA-receptor conjugates in minute ConA-bearing pinosomes that undergo intracellular fusion processes to yield medium size (2–5 μ) and large size (>5 μ) vacuoles. The number and size of the vacuoles depends on ConA concentration and on the composition of the incubation medium. Cytochalasin B (CB) reduces ConA internalization by 30% and the surface bound lectin redistributes to form numerous clumps of fluorescence. Macrophage ConA-induced vacuolation is inhibited by CB both when the incubation is carried out in Dulbecco's modified Eagle's medium (90% inhibition) or in phosphate buffered saline (80% inhibition). Colchicine reduces [³H]ConA internalization by 20% and has no detectable effect on the surface distribution of fluorescein-ConA conjugates. Incubation of macrophages with colchicine prior to or during ConA induction of vacuole formation results in an enhancement of cell vacuolation; the number of the developing large size vacuoles exceeds 4–6-fold that obtained in the absence of the alkaloid. The results suggest the involvement of elements of the cytoskeleton (microfilaments-microtubuli) in regulation of the sequence of events leading to ConA-induced vacuolation and that CB and colchicine have opposite effects on the process.     

Specific interaction of cytochalasins with muscle and platelet actin filaments in vitro

The cytochalasins (CE, CD, CB and H₂CB) inhibit numerous cellular processes which require the interaction of actin with other structural and contractile proteins. In this report we describe the effects of the cytochalasins on the viscosity and morphology of muscle and platelet actin. The cytochalasins decreased the viscosity of F-actin solutions. The effect of H₂CB, CB and CD on F-actin viscosity was maximal at concentrations of 20–50μM and did not increase with time. In contrast, CE caused a progressive decrease in the viscosity of F-actin solutions which was dependent upon the concentration of CE and the duration of incubation of the CE-actin mixture. After two hours of incubation of drug-actin mixtures, the relative effectiveness of the cytochalasins in reducing the viscosity of F-actin was CE > CD > CB = H₂CB. The effects of CD and CE were paralleled by morphologic changes in negatively stained actin filaments. The effects of the cytochalasins on the viscosity and morphology of muscle and platelet actin were the same whether the drugs were added before or after the polymerization of the protein. These studies show that the interaction of the cytochalasins with actin is highly specific. Because the relative potencies of these drugs for affecting motile processes and the relative affinities of the drugs for binding sites within a variety of cells are CE > CD > CB = H₂CB, the effects of cytochalasins on actin described here may contribute to some of the biological effects of the drugs on motile processes.     

Cytochalasin sensitive structures and lymphocyte activation.

In purified human peripheral blood lymphocytes, low (0.01–10 μM) concentrations of cytochalasins A B, E and D (CA, CB, CD, CE) produced marked augmentation of transport and metabolic responses to phytohemagglutinin (PHA) and concanavalin A (ConA) including effects on DNA synthesis, cAMP accumulation, phosphatidylinositol turnover and sodium-dependent amino acid transport. At high concentrations (10–100 μM) these same responses were inhibited. Cytochalasin effects were minimal or absent if lectin was not present indicating that these agents are acting by modulating the action of the lectin rather than through a direct effect on cell metabolism. Using [¹²⁵I]ConA, the number of lectin molecules bound per cell was shown not to be altered by the cytochalasins. Taken together with the previously reported effects of the cytochalasins on calcium uptake in lectin stimulated lymphocytes, these observations suggest that microfilaments (or related cytochalasin sensitive structures) play an important role in the modulation of lymphocyte activation.     

Selective solubilization and purification of cell-surface concanavalin A-binding glycoproteins from Novikoff hepatocellular carcinoma cells

Novikoff hepatocellular carcinoma cells possess cell-surface glycoproteins that bind the lectin, concanavalin A. A subset of Con A-binding plasma membrane glycoproteins was solubilized by addition of n-butanol to a suspension of Novikoff cells. Glycoproteins solubilized into the n-butanol-saturated aqueous phase of the two-phase mixture were purified by sequential chromatography on DEAE-cellulose and Sepharose-conjugated concanavalin A. Glycoproteins specifically bound to the Sepharose-conjugated Con A exhibited apparent M_r = 72,000 to 125,000. The plasma membrane localization of these components was inferred by their isolation from cells surface labeled with NaIO₄/ NaB³H₄. A xenoantiserum, raised against glycoproteins specifically bound to Sepharose-conjugated concanavalin A was employed to identify reactive components in nonionic detergent extracts of Novikoff tumor cells or rat hepatocytes surface labeled using lactoperoxidase-catalyzed iodination (¹²⁵I). Major reactive peptides in extracts of Novikoff cells exhibited apparent M_r = 74,000, 82, 000, 110,000, and 135,000, while those in extracts of hepatocytes possessed apparent M_r = 98,000 and 105,000. The reactivity of the antiserum with extracts of ¹²⁵I-labeled Novikoff cells was abolished by absorption of the antiserum with hepatocytes, indicating that the qualitative differences observed may result from structural modification of one or more cell-surface glycoproteins, rather than the expression of new or inappropriate glycoproteins. This antiserum will provide a useful probe to investigate alterations in the expression or structure of glycoproteins that occur as a consequence of malignant transformation or adaptation of malignant cells to growth in the ascitic form.     

Stem cells from human exfoliated deciduous teeth ameliorate concanavalin A-induced autoimmune hepatitis by protecting hepatocytes from apoptosis

BACKGROUNDAutoimmune hepatitis is a serious autoimmune liver disease that threatens human health worldwide, which emphasizes the urgent need to identify novel treatments. Stem cells from human exfoliated deciduous teeth (SHED), which are easy to obtain in a non-invasive manner, show pronounced proliferative and immunomodulatory capacities.AIMTo investigate the protective effects of SHED on concanavalin A (ConA)-induced hepatitis in mice, and to elucidate the associated regulatory mechanisms.METHODSWe used a ConA-induced acute hepatitis mouse model and an in vitro co-culture system to study the protective effects of SHED on ConA-induced autoimmune hepatitis, as well as the associated underlying mechanisms.RESULTSSHED infusion could prevent aberrant histopathological liver architecture caused by ConA-induced infiltration of CD3⁺, CD4⁺, tumor necrosis-alpha⁺, and interferon-gamma⁺ inflammatory cells. Alanine aminotransferase and aspartate aminotransferase were significantly elevated in hepatitis mice. SHED infusion could therefore block ConA-induced alanine aminotransferase and aspartate aminotransferase elevations. Mechanistically, ConA upregulated tumor necrosis-alpha and interferon-gamma expression, which was activated by the nuclear factor-kappa B pathway to induce hepatocyte apoptosis, resulting in acute liver injury. SHED administration protected hepatocytes from ConA-induced apoptosis. CONCLUSIONSHED alleviates ConA-induced acute liver injury via inhibition of hepatocyte apoptosis mediated by the nuclear factor-kappa B pathway. Our findings could provide a potential treatment strategy for hepatitis.     

Relationship between phosphatidylcholine biosynthesis and cellular commitment in concanavalin A-stimulated lymphocytes

The incorporation of [¹⁴C]choline into phosphatidylcholine was studied in lymphocyte cultures exposed to concanavalin A (ConA). The lectin was found to induce an increase in the incorporation of the label with following features:

1. 1. It occurs very promptly after exposure.

2. 2. It is not elicited by a non-mitogenic lectin.

3. 3. The increase in the early stage is proportional to lectin concentration.

4. 4. It can be terminated by a competitive inhibitor of ConA binding.

5. 5. The extent of the increase shows a correlation with the rate of cellular commitment to initiate DNA synthesis. These results suggest that in the mitogenic stimulation of T lymphocytes enhanced synthesis of membrane phospholipids is a precommitment event.

     

Effect of cytochalasins B and D on the developmental competence of somatic cell nuclear transfer embryos in miniature pigs

In many animals, cytochalasins have generally been used as cytoskeletal inhibitors for the diploid complement retention of somatic cell nuclear transfer (SCNT) embryos. However, limited information is available on the effects of cytochalasins on the in vitro development of SCNT embryos. Hence, we compared the effects of cytochalasin B (CB) and cytochalasin D (CD) on pseudo-polar body (pPB) extrusion, cortical actin filament (F-actin) distribution in porcine parthenogenetic oocytes and in vitro development of SCNT embryos that were reconstructed using foetal fibroblasts in the G0/G1 phase derived from miniature pigs. CB (7.5 microg/ml) and CD (2.5 microg/ml) treatments effectively inhibited pPB extrusion in SCNT embryos. CB (2.5 microg/ml) treatment could not inhibit pPB extrusion and insufficiently destabilized F-actin immediately following artificial activation. In parthenogenetic oocytes treated with 2.5 microg/ml CD, normal reorganization and uniform distribution of cortical F-actin at the cytoplasmic membrane were observed at 8 h after artificial activation; this finding was similar to that of control oocytes. In contrast, parthenogenetic oocytes treated with 7.5 microg/ml CB showed non-uniform distribution of F-actin at 8 h after artificial activation. On day 5 after in vitro cultivation, the blastocyst formation rate of SCNT embryos treated with 2.5 microg/ml CD was significantly higher than that of SCNT embryos treated with 2.5 and 7.5 microg/ml CB (p < 0.05). Hence, the present findings suggest that CD is more effective than CB as the cytoskeletal inhibitor for the production of SCNT embryos in miniature pigs.     

Estrogen target cells. Establishment of a cell line derived from the rat pituitary tumor MtT/F4.

Pretreatment of rabbit kidney cells with cytochalasins B and D (CB, CD) enhanced herpes simplex virus type 2 (HSV-2) DNA infectivity 3- to 6-fold over values obtained using the standard CaCl₂ technique. Cells were pretreated with CB for 4–6 h to achieve infectivity enhancement. A lower concentration of CD, and shorter pretreatment periods, resulted in comparable DNA infectivity. Separate exposure of cells to colchicine, colcemid, or vinblastine increased DNA infectivity 7-, 6-, and 5-fold, respectively, over control values. Additional enhancement was obtained when CD was used together with any one of the aforementioned drugs. Maximal enhancement of HSV-2 DNA infectivity was obtained by pretreating recipient cells with a drug mixture containing colchicine, colcemid, and CD. This treatment maximized infectivity levels 20- to 30-fold over CaCl₂ control values.     

Rearrangement of concanavalin A receptor sites on cells tagged with dinitrofluorobenzene: Evidence for the chemical induction of a change usually associated with malignant transformation

Normal human peripheral blood granulocytes which are tagged with 1-fluoro-2,4-dinitrobenzene (DNFB) are agglutinated by concanavalin A (ConA) in a way which resembles the pattern of reactivity displayed by leukemic cells. The present study further defines this reaction. The binding of ConA to untagged and DNP-tagged granulocytes, treated with DNFB at a ratio of 10¹¹ molecules/cell, was quantified by isotopic dilution experiments employing [³H]ConA. Similar amounts of the lectin were bound to untagged and DNP-tagged cells following incubation for 5 min at 4 °C or 30 min at 24 °C: 1.1 × 10⁵ molecules/cell, 4.6 × 10²/μ² of surface area, and 1.6 × 10³/μg of protein. The binding of [³H]ConA to both untagged and DNP-tagged cells was inhibited to the same degree by α-methylglucopyranoside (α-MG). Fixation with either glutaraldehyde or formaldehyde, which immobilizes ConA receptor sites, completely inhibited the agglutination of both untagged and DNP-tagged cells although lectin binding was unchanged. This suggests that the inhibition of agglutination was not due to the blocking of ConA-binding sites by aldehyde groups but rather to the immobilization of lectin receptors. We conclude that dinitrophenylation of normal granulocytes facilitates the rearrangement of lectin receptors in a way which resembles the ConA-induced clustering of sites which have been observed with malignant and transformed cells.     

Effects of metabolic poisons on rice: the comparative sensitivity of aerobic and anaerobic modes of germination

Rice germinates equally well when incubated in air and in nitrogen. It was therefore chosen for a comparative study of the effect of oxygen status in a single organism upon the activity of conventional metabolic poisons. Inhibitor activity was based upon the concentration required for 50% inhibition of germination. The inhibitors were: AgNO₃, HgCl₂, phenylmercuric acetate, iodoacetamide, KCN, NaN₃, NaF, fluoroacetate, 2,4-dinitrophenol, Na₂HAsO₄ and CO. Only 5 inhibitors out of 11 differed significantly in their effects on aerobic and anaerobic germination. These included phenylmercuric acetate (N₂), sodium fluoroacetate (air), NaN₃ (air), and 2,4-dinitrophenol (N₂). CO activity was manifest only in air and it was photo-reversible. The effects of CO, NaN₃, and fluoroacetate were consistent with their conventional role in aerobic metabolism. The failure of KCN to discriminate was attributed to its relative non-specificity. 2,4-Dinitrophenol behaved anomalously, requiring a 20-fold lower concentration for inhibition under nitrogen than under air. Among sulfhydryl poisons, only phenylmercuric acetate differentiated between germination in air and nitrogen and was nearly 8 times more active under nitrogen. Uptake measurements using phenylmercuric acetate and arsenate showed both compounds to be in greater concentration under aerobic conditions, thereby rendering unlikely any explanation based upon greater permeability under nitrogen. In addition to other anomalies, the fact that NaN₃, fluoroacetate and CO inhibited anaerobic germination at any concentration requires explanation. It was concluded that the general significance and utility of metabolic inhibitor studies in vivo required further evaluation.     

Effect of metabolic inhibitors on the agglutination of tumor cells by concanavalin A and Ricinus communis agglutinin

The agglutinations of rat ascites tumor cells by concanavalin A and by $\text{Ricinus communis}$ agglutinin were inhibited by low temperature, 2,4-dinitrophenol and cytochalasin B but not by cycloheximide. These metabolic inhibitors, however, did not inhibit the binding of the agglutinins to the cells. These results suggest that the agglutination was dependent on an active process; probably on a microfilament system responsible for cell surface movement which requires ATP, but not on protein synthesis.     

Roles of platelets and macrophages in the protective effects of lipopolysaccharide against concanavalin A-induced murine hepatitis

Platelets are reportedly causal in hepatitis. We previously showed that in mice, lipopolysaccharide (LPS) induces a reversible and macrophage-dependent hepatic platelet accumulation (HPA), including translocation of platelets into Disse spaces and their entry into hepatocytes. Concanavalin A (ConA), which induces hepatitis in mice via both T cells and macrophages, also induces HPA. Here, we examined the relationship between HPA and ConA-hepatitis. ConA-hepatitis and HPA were evaluated by serum transaminases, hepatic 5-hydroxytryptamine, and/or electron microscopy. Unlike LPS-induced HPA, ConA-induced HPA was only moderately dependent on phagocytic macrophages. Against expectations, platelet-depletion significantly exacerbated ConA-hepatitis, and anti-P-selectin antibody and P-selectin receptor blockade reduced both ConA-induced HPA and hepatitis. Prior induction of HPA by pretreatment with low-dose LPS powerfully reduced ConA-hepatitis. Such protection by LPS-pretreatment was not effective in mice depleted of phagocytic macrophages. In platelet-depleted mice, LPS-pretreatment severely exacerbated ConA-hepatitis. In mice depleted of both macrophages and platelets, neither ConA nor LPS-pretreatment + ConA induced hepatitis. In mice deficient in IL-1α and IL-1β (but not in TNFα), ConA-induced hepatitis was mild, and a protective effect of LPS was not detected. These results suggest that (i) there are causal and protective types of HPA, (ii) the causal type involves hepatic aggregation of platelets, which may be induced by platelet stimulants leaked from injured hepatocytes, (iii) the protective type is inducible by administration of prior low-dose LPS in a manner dependent on phagocytic (or F4/80-positive) macrophages, and (iv) IL-1 is involved in both the causal and protective types.     

Cellular energy dependent agglutination of rat ascites tumor cells mediated by concanavalin A and Ricinus communis agglutinin.

Effect of various metabolic inhibitors on the agglutination of rat ascites tumor cells mediated by concanavalin A and Ricinus communis agglutinin was studied using a quantitative assay method for agglutination in which turbidity of cell suspension is measured. Cell agglutination was inhibited by low temperature, cytochalasin B and inhibitors of energy generating systems without affecting lectin binding, and agglutination was not affected by hydroxyurea, actinomycin D or cycloheximide. The inhibitors of energy generating systems decreased the cellular ATP level and inhibited macromolecular synthesis under the conditions where they inhibited the agglutinations. In contrast, cytochalasin B did not depress the cellular ATP level nor inhibit RNA and protein syntheses. These results suggest that the agglutination is associated with cellular energy dependent processes other than macromolecular synthesis; probably with some cellular surface movements participated by microfilament activity.     

The effect of inhibitors on the mutagenic activity of N-methyl-N-nitrosourea and N-ethyl-N-Nitrosourea inArabidopsis thaliana

NaN₃—a respiratory inhibitor and anaerobic conditions (nitrogen) decrease the mutagenic activity of N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea inArabidopsis thaliana. DNP-an uncoupling agent increases the effect of both nitrosoureas, but this may not be caused by the specific action of DNP.     

Adhesion of human skin fibroblasts : Effect of tetravalent, divalent and monovalent concanavalin A

Concanavalin A (ConA) pretreatment inhibited the adhesion of fibroblasts to plastic surface in a dose-dependent manner. The ConA effect was reversible and could be inhibited by α-methylmannoside. Pretreatment with cytochalasin B (CB) and colchicine increased the ConA effect. Divalent and monovalent ConA derivatives had no effect on the fibroblast adhesion. This indicates that ligand attachment to ConA receptors is not sufficient to prevent cell adhesion. The requirement of tetravalent ConA for inhibition of cell adhesion suggests that the decrease of lateral mobility of membrane components, which seems to be specific for tetravalent ConA, is responsible for the inhibition of cell adhesion. The enhancement of the ConA effect by colchicine and CB pretreatment suggests an involvement of microtubules and microfilaments in the mobility of ConA receptors of fibroblasts.     

Cytochalasin inhibition of isolated rat gastric parietal cell function

Submicrogram concentrations (0.04-0.29 microM) of the microfilament disrupting agents cytochalasins D, E, and B (CD, CE, CB) were shown to inhibit secretagogue-stimulated 14C-aminopyrine accumulation (AP) in isolated rat gastric mucosal parietal cells. The microtubule disrupting agent colchicine had little influence on AP accumulation. Histamine- and dibutyryl cyclic AMP (DbcAMP)-stimulated AP accumulation was inhibited with an order of potency CD greater than CE approximately equal to CB. CB inhibition of these secretagogue actions was, however, only approximately 65-70% of the maximal stimulated response, whereas CD and CE caused 100% inhibition. On the other hand, carbamylcholine-stimulated AP accumulation was inhibited 100% by all cytochalasins tested with an order of potency CD approximately equal to CE greater than CB. These data are discussed in relation to acid secretagogue-induced morphological changes involving actin filament organization in parietal cells.     

A comparative study on the antimutagenicity of atorvastatin and lovastatin against directly acting mutagens

Elevated levels of oxidative DNA lesions have been noted in many tumors and such damage is strongly implicated in the etiology of cancer. The cumulative risk of cancer increases with the fourth power of age and is associated with an accumulation of oxidative DNA damage. Many agents, synthetic or natural, that can inhibit mutation have been depicted as cancer chemopreventive agents. Antimutagenicity of the 3-hydroxy-3-methylgutaryl-CoA (HMG-CoA) reductase inhibitors atorvastatin and lovastatin was studied using the Ames Salmonella typhimurium assay. Directly acting mutagens, sodium azide (NaN₃) and 4-nitro-o-phenylenediamine (NPDA), were used to induce mutation in Salmonella strains TA98 and TA100. The antimutagenicity of lovastatin and atorvastatin was found to be significant (p < 0.01) and dose-dependent. The percentage inhibition of a 3 mg lovastatin-treated plate was found to be 79.9% and 61.8% against NPDA- and NaN₃-induced mutation to TA98 and TA100, respectively. Atorvastatin (0.5 mg/plate) inhibited NPDA-and NaN₃-induced mutation to TA98 and TA100 by 78.6% and 45.5%, respectively. Atorvastatin showed antimutagenic activity at lower concentrations than lovatstatin. The results of the present study regarding the antimutagenic activity of atorvastatin and lovastatin suggested their therapeutic application as cancer chemopreventive agents.     

The Effects of Cyanide and Azide on the Photoreduction of 3-Phosphoglycerate and Oxaloacetate by Wild Type and Two Reductive Pentose Phosphate Cycle Mutants of Chlamydomonas reinhardtii

3-Phosphoglycerate- and oxaloacetate-dependent O₂ photoevolution by permeabilized cell preparations (Pressates), prepared from wild type (Wt) and two reductive pentose phosphate cycle mutants of Chlamydomonas reinhardtii showed different sensitivities to the inhibitors sodium cyanide and sodium azide. NaCN (1.5 millimolar) severely inhibits both CO₂- and 3-phosphoglycerate-dependent O₂ photoevolution by the Wt Pressate, but does not inhibit 3-phosphoglycerate-dependent O₂ photoevolution by Pressates prepared from the mutants rcl-u-1-10-6C (which lacks ribulose, 1-5, bisphosphate carboxylase activity) and F60 (which lacks phosphoribulokinase activity). NaN₃ (0.5 millimolar) inhibits 3-phosphoglycerate-dependent O₂ photoevolution by the rcl-u-1-10-6C Pressate more severely than in the Pressates prepared from F60 and Wt. A higher concentration of NaN₃ (2.0 millimolar) severely inhibited oxaloacetate-dependent O₂ photoevolution by the rcl-u-1-10-6C, but not by the F60 Pressate. O₂ exchange-dependent upon methyl viologen was not strongly inhibited by 2 millimolar NaN₃ in either of the mutant Pressates. The data suggests that the mutational lesions which resulted in decreased ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase activities effected changes in other photosynthetic reactions, either by direct interactions between component proteins or by causing changes in substrate or cofactor availability to the partial reactions.     

A quantitative evaluation of peroxidase inhibitors for tyramide signal amplification mediated cytochemistry and histochemistry

Many peroxidase inhibitors have been used in horseradish peroxidase (HRP) mediated immunostaining and in situ hybridization to quench background peroxidase activity. However, the efficacy of these inhibitors has been controversial, partially due to the lack of a quantitative study. Tyramide signal amplification (TSA) is much more sensitive than other HRP-mediated methods but its super-sensitivity also demands effective inhibition of background peroxidase activity. In searching for an effective peroxidase inhibitor, we have systematically evaluated the efficacy of several peroxidase inhibitors by quantifying the fluorescence intensity in cultured fibroblasts and tissue sections treated with the inhibitors. For cultured cells, 0.05 mM of phenylhydrazine and 1 unit/ml of glucose oxidase gave only moderate inhibition of HRP activity while 1 mM of sodium azide (NaN₃), 3% of hydrogen peroxide (H₂O₂), NaN₃/H₂O₂ combined and 0.02 N hydrochloric acid (HCl) provided more complete inhibition. However, the inhibitory effect of NaN₃/H₂O₂ is reversible upon removal of the inhibitors and followed by incubation and wash to mimic antibody interactions. Similar results were obtained from rat skin wound tissues that have strong endogenous peroxidase activity. Our results recommend the use of HCl and caution the use of phenylhydrazine, glucose oxidase, NaN₃ and H₂O₂ as potent peroxidase inhibitors.