Protein tyrosine phosphatase 1B is a mediator of cyclic ADP ribose-induced Ca²⁺ signaling in ventricular myocytes

Cyclic ADP-ribose (cADPR) releases Ca²⁺ from ryanodine receptor (RyR)-sensitive calcium pools in various cell types. In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca²⁺ (that is, a rapid increase and decrease of calcium within one second) during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca²⁺ ([Ca²⁺](i)) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca²⁺ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca²⁺ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.

Letalidad de citocalasina B y otros compuestos aislados del hongo Aspergillus spp. (Trichocomaceae) endófito de Bauhinia guianensis (Fabaceae) / Lethality of cytochalasin B and other compounds isolated from fungus Aspergillus sp. (Trichocomaceae) endophyte of Bauhinia guianensis (Fabaceae)

Los hongos endofíticos son hongos que colonizan los tejidos internos de las plantas; varios compuestos biológicamente activos se han aislado a partir de estos hongos. Existen pocos estudios de compuestos aislados de hongos endófitos de plantas amazónicas. Por lo tanto, este estudio tuvo como objetivo el aislamiento y la identificación estructural de ergosterol (1), peróxido de ergosterol (2), mevalonolactona (3), citocalasina B (4) y citocalasina H (5) a partir de Aspergillus spp. EJC 04, un hongo endofítico de Bauhinia guianensis. La citocalasina B (4) y el derivado diacetato de citocalasina B (4a) mostraron una alta letalidad en el ensayo de Artemia salina. Esta es la primera aparición de citocalasinas en hongos endófitos amazónica de B. guianensis

Endophytic fungi are fungi that colonize internal tissues of plants; several biologically active compounds have been isolated from these fungi. There are few studies of compounds isolated from endophytic fungi of Amazon plants. Thus, this study aimed the isolation and structural identification of ergosterol (1), ergosterol peroxide (2), mevalonolactone (3), cytochalasin B (4) and cytochalasin H (5) from Aspergillus sp. EJC 04, an endophytic fungus from Bauhinia guianensis. The cytochalasin B (4) and the diacetate derivative of cytochalasin B (4a) showed high lethality in the brine shrimp assay. This is the first occurrence of cytochalasins in Amazonian endophytic fungi from B. guianensis

Effects of Bisphosphonates on Glucose Transport in a Conditionally Immortalized Rat Retinal Capillary Endothelial Cell Line (TR-iBRB Cells)

The objective of the present study was to elucidate the effect of bisphosphonates, anti-osteoporosis agents, on glucose uptake in retinal capillary endothelial cells under normal and high glucose conditions. The change of glucose uptake by pre-treatment of bisphosphonates at the inner blood-retinal barrier (iBRB) was determined by measuring cellular uptake of [3H]3-O-methyl glucose (3-OMG) using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB cells) under normal and high glucose conditions. [3H]3-OMG uptake was inhibited by simultaneous treatment of unlabeled D-glucose and 3-OMG as well as glucose transport inhibitor, cytochalasin B. On the other hand, simultaneous treatment of alendronate or pamidronate had no significant inhibitory effect on [3H]3-OMG uptake by TR-iBRB cells. Under high glucose condition of TR-iBRB cells, [3H]3-OMG uptake was increased at 48 h. However, [3H]3-OMG uptake was decreased significantly by pre-treatment of alendronate or pamidronate compared with the values for normal and high glucose conditions. Moreover, geranylgeraniol (GGOH), a mevalonate pathway intermediate, increased the uptake of [3H]3-OMG reduced by bisphosphonates pre-treatment. But, pre-treatment of histamine did not show significant inhibition of [3H]3-OMG uptake. The glucose uptake may be down regulated by inhibiting the mevalonate pathway with pre-treatment of bisphosphonates in TR-iBRB cells at high glucose condition.

Cytochalasin B Modulates Macrophage-Mediated Inflammatory Responses

The actin cytoskeleton plays an important role in macrophage-mediated inflammatory responses by modulating the activation of Src and subsequently inducing nuclear factor (NF)-kappaB translocation. In spite of its critical functions, few papers have examined how the actin cytoskeleton can be regulated by the activation of toll-like receptor (TLR). Therefore, in this study, we further characterized the biological value of the actin cytoskeleton in the functional activation of macrophages using an actin cytoskeleton disruptor, cytochalasin B (Cyto B), and explored the actin cytoskeleton's involvement in morphological changes, cellular attachment, and signaling events. Cyto B strongly suppressed the TLR4-mediated mRNA expression of inflammatory genes such as cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-alpha, and inducible nitric oxide (iNOS), without altering cell viability. This compound also strongly suppressed the morphological changes induced by lipopolysaccharide (LPS), a TLR4 ligand. Cyto B also remarkably suppressed NO production under non-adherent conditions but not in an adherent environment. Cyto B did not block the co-localization between surface glycoprotein myeloid differentiation protein-2 (MD2), a LPS signaling glycoprotein, and the actin cytoskeleton under LPS conditions. Interestingly, Cyto B and PP2, a Src inhibitor, enhanced the phagocytic uptake of fluorescein isothiocyanate (FITC)-dextran. Finally, it was found that Cyto B blocked the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at 1 min and the phosphorylation of heat shock protein 27 (HSP27) at 5 min. Therefore, our data suggest that the actin cytoskeleton may be one of the key components involved in the control of TLR4-mediated inflammatory responses in macrophages.

Preparation of cytoplasts from HL-60 cells / 生物医学工程学杂志

This experimental research was aimed to establish an optimum system of enucleation, purification and identification for preparing the cytoplasts of suspension culture cells in order to undertake cell recombination. Human leukemia HL-60 cells in suspension culture were purified by 42% Percoll density gradient centrifugation and low-speed centrifugation at 1 500r/min, respectively. The purified HL-60 cells were treated with cytochalasin B (CB) alone or combined with colcchicine and enucleated by isopycnic gradient centrifugation on 50% Percoll at 25 degrees C and 34 degrees C, respectively. Cytoplasts made from HL-60 cells were purified through gradient centrifugation by 37%, 38% and 40% Percoll, respectively. The final cytoplasts were identified by Wright-Giemsa staining and 4,6-diamidino-2-phenylidole dihydrochloride (DAPI)/5, 6-carboxyflu-orescein diacetate succinimidyl ester (CFSE) double-staining. The phenotype and mitochondrial membrane potential of HL-60 cytoplasts were analyzed by flow cytometry. The results indicated that the enucleation ratio of HL-60 cells induced by CB combined with colcchicine was up to 91. 98% +/-4. 29%, which was significantly higher than that in CB alone group (74. 95% +/- 3. 02%)(P<0. 01). The rates of enucleation and cytoplast with diameter over 5min in 34 degrees C group were higher than those in 25 degrees C group (all P<0. 01). The cytoplast purities were (95.43 +/- 0. 59)% in 38% Percoll groups,which were higher than those of 40% Percoll (P<0. 05). Nucleus and caryoplasm could be clearly distinguished by DAPI and CFSE double labeling. The results further showed that the phenotype of HL-60 cytoplasts had no significant change, and the activity of the cytoplasts was above 80% within 12h. It is concluded that enucleation throuth density gradient centrifugation on 50% Percoll mediated by CB combined with colcchicine, 38%Percoll of purification followed by DAPI/CFSE double labeling and MMP detection is an optimum scheme for preparation and identification of cytoplast from suspension culture cells.

Appropriate In Vitro Methods for Genotoxicity Testing of Silver Nanoparticles

OBJECTIVES: We investigated the genotoxic effects of 40-59 nm silver nanoparticles (Ag-NPs) by bacterial reverse mutation assay (Ames test), in vitro comet assay and micronucleus (MN) assay. In particular, we directly compared the effect of cytochalasin B (cytoB) and rat liver homogenate (S9 mix) in the formation of MN by Ag-NPs. METHODS: Before testing, we confirmed that Ag-NPs were completely dispersed in the experimental medium by sonication (three times in 1 minute) and filtration (0.2 microm pore size filter), and then we measured their size in a zeta potential analyzer. After that the genotoxicity were measured and especially, S9 mix and with and without cytoB were compared one another in MN assay. RESULTS: Ames test using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains revealed that Ag-NPs with or without S9 mix did not display a mutagenic effect. The genotoxicity of Ag-NPs was also evaluated in a mammalian cell system using Chinese hamster ovary cells. The results revealed that Ag-NPs stimulated DNA breakage and MN formation with or without S9 mix in a dose-dependent manner (from 0.01 microg/mL to 10 microg/mL). In particular, MN induction was affected by cytoB. CONCLUSIONS: All of our findings, with the exception of the Ames test results, indicate that Ag-NPs show genotoxic effects in mammalian cell system. In addition, present study suggests the potential error due to use of cytoB in genotoxic test of nanoparticles.

Bovine parthenogenotes produced by inhibition of first or second polar bodies emission

Parthenogenetic embryos are an ethically acceptable alternative for the derivation of human embryonic stem cells. In this work, we propose a new strategy to produce bovine parthenogenetic embryos inhibiting the emission of the first polar body during in vitro maturation, and allowing the extrusion of the second polar body during oocyte activation. Cytochalasin B, an inhibitor of actin microfilaments, was employed during in vitro maturation to inhibit first polar body emission or during parthenogenetic activation to block second polar body emission. Only one polar body was inhibited in each strategy in order to keep the diploid chromosome set. In experiment 1, the effect of cytochalasin B on in vitro maturation of bovine oocytes was evaluated. Most oocytes (77%) were arrested at a meiotic stage characterized by the presence of a large internal metaphase plate and absence of polar body. In experiment 2, development of embryos exposed to cytochalasin B during in vitro maturation (CytoB-IVM) or during activation (CytoB-ACT) was compared. Developmental rates did not differ between diploidization strategies, even when three agents were employed to induce activation. Both groups, CytoB-IVM and CytoB-ACT, tended to maintain diploidy. CytoB-IVM parthenogenesis could help to obtain embryos with a higher degree of homology to the oocyte donor.

Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca²+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca²+]i oscillations similar to those evoked by sperm (95 vs 100 percent; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 µg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6 percent, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83 percent, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7 percent ethanol (62 vs 62 percent, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62 percent, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca²+]i oscillations, completion of meiosis and parthenogenetic development.

Assessment of radioprotective effects of amifostine on human lymphocytes irradiated in vitro by gamma-rays using cytokinesis-blocked micronucleus assay

A radioprotective effect of amifostine as well as its ability to modulate the level of spontaneous and gamma-irradiation-induced genetic changes on human peripheral blood lymphocytes has been investigated. Amifostine, known as a potent radical scavenger, has been introduced as the most effective radioprotector, yet it is not completely approved for the clinical use. However, further in vitro and clinical studies are needed to clarify its mechanisms of action. Whole blood samples from healthy donors were exposed to various doses of gamma-rays. Lymphocytes in cultures were treated with amifostine at different concentrations [2, 4 and 6 mm] in the presence or in the absence of 1 IU/ml alkaline phosphatase before or after gamma-irradiation. Standard procedure for the cytokinesis-block micronucleus [CBMN] assay was used to assess the effect of amifostine on radiation induced micronucleus in binucleate lymphocytes. Irradiated blood samples showed an increase in the total number of micronuclei [MN] significantly different from controls [p<0.05]. However, pre-treatment of lymphocytes with amifostine in the presence of alkaline phosphatase, 15 minutes before irradiation, led to a significant decrease in the frequencies of MN and cells with more than one MN [p<0.05]. Amifostine, in its own, produced little or no protection. However, the addition of amifostine with alkaline phosphatase to the cell cultures 15 minutes after irradiation produced substantial radioprotection significantly different from the frequencies of MN induced by radiation alone [p<0.05]. Results clearly indicated that gamma-rays induced MN in lymphocytes in a dose dependent manner. The highest protective effect was achieved when amifostine was phosphorilated by alkaline phosphatase and present before irradiation in the cellular environment, indicating its radical scavenging mechanism of radioprotection. Since the administration of amifostime after irradiation also led to a considerable decrease in the frequency of radiation induced MN, which might be possible for other mechanisms such as induction of cell cycle delay and hence influencing DNA repair, are involved in radioprotection by amifostine

Enucleated L929 mouse fibroblasts support invasion and multiplication of Shigella flexneri 5a

Invasive bacteria can induce their own uptake and specify their intracellular localization; hence it is commonly assumed that proximate modulation of host cell transcription is not required for infection. However, bacteria can also modulate, directly or indirectly, the transcription of many host cell genes, whose role in the infection may be difficult to determine by global gene expression. Is the host cell nucleus proximately required for intracellular infection and, if so, for which pathogens and at what stages of infection? Enucleated cells were previously infected with Toxoplasma gondii, Chlamydia psittaci, C. trachomatis, or Rickettsia prowazekii. We enucleated L929 mouse fibroblasts by centrifugation in the presence of cytochalasin B, and compared the infection with Shigella flexneri M90T 5a of nucleated and enucleated cells. Percent infection and bacterial loads were estimated with a gentamicin suppression assay in cultures fixed and stained at different times after infection. Enucleation reduced by about half the percent of infected cells, a finding that may reflect the reduced endocytic ability of L929 cytoplasts. However, average numbers of bacteria and frequency distributions of bacterial numbers per cell at different times were similar in enucleated and nucleated cells. Bacteria with actin-rich tails were detected in both cytoplasts and nucleated cells. Lastly, cytoplasts were similarly infected 2 and 24 h after enucleation, suggesting that short-lived mRNAs were not involved in the infection. Productive S. flexneri infection could thus take place in cells unable to modulate gene transcription, RNA processing, or nucleus-dependent signaling cascades.

Screening differentially expressed genes in denucleated K562 cells with restriction display technique / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen differentially expressed genes in cytochalasin B (CB)-induced denucleated K562 cells by restriction display (RD) technique.</p><p><b>METHODS</b>The total RNA was isolated and purified from K562 cells before and after CB (10 mug/ml) treatment. The mRNA from both treated and untreated K562 cells were reversely transcribed into cDNA, and the differentially expressed genes were separated using RD technique combined with polyacrylamide gel electrophoresis and sliver staining, followed by cloning, sequencing and homology analysis against GenBank database of these genes.</p><p><b>RESULTS</b>Seven differentially expressed genes were identified in CB-treated cells including aquaporin 1 (AQP1) gene, which was verified to be up-regulated after CB treatment by RT-PCR.</p><p><b>CONCLUSION</b>AQP1 gene might be in close association with the regulation of denucleation processes and CB-induced proliferation inhibition of K562 cells.</p>

Effect of Cytochalasin B on Survivability and in Vitro Development of Mouse Oocyte Frozen by Vitrification / 대한산부인과학회잡지

OBJECTIVE: The purpose of this study was to evaluate the effect of CCB treatment on the survivability and in vitro development of mouse oocyte frozen by vitrification. METHODS: Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then exposed to EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes on EM grid was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M sucrose at 37degrees C for 3 minutes. This was followed by 0.5 M and 0.25 M sucrose for 3 minutes, each. We compared the survivability, cleavage and developmental rate of mouse frozen by vitrification between CCB treated and non-treated groups. Chi-square was used to determine statistical significance. statistical significance was defined as p<0.05. RESULTS: Survivability (79.3%) and developmental rate into blastocyst (52.3%) of mouse oocyte were markedly decreased after vitrification. There were no significant differences between CCB treated and non- treated groups regarding survivability of oocyte frozen by vitrification (80.3% vs 78.5%). The developmental rate into 2-cell in CCB treated group was significantly higher than that in non-treated group (69.7% vs 61.9%, p<0.05). The developmental rate into blastocyst in CCB treated group was higher than that in non-treated group (54.9% vs 51.5%), but the difference was not significant. CONCLUSION: Survivability of mouse oocyte could not be affected by CCB treatment and developmental rate into 2-cell was improved in CCB treated group. It is suggested that CCB treatment prior vitrification improve stability of cytoskeleton and then improve fertilization and early stage embryo development.

The Effect of Cytochalasin B on Cytoskeletal Stability of Mouse Oocyte Frozen by Vitrification / 대한불임학회지

OBJECTIVE: The purpose of this study was to evaluate the effect of Cytochalasin B (CCB) on the cytoskeletal stability of mouse oocyte frozen by vitrification. METHODS: Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then vitrified in EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M, 0.5 M and 0.25 M sucrose at 37degress C for 3 minutes, each. These oocytes were fixed in 4% formaldehyde for an hour and then washed in PPB for 15 minutes 3 times, then incubated in PPB containing anti-tubulin monoclonal antibody at 4degress C overnight. And then, the oocytes were incubated with FITC-conjugated anti-mouse IgG and propidium iodide (PI) for 45 minutes. Pattern of microtubules and microfilaments of oocytes were evaluated with a confocal microscope. RESULTS: The rate of oocytes containing normal microtubules and microfilaments was significantly decreased after vitrification. The rate of oocyte containing normal microtubules in CCB treated group was higher than those in non-treated group (53.7% vs. 48.9%), but the difference was not significant. The rate of oocyte containing normal microfilaments in CCB treated group was significantly higher than those in non-treated group (64.5% vs. 38.3%, p<0.05).CONCLUSION: Microfilaments stability could be improved by CCB treatment prior to vitrification. It is suggested that CCB treatment prior to vitrification improve stability of cytoskeleton and then increase success rate in IVF-ET program using vitrification and thawing oocyte.

The Effect of Cytochalasin B on Cytoskeletal Stability of Mouse Oocyte Frozen by Vitrification / 대한불임학회지

OBJECTIVE: The purpose of this study was to evaluate the effect of Cytochalasin B (CCB) on the cytoskeletal stability of mouse oocyte frozen by vitrification. METHODS: Mouse oocytes retrieved from cycle stimulated by PMSG and hCG were treated by CCB and then vitrified in EFS-30. These oocytes were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing of the oocytes was carried out at room temperature for 5 seconds, then the EM grid was placed into 0.75 M, 0.5 M and 0.25 M sucrose at 37degress C for 3 minutes, each. These oocytes were fixed in 4% formaldehyde for an hour and then washed in PPB for 15 minutes 3 times, then incubated in PPB containing anti-tubulin monoclonal antibody at 4degress C overnight. And then, the oocytes were incubated with FITC-conjugated anti-mouse IgG and propidium iodide (PI) for 45 minutes. Pattern of microtubules and microfilaments of oocytes were evaluated with a confocal microscope. RESULTS: The rate of oocytes containing normal microtubules and microfilaments was significantly decreased after vitrification. The rate of oocyte containing normal microtubules in CCB treated group was higher than those in non-treated group (53.7% vs. 48.9%), but the difference was not significant. The rate of oocyte containing normal microfilaments in CCB treated group was significantly higher than those in non-treated group (64.5% vs. 38.3%, p<0.05).CONCLUSION: Microfilaments stability could be improved by CCB treatment prior to vitrification. It is suggested that CCB treatment prior to vitrification improve stability of cytoskeleton and then increase success rate in IVF-ET program using vitrification and thawing oocyte.

Effects of glutamate on dehydroascorbate uptake and Its enhanced vulnerability to the peroxidation in cerebral cortical slices

Pro-oxidant properties of ascorbate have been studied with uses of brain tissues and neuronal cells. Here we address potential mechanism of ascorbate coupling with glutamate to generate oxidative stress, and the role which oxidized ascorbate (dehydroascorbate) transport plays in oxidative neuronal injury. Ascorbate in neurones can be depleted by adding glutamate in culture medium since endogenous ascorbate can be exchanged with glutamate, which enhances ascorbate/ dehydroascorbate transport by depleting ascorbate in the neurons with the glutamate-heteroexchange. However, ascorbate is known readily being oxidized to dehydroascorbate in the medium. Glutamate enhanced the dehydroascorbate uptake by cells via a glucose transporter (GLUT) from extracellular region, and cytosolic dehydroascorbate enhanced lipid peroxide production and reduced glutathione (GSH) concentrations. Iso-ascorbate, the epimer of ascorbate was ineffective in generating the oxidative stress. These observations support the current concept that the high rates of dehydroascorbate transport via a GLUT after the release of ascorbate by glutamate leads to peroxidation, the role of glutamate on ascorbate/ dehydroascorbate recycling being critical to induce neuronal death via an oxidative stress in the brain injury.

Anticonvulsant effect of cytoskeletal depolymerizers in combination with potassium channel opener and adenylate cyclase activator; a causative link with nerve growth factor?

Anticonvulsant effect of cytoskeletal depolymerizing drugs in combination with potassium channel (KATP) opener and adenylate cyclase activator was evaluated in animal models of epilepsy. Seizures were induced in the animals by subjecting them to maximal electroshock (MES) or by injecting a chemical convulsant, pentylenetetrazole (PTZ). Moreover a correlation with the nerve growth factor (NGF) was also investigated. The anticonvulsant effect of minoxidil (1200 micrograms/kg i.p.) and Deacetylforskolin (600 micrograms/kg i.p.) was significantly enhanced in the mice pre-treated with cytoskeletal depolymerizing drugs. On the other hand nerve growth factor potentiated the convulsive phenomenon and decreased the seizure threshold in both the electroshock and chemically induced convulsions. Another interesting feature was the interaction of cytochalasin B, a microfilament disrupter in preventing the action of mNGF and PTZ. This study demonstrates the importance of interaction between cytoskeletal structures and signalling molecules in determining the convulsive threshold. This study clearly points to the importance of the nerve growth factor in convulsive phenomenon.

Myosin V and iNOS expression is enhanced in J774 murine macrophages treated with IFN-gamma

Actin-based motor protein requirements and nitric oxide (NO) production are important features of macrophage activity during phagocytosis or microbicidal processes. Different classes of myosins contribute directly or indirectly to phagocytosis by providing mechanical force for phagosome closure or organelle movement. Recent data have shown the presence of myosins IC, II, V and IXb in phagosomes of bone marrow-derived murine macrophages. In our investigation we demonstrated the presence of different classes of myosins in J774 macrophages. We also analyzed the effect of gamma interferon (IFN-gamma), with or without calcium ionophore or cytochalasin B, on myosins as well as on inducible nitric oxide synthase (iNOS) expression and NO production. Myosins IC, II, Va, VI and IXb were identified in J774 macrophages. There was an increase of myosin V expression in IFN-gamma-treated cells. iNOS expression was increased by IFN-gamma treatment, while calcium ionophore and cytochalasin B had a negative influence on both myosin and iNOS expression, which was decreased. The increases in NO synthesis were reflected by increased iNOS expression. Macrophages activated by IFN-gamma released significant amounts of NO when compared to control groups. In contrast, NO production by calcium ionophore- and cytochalasin B-treated cells was similar to that of control cells. These results suggest that IFN-gamma is involved in macrophage activation by stimulating protein production to permit both phagocytosis and microbicidal activity

Chromosome aberrations and micronuclei in lymphocytes of medical radiation workers exposed to ionizing radiation below dose limit

Ionizing radiation induces various kinds of DNA damage in which may lead to chromosomal aberrations [CA]. Inspite of growing importance in the risk assessment, the dose yield kinetics of CA and their implications for dose assessment are not well established in exposures to low level radiation. In the present study, cytochalasin-B blocked micronucleus assay and metaphase analysis were used as test systems to monitor hospital radiation workers who received chronic low dose ionizing radiation below dose limit. Heparinized blood samples were taken from healthy non-smoker radiology and radiotherapy workers occupationally exposed to X and gamma rays and healthy population whose duties do not expose them to radiation sources and chemical agents. The whole body dose was measured by film badge. Lymphocytes were cultured in RPMI-1640 supplemented with 15 percent FBS, and metaphase spread was prepared using standard cytogenetic method. Cytochalasine-B [3 micro g/ml] treatment was used for binuclei micronuclei assay. 100 mitoses and 1000 binuclei lymphocytes were scored for CA and micronuclei [MN] respectively. Results show a high frequency of CA mainly deletions and simple breaks in radiation workers compared to control [p<0.001].Also results show the mean MN/cell was significantly [p<0.001] higher in radiation workers [0.035] when compared to non exposed individuals [0.022]. A relatively high frequency of MN and CA formation in lymphocytes of radiation workers compared to non exposed individuals might be due to an accumulation of initial DNA damage in people exposed to chronic doses of radiation leading to detectable genetic damages. These observations might imply that the current occupational exposure levels might be inadequate to prevent an increase in chromosome damage rate

The Effect of Antiproliferative Drugs on the Collagen Matrix Cultured with Retinal Pigment Epithelial Cell and Choroidal Fibroblast

PURPOSE: Epiretinal membrane in proliferative vitreoretinopathy (PVR) may cause tractional retinal detachment after vitreoretinal surgery. It has been thought that the proliferative membrane is mainly composed of choroidal fibroblasts and retinal pigment epithelial cells. Inspite of the technical advances, the treatment of PVR is still difficult. Therefore, the need for phamarcologic treatment of proliferative vitreoretinopathy is increasing. METHODS: In vitro models of proliferative vitreoretinopathy allow to identify the factors which may inhibit proliferation and contraction of collagen matrix by choroidal fibroblast and retinal pigment epithelial cells. Cultured choroidal fibroblasts and the RPE cells were plated to the collagen matrix and antiproliferative drugs was tested. RESULTS: Each antiproliferative drug showed the inhibition of collagen matrix contraction at following concentration: colchicine(0.1 microgram/ml), puromycin(1~10 microgram/ml), cytochalasin B(0.05 microgram/ml). Transmission electron micrograph of collagen matrices showed dense collagen fibers surrounding choroidal fibroblast and fine collagen fibers surrounding RPE cell. Scanning electron micrograph of collagen matrices contaning colchicine, puromycin, or cytochalasin B showed that collagen fibers were well preserved without distortion. All collagen matrices containing RPE cells showed more fine collagen fibers than those containing choroidal fibroblasts. CONCLUSION: Colchicine, puromycin, cytochalasin B showed inhibitory effect on cell mediated contraction in addition to potent antiproliferative effect. Retinal pigment epithelial cell played less significant role in causing PVR than choroidal fibroblast.