Combination Of Ionomycin Research Articles

HL‐60 Cells Alternative Response to an Ionomycin PMA Cocktail

The combination of Ionomycin and phorbol 12‐myristate 13‐acetate (PMA) cocktail has been used to increase the proliferation of normal mouse and human T cells and PMA alone has been used to cause the differentiation of the myeloid leukaemia HL‐60 cells in culture to a macrophage‐like adherent population. Contrariwise, the cocktail was linked to apoptosis in acute HL‐60 cells. This distinction has also been noted in the literature by Wang, et al. (2014), who concluded that Jurkat T cells were inhibited at S‐phase and G2/M‐phase by the cocktail. When we cultured the HL‐60 cells for 48 hours in the presence of the cocktail, we also saw no growth and significant death. Originally, we suspected this distinction was due to the biological differences between normal cells and tumor cells; however, further investigation revealed a discrepancy within the literature. While HL‐60, Jurkat, and Glioma cells (Han 2013) are inhibited by the cocktail, breast cancer cell lines were MDA‐MB‐435 and MDA‐MB‐231 had enhanced invasion (Yiu 2006). Future experiments including a cell cycle analysis and apoptosis assay will be used to test other cancerous and non‐cancerous cell lines in hopes of elucidating a pattern for the effects of the ionomycin PMA cocktail.Support or Funding InformationCarl Savino and Olga Gerych‐Savino Endowment, The Geneseo Foundation, and David Wolf, D.O.

Production of collared peccary (Pecari tajacu Linnaeus, 1758) parthenogenic embryos following different oocyte chemical activation and in vitro maturation conditions

To optimize the protocols for assisted reproductive techniques (ARTs) in collared peccary (Pecari tajacu Linnaeus, 1758), we evaluated various conditions for oocyte in vitro maturation (IVM) and chemical activation. Initially, we assessed the IVM rates, cumulus-oocyte complex (COC) quality, and oocyte morphometry in the absence or presence of epidermal growth factor (EGF). There was no difference between the COCs matured in absence or presence of EGF for the expansion of cumulus cells (97.6% ± 1.2 vs. 100% ± 0.0), presence of first polar body (65.9% ± 1.2 vs. 70.5% ± 1.8), nuclear status in second metaphase (62.5% ± 11.6 vs. 68.4% ± 4.9), cytoplasmic maturation (100.0% ± 0.7 vs. 75.0% ± 0.7), reactive oxygen species levels (0.5 ± 0.2 vs. 0.3 ± 0.1), and mitochondrial membrane potential (1.1 ± 0.2 vs. 1.1 ± 0.1). However, the zona pellucida thickness of matured COCs was reduced in the presence of EGF. Thus, the EGF group was used for further experiments. The oocytes were artificially activated with ionomycin and four secondary activator combinations [6-dimethylaminopurine (6D), 6D and cytochalasin B (6D + CB), cycloheximide (CHX), and CHX and CB (CHX + CB)]. The effect of immature COCs based on cumulus cell layers and cytoplasm homogeneity (GI and GII or GIII COCs) on embryonic development and quality was evaluated. There was no difference in the cleavage rates among the groups of secondary activators. The cleavage rates of embryos derived from GI/GII and GIII COCs were greater than 72.2% and 25.0%, respectively. Moreover, treatment with CHX showed a reduction in the cleavage rate of embryos derived from GIII COCs when compared to the cleavage rate of embryos derived from GI/GII COCs (P < 0.05). Nevertheless, higher rates of blastocyst/total GI and GII COCs were observed in the 6D group (27.6% ± 0.3) compared to CHX group (6.9% ± 0.3). Additionally, only 6D treatment resulted in the production of embryos derived from GIII COCs (25.0% ± 0.2). The percentage of the ICM/total cell ratio was also greater in blastocysts derived from 6D (42.5% ± 19.0), 6D + CB (37.9% ± 21.9), and CHX + CB (43.8% ± 19.6) groups when compared to CHX (3.6% ± 0.1) group. Thus, the combination of ionomycin and 6D could produce collared peccary embryos by activation of both GI/GII COCs and GIII COCs. These optimized IVM conditions using EGF and chemical activation using ionomycin and 6D in collared peccaries form the first steps for establishing ARTs to conserve this species.

Effect of immunosuppressive drugs on cytokine production in canine whole blood stimulated with lipopolysaccharide or a combination of ionomycin and phorbol 12-myristate 13-acetate.

A pharmacodynamic assay has been previously developed to monitor ciclosporin treatment in dogs by assessing inhibition of cytokine transcription after whole blood stimulation with 12‐myristate 13‐1 acetate and ionomycin (PMA/I). In this study, whole blood stimulation with either PMA/I or lipopolysaccharide (LPS) was used to assess the effect of multiple drugs (azathioprine, ciclosporin, mycophenolate, leflunomide and prednisone) after a 7‐day treatment course on production of cytokines measured with a multiplex assay in healthy dogs (n = 4 for each treatment). Interleukin‐10 (IL‐10), interferon gamma (IFN γ) and tumour necrosis factor alpha (TNF α) were significantly activated by PMA/I stimulation and IL‐6, IL‐10 and TNF α by LPS stimulation, in the absence of immunosuppressive drugs. After ciclosporin treatment, IL‐10, IFN γ and TNF α production was significantly reduced after stimulation with PMA/I compared to pre‐treatment. After prednisone treatment, TNF α production was significantly reduced after stimulation with PMA/I or LPS compared to pre‐treatment. No significant change was observed after treatment with azathioprine, leflunomide or mycophenolate. This methodology may be useful to monitor dogs not only treated with ciclosporin, but also with prednisone or a combination of both. Further studies are needed to assess the use of this assay in a clinical setting.

Artificial activation of bovine and equine oocytes with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine in low or high calcium concentrations

Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the parthenogenetic activation of oocytes in these animals. The oocytes were collected, in vitro matured for 24 to 30 h and activated artificially, in the presence of low or high concentrations of calcium, with combinations of calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP). For assessment of activation rates, oocytes were stained with Hoechst 33342 and observed under an inverted microscope. We showed that all combinations of drugs were equally efficient in activating bovine oocytes, with the best results obtained when high concentrations of calcium were adopted. For equine oocytes, high concentrations of calcium were not beneficial for the parthenogenetic activation and the combination of ionomycin with either 6-DMAP or roscovitine was effective in inducing artificial activation of oocytes. We believe that our preliminary findings provide some clues for the development of a better AOA protocol to be used with these species.

Chemical Activation with a Combination of Ionomycin and Dehydroleucodine for Production of Parthenogenetic, ICSI and Cloned Bovine Embryos

The aim of this study was to evaluate the potential of dehydroleucodine (DhL), a new drug isolated from a medicinal herb used in Argentina, for activation of bovine oocyte. Several DhL concentrations and exposure times after ionomycin (Io) treatment were tested. The optimal DhL treatment, found for parthenogenetic development, was employed to produce bovine embryos by intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). The best parthenogenic embryo developments were observed with 5 μM Io for 4 min followed by 5 μM DhL concentration and after 3-h exposure time (52.3% cleavage; 17.4% morulae; 7.3% blastocyst; n = 109). This treatment generated no significant differences with standard Io plus 6-dimethylaminopurine (DMAP) treatment in preimplantation embryo development. In our conditions, the embryo development reached after ICSI and SCNT assisted by the DhL treatment did not differ in terms of cleavage and blastocyst development from activation with standard Io plus DMAP treatment (p > 0.05). In conclusion, DhL utilization to activate oocytes and induce development of parthenogenotes, ICSI-embryos or SCNT-embryos is reported here for first time.

Salbutamol Modulates the Balance of Th1 and Th2 Cytokines by Mononuclear Cells from Allergic Asthmatics

Background: There is evidence that excessive use of inhalational β₂-agonists induces the deterioration of asthma. Although the exact mechanism of this remains to be elucidated, overuse of β₂-agonists may impair the Th1/Th2 balance in asthmatic airways. The aim of the present study was to evaluate whether salbutamol, a representative inhalational β₂-agonist, modifies the production of Th1- and Th2-type cytokines by mononuclear cells separated from patients with asthma and healthy volunteers. Methods: Peripheral blood mononuclear cells (PBMCs) obtained from 8 healthy volunteers and 10 patients with mild persistent asthma allergic to house dust mites were treated with either salbutamol or medium alone. PBMCs were then stimulated with either medium alone, house dust mite (Dermatophagoides farina, Df) allergen or a combination of ionomycin plus phorbol 12-myristate 13-acetate ester (PMA). Concentrations of IFN-γ, IL-13, TNF-α and RANTES in the cell supernatants were measured using ELISA. Results: In PBMCs from healthy volunteers, salbutamol did not modify IFN-γ production, but increased the spontaneous production of IL-13. In contrast, salbutamol significantly inhibited the spontaneous and ionomycin- plus PMA-stimulated production of IFN-γ by PBMCs from asthmatics. Salbutamol significantly enhanced both spontaneous and Df-induced production of IL-13 by PBMCs from asthmatics. Salbutamol did not modify the production of TNF-α. Finally, salbutamol enhanced the production of RANTES induced by Df allergen in asthmatics. Conclusions: Salbutamol inhibits IFN-γ and enhances IL-13 production by PBMCs from asthmatics. These effects would promote a Th1/Th2 imbalance in the airways and may therefore contribute to the deterioration of asthma.

27 EFFECT OF ACTIVATION METHODS AND BIOPSY SAMPLING ON NUCLEAR TRANSFER BLASTOCYST PRODUCTION AND CLONING EFFICIENCY IN A HORSE

In the horse, rates of blastocyst production after nuclear transfer are low. This study was conducted to examine the effect of activation via injection of different volumes of sperm extract or via injection of murine mRNA for PLC-ζ, a sperm-specific protein which induces Ca2+ oscillations in all species thus far studied, on blastocyst development after nuclear transfer. Two biopsy samples from the same horse were also examined for cloning efficiency. Donor fibroblasts cultured from a skin biopsy taken from a 19-year-old mare were treated with 15 µm R-roscovitine for 18 to 24 h before direct injection into enucleated ooplasts. Ionomycin treatment, 5 µm for 4 min, was used in all activation treatments, as the combination of ionomycin with sperm extract provided the highest blastocyst development and foaling rates in our previous report (Hinrichs et al. 2007 Reproduction 134, 319–325). All treatments were followed by incubation in 2 mm 6-dimethylaminopurine for 4 h. Differences in blastocyst development between treatments were analyzed using Fisher's exact test. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 s at a fixed pipette diameter and injection pressure, and then treated with ionomycin. The blastocyst rate (9.8%) for 0.1 s was significantly higher than that for 0.2 s (0%) or 0.8 s (1.4%). In Experiment 2, murine PLC-ζ mRNA (0.25 µg µL–1) was injected into reconstructed oocytes 20 to 30 min before or after ionomycin treatment and compared with a control treatment (injection of 2 to 4 pL sperm extract 20 to 50 min after ionomycin exposure). There were no differences in blastocyst development among treatments (0, 4.5, and 5.6%, respectively). Transfer of 10 blastocysts produced in Experiments 1 and 2 resulted in 5 pregnancies: however, all were lost before 70 days of gestation. In Experiment 3, a second skin biopsy was obtained from the same mare and cells from this tissue sample were used for nuclear transfer concurrently with cells from the first sample, using the control method above. Blastocyst production was higher using cells from the second biopsy sample (4/23 v. 0/23; P = 0.05). Transfer of these four blastocysts yielded four pregnancies, two of which continued to term and produced viable foals. These results indicate that blastocyst development after injection of sperm extract is dependent upon the volume injected, that injection of murine PLC-ζ mRNA does not improve blastocyst formation under the given conditions, and that the efficiency of cloning may vary with biopsy sample even from the same animal.

232 PARTHENOGENETIC DEVELOPMENT AND PLOIDY OF BOVINE OOCYTES FOLLOWING VARIOUS CHEMICAL ACTIVATION REGIMENS

Bovine oocytes treated using various combinations of ionomycin (Ion), cycloheximide (CHX), and cytochalasin B (CCB) were evaluated for developmental rates and ploidy status. Metaphase II oocytes were allocated 5 treatment groups, and the groups were treated as follows: Group 1: 5 µm Ion for 5 min; Group 2: Ion + 10 µg mL–1 CHX for 5 h; Group 3: Ion + 10 µg mL–1 CHX + 5 µg mL–1 CCB for 1 h + 10 µg mL–1 CHX for 4 h; Group 4: Ion + 10 µg mL–1 CHX + 5 µg mL–1 CCB for 3 h + 10 µg mL–1 CHX for 2 h; and Group 5: Ion + 10 µg mL–1 CHX + 5 µg mL–1 CCB for 5. Difference among groups was analyzed using one-way ANOVA by SPSS 10.0 (SPSS, Inc., Chicago, IL, USA). In Experiment 1, 430 oocytes in 4 replicates were compared for the extrusion rate of the second polar body (PB) at 8 h after Ion treatment among groups. Group 5 exhibited significantly (P &lt; 0.05) lower rates of second PB extrusion than did Groups 1–4 (22% v. 53–67%). Experiment 2 compared the rates of cleavage at 48 h and development to the blastocyst stage at 216 h after Ion treatment among groups. A total of 536 oocytes were used in 5 replicates. Parthenotes in Group 1 showed lower rates of cleavage and blastocyst development than those in other groups (20% and 1% v. 53–67% and 6–31%). Among the groups, parthenotes in Group 5 showed significantly (P &lt; 0.05) higher blastocyst development. In Experiment 3, at 8 h after Ion treatment, oocytes from Groups 2, 3, and 5 were divided into two subgroups based on the presence or absence of the second PB, and assessed for cleavage rates and ploidy in 239 2-cell-stage parthenotes in 4 replicates, as described earlier by King et al. (1979 Vet. Sci. Commun. 3, 51–56). The cleavage rates did not differ among activation treatments, or by the presence or absence of the second PB in any activation group. The haploid rate was significantly (P &lt; 0.05) higher in Group 2 than in Groups 3 and 5 (38% v. 19% and 0%, respectively). The diploid rate was significantly (P &lt; 0.05) higher in Group 5 than in Groups 2 and 3 (88% v. 69% and 45%, respectively). In Experiment 4, the diploid rate of Group 2 blastocyst-stage parthenotes was 100% (4/4), whereas the diploid rates of Groups 3 and 5 blastocyst-stage parthenotes were 50% (6/12) and 71% (17/24), respectively, but the rates did not differ among groups. These results indicate that oocyte activation by CHX/CCB for 5 h after Ion treatment could enhance parthenogenetic development in bovines with higher rates of diploidy by preventing the extrusion of the second PB.

Intracytoplasmic sperm injection of frozen-thawed bovine oocytes and subsequent embryo development.

Oocyte cryopreservation and intracytoplasmic sperm injection (ICSI) are advantageous to expand their usefulness in genetic engineering. Oocytes matured for 22 hr were vitrified in droplets of cryoprotectants (3.2 M ethylene glycol (EG), 2.36 M dimethyl sulfoxide (DMSO), 0.6 M sucrose) on copper electron microscope (EM) grids. After being warmed, the oocytes were cultured in IVM medium for an additional 2 hr. Sperm treated with dithiothreitol were utilized for ICSI. Oocytes injected with sperm were activated by combination of ionomycin with cycloheximide (CHX). The ICSI oocytes were compared for the rates of pronuclear formation, development, cell number, and the ratio of ICM to those of fresh ICSI and IVF control. The proportion of 2PN formation was significantly higher in IVF control (Group 1) than those in other treated groups. Among the treated groups a significant lower 2PN formation was observed in IVF-frozen-thawed than in ICSI-fresh and frozen-thawed groups. Cleavage rates in IVF-frozen-thawed and ICSI-frozen-thawed groups were significantly lower than those of IVF control and ICSI-fresh groups. In ICSI groups, the rates of cleavage and blastocyst in fresh oocytes were significantly higher than in frozen-thawed. Development rates into blastocysts in the ICSI-fresh and frozen-thawed groups were significantly lower than that of IVF control. Total cell number was significantly lower in both frozen-thawed IVF and ICSI groups than those in IVF-control and ICSI-fresh groups. However, the rates of the remaining cells that were found in the ICM were significantly higher in both frozen-thawed IVF and ICSI than in the IVF-control and ICSI-fresh groups. The results indicated that frozen-thawed bovine oocytes were suitable for ICSI procedure.

Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14

We investigated the properties of metacyclic trypomastigotes of non-virulent Trypanosoma cruzi clone CL-14, as compared to the parental isolate CL. In contrast to the CL isolate, which produces high parasitemias in mice, metacyclic forms of clone CL-14 failed to produce patent infection. In vitro, the number of clone CL-14 parasites that entered epithelial HeLa cells, after 1 h incubation, was approximately four-fold lower than that of the CL isolate and at 72 h post-infection intracellular replication was not apparent whereas cells infected with the CL isolate contained large number of parasites replicating as amastigotes. CL isolate metacyclic forms were long and slender, with the kinetoplast localised closer to the nucleus than to the posterior end, whereas clone CL-14 parasites were shorter, with the kinetoplast very close to the posterior end. Cysteine proteinase cruzipain and trans-sialidase activities were lower in CL isolate than in clone CL-14. The surface profile was similar, except that the expression of gp82, the stage-specific glycoprotein that promotes CL isolate mucosal infection in vivo and host cell invasion in vitro, was greatly reduced on the surface of clone CL-14 metacyclic forms. Genistein, a specific inhibitor of protein tyrosine kinase, which is activated in CL isolate by binding of gp82 to its host cell receptor, did not affect host cell entry of clone CL-14. In contrast with CL isolate, the infectivity of clone CL-14 was not affected by phospholipase C inhibitor U73122 but was diminished by a combination of ionomycin plus NH4Cl, which releases Ca2+ from acidic vacuoles. Internalisation of clone CL-14, but not of CL isolate, was significantly increased by treating parasites with neuraminidase, which removes sialic acid from the mucin-like surface molecule gp35/50. Taken together, our data suggest an association between the non-virulence of clone CL-14 metacyclic forms and the reduced expression of gp82, which precludes the activation of signal transduction pathways leading to effective host cell invasion.

Assessment of the cell-mediated immune response in chickens by detection of chicken interferon-γ in response to mitogen and recall Newcastle disease viral antigen stimulation

The potential of a capture enzyme-linked immunosorbent assay (ELISA) specific for chicken interferon-γ (ChIFN-γ) has been evaluated as a tool to assess cell-mediated immunity (CMI) in the chicken. In a first step, ChIFN-γ production and cell proliferation of mitogen-activated chicken splenocytes have been compared. In general, for each of the stimulation conditions where significant proliferation was observed, production of ChIFN-γ could be measured by ELISA. In our hands, the combination of ionomycin and phorbol-12-myristate 13-acetate or the use of recombinant chicken interleukin-2 gave the most satisfactory results. Then, the CMI response induced by live or killed Newcastle disease virus (NDV) vaccines has been evaluated sequentially by ex vivo antigen-specific ChIFN-γ production and cell proliferation of splenocytes from immune chickens. The ex vivo data showed that both types of NDV vaccines are capable of stimulating CMI responses to NDV in chickens as measured by the ChIFN-γ ELISA. However, most of the chickens vaccinated with the live vaccine produced ChIFN-γ after antigen recall stimulation, from 2 to 4 weeks after vaccination, when only some chickens vaccinated with the inactivated vaccine showed a specific response 4 weeks after vaccination. No significant proliferative responses to either NDV vaccine were detectable during the 4 weeks of the study. From our results, it appears that antigen-specific ChIFN-γ production can be used as a good indicator of actively acquired immunity to NDV and that the sensitivity range of the capture ELISA test is well adequate to measure ex vivo release of ChIFN-γ.

Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14

We investigated the properties of metacyclic trypomastigotes of non-virulent Trypanosoma cruzi clone CL-14, as compared to the parental isolate CL. In contrast to the CL isolate, which produces high parasitemias in mice, metacyclic forms of clone CL-14 failed to produce patent infection. In vitro, the number of clone CL-14 parasites that entered epithelial HeLa cells, after 1 h incubation, was approximately four-fold lower than that of the CL isolate and at 72 h post-infection intracellular replication was not apparent whereas cells infected with the CL isolate contained large number of parasites replicating as amastigotes. CL isolate metacyclic forms were long and slender, with the kinetoplast localised closer to the nucleus than to the posterior end, whereas clone CL-14 parasites were shorter, with the kinetoplast very close to the posterior end. Cysteine proteinase cruzipain and trans-sialidase activities were lower in CL isolate than in clone CL-14. The surface profile was similar, except that the expression of gp82, the stage-specific glycoprotein that promotes CL isolate mucosal infection in vivo and host cell invasion in vitro, was greatly reduced on the surface of clone CL-14 metacyclic forms. Genistein, a specific inhibitor of protein tyrosine kinase, which is activated in CL isolate by binding of gp82 to its host cell receptor, did not affect host cell entry of clone CL-14. In contrast with CL isolate, the infectivity of clone CL-14 was not affected by phospholipase C inhibitor U73122 but was diminished by a combination of ionomycin plus NH 4Cl, which releases Ca 2+ from acidic vacuoles. Internalisation of clone CL-14, but not of CL isolate, was significantly increased by treating parasites with neuraminidase, which removes sialic acid from the mucin-like surface molecule gp35/50. Taken together, our data suggest an association between the non-virulence of clone CL-14 metacyclic forms and the reduced expression of gp82, which precludes the activation of signal transduction pathways leading to effective host cell invasion.

70PRODUCTION OF CLONES BY FIBROBLAST NUCLEAR TRANSFER FROM AN X-AUTOSOME TRANSLOCATION CARRIER COW

Poor reproductive outcome associated with chromosome anomalies is well documented in humans and domestic animals. In cattle, female carriers of Robertsonian and X-autosome translocations tend to be repeat breeders, probably due to synaptic difficulties during meiotic prophase of gametogenesis. Although viable offspring have been obtained through somatic cell nuclear transfer (NT) using adult or fetal cells from normal animals in various species, there have been no reports to date on the application of this technology to translocation carriers. Since NT circumvents meiotic problems encountered by translocation carriers, we used this approach to generate cloned embryos, fetuses and calves from a subfertile Limousin-Jersey crossbred cow previously identified as a carrier of an X-autosome translocation. Primary cultures were established from ear skin biopsies, and used at the 5th or 6th passage for NT. Recipient oocytes were enucleated at 19h post-maturation (hpm), fused with individual fibroblasts by a single electrical pulse (1.4KVcm−1, 40μs) and activated at 24hpm with a combination of ionomycin (5μM, 5min) and cycloheximide (10μgmL−1). Reconstructed eggs were cultured in SOF at 39°C in a humidified low oxygen atmosphere. In 33 runs involving 2470 oocyte-donor cell complexes, cleavage and blastocyst rates were 88% (2173/2470) and 36% (889/2470), respectively. Two or three blastocysts (Day 7±1) were transferred into each recipient, previously synchronized with a combination of CIDR, GnRH and PGF2α. Ultrasonography was performed at Days 28 to 60 and at Days 90 and 150. Pregnancy was confirmed on Day 28 in 10 of a total of 22 recipients, 2 of which were later found to be carrying twin fetuses. Of 60 embryos transferred, 11 (18.3% of embryos) survived to Day 42, 6 (10%) to Day 60, and 4 (6.6%) to Day 90. A Day-94 fetus was surgically retrieved to examine the synaptic pattern of meiocytes in fetal ovaries. The fetus and internal organs were normal in appearance, and of normal size (16.5cm C-R length). The X-autosome translocation was confirmed in blood cultures, and synaptic anomalies involving chromosome 23 and the X chromosome were detected in fetal ovaries. Another clone was delivered by C-section at 276 days but died within 1h of delivery, while one singleton pregnancy is still ongoing at &amp;gt;200 days. These results demonstrate that NT can be used to produce embryos, fetuses and offspring from an X-autosome translocation carrier, with the potential to facilitate study of synaptic behaviour of female germ cells, and X-inactivation in different cell lineages of cloned blastocysts, and to generate individuals with otherwise poor reproductive prospects. [Supported by NSERC, Canada and OMAFRA]

341APOPTOSIS IN SOMATIC CELL CLONED AND EGFP TRANSGENIC BOVINE EMBRYOS

The overall success rate achieved by cloning techniques to date is low, mainly due to deficiencies in nuclear reprogramming, gene expression and DNA fragmentation, which result in early and late embryonic losses. This study was carried out to compare the incidences of DNA fragmentation during development of IVF, parthenote (PT), nuclear transfer (NT) and transgenic-cloned (TG) embryos. Terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL) with propidium iodide counterstaining was used for determination of DNA fragmentation and total cell number. Donor cells were fetal fibroblasts with or without transfection with EGFP, and cultured in DMEM+15% FCS until confluent, for up to 5 days. At 19h post maturation (hpm), enucleated oocytes were reconstructed with donor cells and activated at 24hpm with the combination of ionomycin (5μM, 5min) and cycloheximide (10μg/mL, 5h) after electric fusion by a single DC pulse (1.6KV/cm, 60μs) delivered by a BTX 200. Parthenotes were produced by the same activation protocol at 24hpm. The eggs and control IVF embryos were cultured in CR1aa at 39°C in a humidified atmosphere of 5% CO2 in air. Differences among groups were analyzed using one-way ANOVA after arc-sine transformation of proportional data. Embryos at the 8-cell stage in all treatments, IVF, PT, NT and TG, showed DNA fragmentation. The apoptotic cell index (total number of apoptotic nuclei/total number of nuclei) of Day 7 blastocysts was significantly (P&amp;lt;0.05) higher in TG and NT embryos (17/91, 18.6±4.0% and 13/94, 13.8±4.7%, respectively) compared to IVF and PT embryos (9/122, 7.4±3.4% and 8/93, 8.6±2.9%, respectively). TUNEL positive cells were detected in almost all blastocysts at Day 7 and were mainly observed in the ICM. The DNA fragmentation ratio of the ICM in the blastosysts at Day 7 (number of apoptotic nuclei in the ICM/total number of apoptotic nuclei in the blastocyst) was significantly (P&amp;lt;0.05) higher in TG embryos (64.7±21.4%) than in IVF (44.4±28.0%), PT (50.0±18.6%) or NT (53.0±32.5%) embryos. These results indicate a higher occurrence of DNA fragmentation observed in NT and TG embryos when compared to IVF and PT embryos. In addition, ICM of TG blastocysts revealed a high DNA fragmentation ratio, which may be related to early embryonic loss after transfer of the resulting embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 300012-05-3-SB010 and Cho-A Pharm. Co. LTD.]

33DEVELOPMENT OF BOVINE EMBRYOS CLONED WITH FETAL FIBROBLASTS ARRESTED AT G0/G1 PHASE BY DIFFERENT TREATMENTS

Numerous factors have an effect on the development of cloned embryos, and one of the most important might be the synchronization between donor nuclei and recipient ooplasts. The objective of this study was to examine the effect of donor cell treatments for G0/G1 synchronization and the donor cell type on development and incidence of apoptosis in cloned cattle embryos. Primary cultures were established from a female fetus on Day 50 of gestation and adult ear skin biopsies. The cells were used for assessements of cell cycle and apoptosis, and for nuclear transfer. Cells were randomly allocated into 3 experimental treatment groups after 6–8 passages: Group 1 (confluent), cells were cultured in DMEM supplemented with 10% FBS until 90% confluent; Group 2 (serum-starvation), cells were cultured in DMEM supplemented with 0.5% FBS for 5 days; Group 3 (Roscovitine), cells were cultured in DMEM supplemented with 10% FBS and 30μM Roscovitine for 12h. Cell cycle and apoptosis were analyzed using flow cytometry after labelling with DAPI and YO-PRO-1, respectively. At 19h post-maturation (hpm), enucleated oocytes were reconstructed with donor cells and fused by a single DC pulse (1.6kV/cm, 60μs) delivered by a BTX 200. After activation with the combination of ionomycin (5μM, 5min) and cycloheximide (10μgmL−1, 5h), the eggs were cultured in CR1aa medium for 3 days and additionally cultured in CR1aa medium supplemented with 30mgmL−1 BSA for 5 days at 39°C in a humidified atmosphere of 5% CO2 in air. Differences between groups were analyzed using one-way ANOVA after arc-sine transformation of the proportional data. There were no significantly differences in the incidence of cells arrested at G0/G1 for fetal fibroblasts cultured in the three treatment groups (87%, 83% and 80%; confluent, serum starvation and Roscovitine, respectively). More cells were apoptotic in Group 2 compared to the cells in Groups 1 and 3 (12% v. 6 and 6%, respectively) (P&amp;lt;0.05). Blastocyst development of cloned embryos was significantly (P&amp;lt;0.05) higher when fetal fibroblasts from Group 1 were used, compared to Groups 2 and 3 (35.1%, v. 31 and 29.7%, respectively). Similar results were observed in the use of ear skin fibroblasts as nuclear transfer donor cells (32.7%, v. 24 and 24%, respectively). These results suggest that fetal fibroblasts can be effectively synchronized at G0/G1 by three different treatments, including growth to confluence, serum-starvation and Roscovitine treatment. However, based on blastocyst development and levels of apoptosis, the use of confluent fetal fibroblasts as donor cells is more effective than using cells synchronized by serum-starvation or Roscovitine treatment in the production of cloned bovine embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 30012-05-3-SB010 and Cho-A Pharm. Co. LTD.]

Activation of monocytic cells through Fc gamma receptors induces the expression of macrophage-inflammatory protein (MIP)-1 alpha, MIP-1 beta, and RANTES.

Monocytic cells were stimulated with IgG-OVA equivalence immune complexes, mAb reacting with FcgammaRI, FcgammaRIIA, and FcgammaRIII, LPS, TNF-alpha, and the combination of ionomycin and phorbol ester, to address their effects on the expression of the mRNAs encoding for chemokines. Stimulation of monocytes with immune complexes induced a rapid expression of macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, and IL-8 mRNAs. In contrast, RANTES mRNA was already detectable in resting cells and only increased after 16 h of stimulation. A similar pattern was observed following homotypic stimulation of FcgammaR with mAb reacting with FcgammaRI and FcgammaRIIA, but not with a mAb reacting with FcgammaRIII, a subtype of receptor not expressed in THP-1 cells, thus indicating that both FcgammaRI and FcgammaRIIA are involved in the response. The pattern of chemokine induction elicited by LPS and the combination of ionomycin and PMA showed some similarities to those produced by FcgammaR cross-linking, although expression of IFN-gamma-inducible protein 10 mRNA was also observed in response to those agonists. The production of MIP-1alpha, MIP-1beta, and RANTES proteins encompassing the induction of their mRNAs was confirmed by specific ELISA. Experiments to address the transcription factors involved in the regulation of MIP-1alpha using pharmacological agents and EMSA showed the possible involvement of CCAAT/enhancer-binding protein beta sites and ruled out the functional significance of both NF-AT and AP-1 sites.

Activation of distinct signal transduction pathways in Trypanosoma cruzi isolates with differential capacity to invade host cells

Mammalian cell invasion by Trypanosoma cruzi requires the activation of signal transduction pathways that result in a Ca 2+ response both in the parasite and the host cell. By using drugs that interfere with the signalling processes, we investigated if the difference in the ability of T. cruzi isolates to invade host cells was associated with the activation of distinct signalling routes in the parasites. Experiments were performed with metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, using the highly invasive isolate CL and the poorly infective isolate G, which belong to distinct phylogenetic lineages. Treatment of parasites with adenylyl cyclase activator forskolin increased the infectivity of the G but not of the CL isolate towards HeLa cells. On the other hand, a specific protein tyrosine kinase inhibitor genistein reduced by ∼75% the penetration of CL but not of G isolate into HeLa cells. In the CL but not in the G isolate, protein tyrosine kinase mediated the phosphorylation of a 175 kDa protein in a manner inducible by a HeLa cell extract. Upon treatment with the phospholipase C inhibitor U73122, or with drugs such as caffeine, which affects Ca 2+ release from inositol-1,4,5-triphosphate-sensitive stores, or thapsigargin, an inhibitor of intracellular Ca 2+ transport ATPases, the infectivity of the CL but not of the G isolate diminished significantly ( P<0.005). In both isolates, a combination of ionomycin plus NH 4Cl or nigericin released Ca 2+ from acidic vacuoles containing a Ca 2+/H + exchange system. This treatment reduced the infectivity of metacyclic forms of the G but not of the CL isolate. Taken together, these data suggest that, for host cell invasion, distinct signalling pathways are activated in metacyclic trypomastigotes of the two isolates, leading to Ca 2+ release from different intracellular compartments.

T Cell Activation Signals Upregulate CBP-Dependent Transcriptional Activity

The transcriptional coactivator CREB-binding protein (CBP) is known to play an important role in coupling signal transduction pathways to changes in gene expression. In many cases, this is achieved by the stimulus-specific recruitment of CBP to promoter-bound transcription factors. However, a number of recent studies have suggested that signal transduction pathways can also directly influence CBP-mediated transcriptional activity. Here we show that in Jurkat cells the activity of the CBP C-terminal transactivation domain is strongly upregulated in response to either T cell receptor stimulation or the combination of ionomycin and phorbol ester. We further show that maximal stimulation of CBP-mediated transcription requires the synergistic activation of both the calcineurin and Ras-MAPK signaling pathways. These results indicate that CBP can function as a T cell activation-inducible transcriptional coactivator and is therefore likely to play an important role in T cell activation-induced gene expression.

Murine bone marrow-derived mast cells as potent producers of IL-9: costimulatory function of IL-10 and kit ligand in the presence of IL-1.

Recently, the Th2-type cytokine IL-9 was identified by genetic mapping analyses as a key mediator that determines the susceptibility to asthma. This has been further supported by data from IL-9-transgenic mice in which the overexpression of IL-9 in the lung causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness. In an accompanying paper, we demonstrate that murine bone marrow-derived mast cells (BMMC) after stimulation with either ionomycin, a combination of ionomycin and IL-1, or via IgE-Ag complexes and IL-1 are very potent producers of IL-9. Herein we show that a dramatic increase of IL-9 production is observed when BMMC activated with ionomycin/IL-1 or with IgE-Ag complexes/IL-1 are treated with either additional kit ligand (KL) or IL-10. Both KL and IL-10 considerably enhance the production of IL-9 mRNA and protein. We were also able to demonstrate that the production of endogenous IL-10 by activated mast cells acts on the production of IL-9. Half-life measurements of IL-9 mRNA revealed no significant effect by KL, but a 2-fold increase of mRNA stability under the influence of IL-10. Reporter gene assays of transfected BMMC showed an enhanced transcriptional activity of the IL-9 promoter in the presence of either IL-10 or KL compared with cells stimulated only with a combination of IL-1 and ionomycin. The influence of KL and IL-10 might be of physiological importance, because it is known that both cytokines are produced by bronchial epithelial cells.

Involvement of FK506-sensitive and insensitive granule exocytosis pathways in perforin-dependent target cell lysis mediated by a CD8 + CTL clone

The release of granzyme A and B through granule exocytosis by CD8 + CTL clone OE4 upon T cell receptor (TCR) activation was blocked by FK506 in a dose-dependent manner (IC 50=3 nM), whereas a significant granzyme release was still detectable even in the presence of excess FK506. In contrast, the production of IFN-γ was highly sensitive to FK506 (IC 50=0.01 nM) and could be completely blocked by FK506. Both FK506-sensitive and insensitive granule exocytosis pathways were involved in the actual perforin-dependent killing toward different target cells. The combination of ionomycin and phorbol ester was able to mimic TCR stimulation to induce IFN-γ production, although the same treatment triggered granule exocytosis inefficiently. Granule exocytosis and IFN-γ production following TCR activation were profoundly prevented by calphostin C. Thus, these results demonstrate that the granule exocytosis pathway in this CD8 + CTL clone depends on the activation of protein kinase C, and requires either calcineurin-dependent or independent additional signals downstream of TCR activation.