FBS Medium Research Articles

Ca2+ Entry via TRPC Channels Is Necessary for Thrombin-induced NF-κB Activation in Endothelial Cells through AMP-activated Protein Kinase and Protein Kinase Cδ

The transient receptor potential canonical (TRPC) family channels are proposed to be essential for store-operated Ca2+ entry in endothelial cells. Ca2+ signaling is involved in NF-kappaB activation, but the role of store-operated Ca2+ entry is unclear. Here we show that thrombin-induced Ca2+ entry and the resultant AMP-activated protein kinase (AMPK) activation targets the Ca2+-independent protein kinase Cdelta (PKCdelta) to mediate NF-kappaB activation in endothelial cells. We observed that thrombin-induced p65/RelA, AMPK, and PKCdelta activation were markedly reduced by knockdown of the TRPC isoform TRPC1 expressed in human endothelial cells and in endothelial cells obtained from Trpc4 knock-out mice. Inhibition of Ca2+/calmodulin-dependent protein kinase kinase beta downstream of the Ca2+ influx or knockdown of the downstream Ca2+/calmodulin-dependent protein kinase kinase beta target kinase, AMPK, also prevented NF-kappaB activation. Further, we observed that AMPK interacted with PKCdelta and phosphorylated Thr505 in the activation loop of PKCdelta in thrombin-stimulated endothelial cells. Expression of a PKCdelta-T505A mutant suppressed the thrombin-induced but not the TNF-alpha-induced NF-kappaB activation. These findings demonstrate a novel mechanism for TRPC channels to mediate NF-kappaB activation in endothelial cells that involves the convergence of the TRPC-regulated signaling at AMPK and PKCdelta and that may be a target of interference of the inappropriate activation of NF-kappaB associated with thrombosis.

50 FLOW CYTOMETRY-MEDIATED DETECTION OF LATE-APOPTOTIC HYPODIPLOID CELL FRACTIONS IN LIPOFECTED PORCINE ADULT DERMAL FIBROBLAST CELL LINES SELECTED FOR SOMATIC CELL NUCLEAR TRANSFER

Analysis of nuclear DNA (nDNA) content of in vitro cultured somatic cells undergoing apoptosis became one of the most common methods for single-parameter flow cytometric measurement of this process. Apoptosis assessment is performed by quantification of hypodiploid cells. The cell fractions with hypodiploid (<2C) nDNA molecule number, which involve the so-called sub-G1 peak in DNA histograms are identified as late-apoptotic subpopulations. Advantage of this method is the possibility of simultaneous cell cycle measurement. The present study was conducted to investigate the preimplantation developmental outcome of porcine transgenic NT embryos reconstituted with non-apoptotic gilt ear skin-derived fibroblast cells that had been lipofected with pWAPhGH-GFPBsd gene construct. The nuclear donor cells were derived from such cell line populations whose representative random samples had been analyzed on both cell cycle and apoptosis through the non-vital nDNA fluorescent dyeing and subsequent flow cytometry (FACS). Frozen/thawed fibroblast cells, which had been cultured up to a total confluency after 2–3 passages, were used for the diagnostics. The fixed dermal fibroblasts were exposed to nDNA extraction buffer for 5 min and incubated in DNA staining solution (propidium iodide and RNAse) for 30 min. After fluorescent labeling, the cells were analyzed in the flow cytometer by reading nDNA fluorescence in the red band. Somatic cell cloned embryos, which had been created by simultaneous fusion and electrical activation, followed by delayed chemical activation of reconstructed oocytes, were cultured in NCSU-23/FBS medium for 6 to 7 days up to morula/blastocyst stages (Skrzyszowska et al. 2008 Theriogenology 70, 248–259). The FACS analysis revealed that out of all the fibroblast cells diagnosed, 94.9% were cycling and 5.1% were late-apoptotic. In turn, from among the non-apoptotic cells, an average of 92.7% were at G1/G0 stages of cell cycle, 3.1% were at S stage and 4.2% were at G2/M stages. A total of 294/348 (84.5%) enucleated oocytes were successfully fused with non-apoptotic nuclear donor cells. Out of 294 cultured NT embryos, 199 (67.7%) were cleaved. The rates of cloned embryos that reached the morula and blastocyst stages yielded 165/294 (56.1%) and 57/294 (19.4%), respectively. In conclusion, the FACS analysis for mitotic cycle of 100%-confluent lipofected adult dermal fibroblasts confirmed that the cell cycle synchronization at G1/G0 phases was highly efficient, while the frequency of late-apoptotic cells was low. It was also found that the relatively high percentages of pWAPhGH-GFPBsd transgenic blastocysts developed in vitro from NT embryos reconstructed with fibroblast cells undergoing lipofection. Furthermore, porcine cloned blastocysts exhibited approximately 100% index of reporter eGFP transgene expression, which was visually confirmed by their live-fluorescent evaluation. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.

Intracellular GSH and ROS levels may be related to galactose-mediated human lens epithelial cell apoptosis: Role of recombinant hirudin variant III

Oxidative processes in the lenses are the most commonly found damaging factor for the development of cataracts. Hirudin, a most potent inhibitor of thrombin as an antithrombic drug, also have potential use in cataracts. In order to investigate the mechanisms of hirudin against galactose-induced cataract at the cellular level. We used recombinant hirudin variant III (rHV3) to study the protective effect of hirudin on galactose-mediated human lens epithelial cells injury. The human lens epithelial cells (hLECs) were cultured in D/F 12–10% FBS medium containing 125 mM d-galactose with or without rHV3. Cell viability was assessed by methylthiazol tetrazolium (MTT) assay and propidium iodide (PI) staining in situ. Cell apoptosis was elevated with comet assay (single cell gel electrophoresis, SCGE), AO/EB double staining and Annexin-V/PI double staining assay. Reactive oxygen species (ROS) were quantified with 2′,7′-dichlorofluorescein (DCF), and free glutathione (GSH) levels were measured with a commercial GSH quantification kit. Decreased viability and increased apoptosis of the hLECs were observed when incubated with 125 mM galactose. These hLECs also demonstrated the increased presence of ROS, whereas GSH was reduced. rHV3 blocked the induction of cell death, apoptosis and oxidative stress in hLECs. One mechanism may be through regulating intracellular ROS and GSH levels to inhibit apoptosis of the human lens epithelial cells.

MCF-7aro/ERE, a novel cell line for rapid screening of aromatase inhibitors, ERα ligands and ERRα ligands

We have previously generated a breast cancer cell line, MCF-7aro, which over-expresses aromatase and is also ER positive. Recently, this MCF-7aro cell line was stably transfected with a promoter reporter plasmid, pGL3-Luc, containing three repeats of estrogen responsive element (ERE). Experiments using MCF-7aro/ERE have demonstrated that it is a novel, non-radioactive screening system for aromatase inhibitors (AIs), ERα ligands and ERRα ligands. The screening is carried out in a 96-well plate format. To evaluate this system, the cells were cultured overnight in charcoal–dextran stripped FBS medium supplemented with 0.1 nM testosterone or 17β-estradiol, and various concentrations of antiestrogens or AIs. We found that the luciferase activity was induced when the cells were cultured either in the presence of testosterone or 17β-estradiol. Furthermore, a 50% decrease in luciferase activity could be achieved when the cells were cultured in the presence of testosterone together with letrozole, anastrozole, tamoxifen or fulvestrant (concentrations being 75 nM, 290 nM, 100 nM, and 5 nM, respectively), compared to the testosterone-only cultured cells. Using this assay system, we confirmed that 3(2′-chlorophenyl)-7-methoxy-4-phenylcoumarin is an agonist of ER. Furthermore, genestein has been shown to be a ligand of ERRα because its binding could be blocked by an ERRα inverse agonist, XCT790. These results indicate that MCF-7aro/ERE is a novel cell line for rapid screening of AIs, ERα ligands and ERRα ligands.

Physico-chemical and thermodynamic aspects of fibroblastic attachment on RGDS-modified chitosan membranes

In the present study, the cell attachment/spreading behaviour of L929 mouse fibroblasts on chitosan membranes was evaluated by using physico-chemical properties. For this purpose chitosan membranes were prepared and then photochemically modified with the cell adhesive peptide RGDS (Arg-Gly-Asp-Ser). The physico-chemical properties of unmodified (CHI) and RGDS-modified chitosan (CHI-RGDS) membranes were evaluated by calculating surface free energy ( γ sv) and interfacial free energy ( γ sw) values using captive bubble contact angle measurements and harmonic mean equation. The cell attachment experiments were performed both in 10% FBS containing and serum-free media with CHI and CHI-RGDS membranes. Eventually, it was not possible to predict a direct relationship between the change in physico-chemical properties and L929 cell attachment behaviour. The experimental results obtained from cell attachment agree with the theoretical prediction for the free energy of adhesion except for the cell attachment on CHI membrane in serum-free medium. Although a negative interfacial free energy of adhesion was calculated for CHI membrane in serum-free medium (Δ F adh = −2.19 ergs/cm 2), the cell attachment was poor (∼70%) compared to CHI-RGDS (∼90%) and none of the cells were spread on CHI surface to gain a fibroblastic morphology. Negative energy of adhesion was calculated for CHI and CHI-RGDS in 10% FBS medium, in which ∼100% of cells were attached on the membranes correlating with the thermodynamic approach. It can be suggested that, adsorption of serum proteins strongly affected the cell attachment meanwhile the presence of biosignal RGDS molecules triggered the cell spreading in serum medium.

Antiangiogenic Antithrombin Blocks the Heparan Sulfate-dependent Binding of Proangiogenic Growth Factors to Their Endothelial Cell Receptors

The anticoagulant serpin antithrombin acquires a potent antiangiogenic activity upon undergoing conformational alterations to cleaved or latent forms. Here we show that antithrombin antiangiogenic activity is mediated at least in part through the ability of the conformationally altered serpin to block the proangiogenic growth factors fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) from forming signaling competent ternary complexes with their protein receptors and heparan sulfate co-receptors on endothelial cells. Cleaved and latent but not native forms of antithrombin blocked the formation of FGF-2-FGF receptor-1 ectodomain-heparin ternary complexes, and the dimerization of these complexes in solution and similarly inhibited the formation of FGF-2-heparin binary complexes and their dimerization. Only antiangiogenic forms of antithrombin likewise inhibited (125)I-FGF-2 binding to its low affinity heparan sulfate co-receptor and blocked FGF receptor-1 autophosphorylation and p42/44 MAP kinase phosphorylation in cultured human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with heparinase III to specifically eliminate the FGF-2 heparan sulfate co-receptor suppressed the ability of antiangiogenic antithrombin to inhibit growth factor-stimulated proliferation. Antiangiogenic antithrombin inhibited full-length VEGF(165) stimulation of HUVEC proliferation but did not affect the stimulation of cells by the heparin-binding domain-deleted VEGF(121). Taken together, these results demonstrate that antiangiogenic forms of antithrombin block the proangiogenic effects of FGF-2 and VEGF on endothelial cells by competing with the growth factors for binding the heparan sulfate co-receptor, which mediates growth factor-receptor interactions. Moreover, the inability of native antithrombin to bind this co-receptor implies that native and conformationally altered forms of antithrombin differentially bind proangiogenic heparan sulfate domains.

Cryopreservation of adherent neuronal networks

Neuronal networks have been widely used for neurophysiology, drug discovery and toxicity testing. An essential prerequisite for future widespread application of neuronal networks is the development of efficient cryopreservation protocols to facilitate their storage and transportation. Here is the first report on cryopreservation of mammalian adherent neuronal networks. Dissociated spinal cord cells were attached to a poly- d-lysine/laminin surface and allowed to form neuronal networks. Adherent neuronal networks were embedded in a thin film of collagen gel and loaded with trehalose prior to transfer to a freezing medium containing DMSO, FBS and culture medium. This was followed by a slow rate of cooling to −80 °C for 24 h and then storage for up to 2 months in liquid nitrogen at −196 °C. The three components: DMSO, collagen gel entrapment and trehalose loading combined provided the highest post-thaw viability, relative to individual or two component protocols. The post-thaw cells with this protocol demonstrated similar neuronal and astrocytic markers and morphological structure as those detected in unfrozen cells. Fluorescent dye FM1-43 staining revealed active recycling of synaptic vesicles upon depolarizing stimulation in the post-thaw neuronal networks. These results suggest that a combination of DMSO, collagen gel entrapment and trehalose loading can significantly improve conventional slow-cooling methods in cryopreservation of adherent neuronal networks.

Molecular Determinants of Kinase Pathway Activation by Apo2 Ligand/Tumor Necrosis Factor-related Apoptosis-inducing Ligand

Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) mainly activates programmed cell death through caspases. By contrast, TNF primarily induces gene transcription through the inhibitor of kappaB kinase (IKK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. Apo2L/TRAIL also can stimulate these kinases, albeit less strongly; however, the underlying mechanisms of this stimulation and its relation to apoptosis are not well understood. Here we show that Apo2L/TRAIL activates kinase pathways by promoting the association of a secondary signaling complex, subsequent to assembly of a primary, death-inducing signaling complex (DISC). The secondary complex retained the DISC components FADD and caspase-8, but recruited several factors involved in kinase activation by TNF, namely, RIP1, TRAF2, and NEMO/IKKgamma. Secondary complex formation required Fas-associated death domain (FADD), as well as caspase-8 activity. Apo2L/TRAIL stimulation of JNK and p38 further depended on RIP1 and TRAF2, whereas IKK activation required NEMO. Apo2L/TRAIL induced secretion of interleukin-8 and monocyte chemoattractant protein-1, augmenting macrophage migration. Thus, Apo2L/TRAIL and TNF organize common molecular determinants in distinct signaling complexes to stimulate similar kinase pathways. One function of kinase stimulation by Apo2L/TRAIL may be to promote phagocytic engulfment of apoptotic cells.

492. Harvest Timing and Media Composition Effects on Lentiviral Vector Production

The Indiana University Vector Production Facility (IUVPF), in conjunction with the National Gene Vector Laboratories, has been developing the capability to produce and process a volume of lentiviral vector sufficient for phase I/II clinical trials. To date, the facility has been able to produce vector in Nunc Cell Factories in unprocessed volumes from 120mL to 20L in a single production run. In the course of developing this capability, we have looked at many production parameters and here report on the effects of harvest timing and media composition. The general production scheme had cells seeded into appropriate tissue culture flasks on day 1, cells were transfected by CaPO4 on day 2, DMEM+10% FBS medium (D10) was changed to harvest medium on day 3 followed by harvest of vector supernatant at indicated time points thereafter. Results are primarily reported as relative comparisons within each experiment. We have used p24 capsid protein ELISA as a physical method of estimating titer. For production using transgene vectors containing the GFP gene, the p24 data was supplemented with GFP expression titers determined by limiting dilution of vector on 293 cells. Harvest timings of 12, 24, 48 and 72 and multiple harvests at 12 and 24 hours after the post transfection media change had a range of GFP expression titers from 1.2|[times]|105 to 2.7|[times]|107 TU/mL. The initial 12 hour harvest had the highest titer, 2.7|[times]|107, but multiple harvests at 12 hour intervals resulted in a rapid drop in titer. The 24 hour harvests were found to be relatively consistent, usually only differing by a factor of 2.

Two Related Low Molecular Mass Polypeptide Isoforms of Amelogenin Have Distinct Activities in Mouse Tooth Germ Differentiation In Vitro

Embryonic mouse tooth germs were cultured in vitro in the presence of two related amelogenin isoforms to determine their effects on tooth development. Our results show that these individual proteins have specific but quite different effects on epithelial-derived ameloblasts versus mesenchymal-derived odontoblasts. Amelogenins, the main protein components of enamel matrix, have been shown to have signaling activity. Amelogenin isoforms differing only by the presence or exclusion of exon 4, designated [A+4] (composed of exons 2, 3, 4, 5, 6d, and 7) and [A-4] (composed of exons 2, 3, 5, 6d, and 7), showed similar, but different, effects both in vitro and in vivo on postnatal teeth. Lower first molar tooth germs of E15/16 CD1 mice were microdissected and cultured in vitro in a semisolid media containing either 20% FBS, 2% FBS, or 2% FBS with either 1.5 nM [A+4], [A-4], or both for 6 days. Tooth germs were analyzed by H&E staining and immunohistochemistry for collagen I, dentin matrix protein 2, and DAPI nuclear staining. Teeth cultured in media containing 20% FBS showed normal development with polarized ameloblasts, and odontoblasts producing dentin matrix, and DMP2 expression in odontoblasts and pre-ameloblasts. Culture in 2% FBS media resulted in no ameloblast polarization and modest odontoblast differentiation with scant dentin matrix. Tooth germs cultured with [A+4] in 2% FBS media had well-polarized odontoblasts with robust dentin production and concomitant ameloblast polarization. DMP2 expression was equal to or greater than seen in the 20% FBS culture condition. In cultures with [A-4] in 2% FBS media, odontoblast polarization and dentin production was reduced compared with [A+4]. However, the pre-ameloblast layer was disorganized, with no ameloblast polarization occurring along the dentin surface. DMP2 expression was reduced in the odontoblasts compared with the 20% FBS and [A+4] conditions and was almost completely abrogated in the pre-ameloblasts. These data show different signaling activities of these closely related amelogenin isoforms on tooth development. Here we make the novel observation that [A-4] has an inhibitory effect on ameloblast development, whereas [A+4] strongly stimulates odontoblast development. We show for the first time that specific amelogenin isoforms have effects on embryonic tooth development in vitro and also hypothesize that DMP2 may play a role in the terminal differentiation of both ameloblasts and odontoblasts.

Heme: a determinant of life and death in renal tubular epithelial cells.

Heme oxygenase-1 (HO-1) and p21 influence cell fate, and genetic HO-1 overexpression upregulates p21 and confers resistance to apoptosis. The present study examined the effects of heme, a metabolite incriminated in renal injury, on sensitivity to apoptosis and cell growth in conjunction with cellular expression of HO-1 and p21. Immortalized rat proximal tubular epithelial cells (IRPTCs) were exposed to hemin (10 microM) in serum-deplete media (0.1% FBS) and in standard cell culture media (5.0% FBS). In the presence of 0.1% FBS media, hemin induced p21 through an HO-dependent, p53-independent mechanism; certain products of HO activity (iron and carbon monoxide), but not others (ferritin, apoferritin, bilirubin), recapitulated these inductive effects on p21 expression. Along with this inductive effect on HO-1 and p21, hemin worsened apoptosis, the latter exacerbated by the inhibition of HO activity and loss of p21 expression. In IRPTCs maintained in 5% FBS, hemin induced HO-dependent p21 expression, provoked cell cycle arrest, and inhibited cell growth without inducing apoptosis; this inhibitory effect of hemin on cell growth was blocked by the concomitant inhibition of HO activity and loss of p21 expression. We conclude that hemin is a potent HO-dependent inducer of p21 and that hemin increases the sensitivity to apoptosis in serum-deplete conditions and decreases cell growth in serum-replete conditions; inhibiting HO activity and concomitantly ablating p21 expression exacerbate apoptosis and reverse the growth-inhibitory actions of hemin. We suggest that these effects of heme may influence the nature of, and recovery from, ischemic and nephrotoxic insults to the kidney.

174EMBRYO TRANSFER OF VITRIFIED IVF EMBRYOS IN CATTLE: PREGNANCY COMPARISON AFTER SINGLE AND DOUBLE TRANSFER

Advancement in vitrification of in vitro-produced bovine embryos will benifit the cattle breeding and production industry. The objective was to evaluate whether bilateral (double) embryo transfers (ET) can improve pregnancy rate compared to ipsilateral (single) transfers. Bovine cumulus-oocyte complexes collected from slaughterhouse ovaries were matured for 20–22h, and subsequently subjected to a standard Brackett and Oliphant in vitro fertilization (IVF). Six hours after IVF, embryos denuded of cumulus were cultured in defined CR1 medium supplemented with essential and non-essential amino acids (CR1aa), plus 6mgmL−1 BSA for 2 days at 39°C under 5% CO2, 5% O2 and 90% N2, and then cultured in CR1aa medium supplemented with 7.5% FBS for a further 5 days on bovine cumulus monolayers. Expanded blastocysts with tighter compaction of the inner cell mass (quality 1) were selected on Day 7 for cryopreservation via modified solid surface vitrification (Dinnyes et al., 2000 Biol. Reprod. 513–8). Vitrification solution contained HEPES-buffered TCM199 supplemented with 20% FBS, ethylene glycol and dimethylsulphoxide. A droplet of 1–2μL vitrification solution containing 4–5 blastocysts was dropped directly onto a cooled surface within 30s after 3-min incubation in equilibration solution. Prior to ET, embryos were warmed and subsequently washed several times in 0.25M sucrose rehydration solution and M199+7.5% FBS medium. The warmed embryos from initial trials were cultured for 2 to 72h to evaluate their viability after vitrification. During ET trails, vitrified embryos were loaded into transfer straws (one embryo per straw) after warming. The treatments were as following, (1) single transfers, one embryo was transferred into the horn ipslateral to CL; (2) double transfers, one embryo was transferred by non-surgical means into each uterine horn of a synchronous recipient on Day 7. ET trails were conducted in both the USA (double transfers) and China (single v. double transfers). Pregnancy was determined by palpation per rectum around Day 70 after transfer. The data were compared by Student’s t-test. The survival rate of vitrified IVF embryos reached as high as 91.4% (n=256) 2h post-warming, and hatching rate was 70.7% (n=154) 72h after culture in vitro, respectively. The data (Table 1) show that double transfers resulted in a significantly higher pregnancy rate than did single transfers (P<0.05). With double transfers, a higher pregnancy rate was achieved in the USA than in China (76.2% v. 45.6%, P=0.079). This study confirms that double embryo transfers can improve the pregnancy outcome after ET, perhaps because bilateral placement of embryos may increase embryonic signals to the maternal environment. Further evaluation of gestation length, single/twin conception and calving difficulty is under investigation. Table 1 Pregnancy rate (Day 70) of vitrified bovine IVF embryos following single and double transfer

74PREGNANCY ESTABLISHMENT OF NUCLEAR TRANSFERRED EMBRYOS AFTER VITRIFICATION AND EMBRYO TRANSFER IN CATTLE

The success of cryopreservation of bovine cloned embryos is important to commercial application of somatic nuclear transfer (NT) in the dairy and beef industry, especially for solving the problem of embryo transfer (ET) between fresh NT embryos and recipients. This experiment was conducted to test the possibility of establishing pregnancy by embryo transfer of vitrified bovine NT embryos. Oocytes were aspirated from antral follicles of slaughterhouse ovaries, and subsequently cultured in maturation medium for 20h. Cumulus cells were then denuded from the oocytes by vigorous vortexing for later enucleation and donor nuclear transfer. Skin fibroblast cells used for NT were derived from cultured ear explants taken from an elite dairy cow, and cumulus cells were cultured from the cumulus-oocytes complexes collected by ultrasound-guided transvaginal retrieval. Fibroblasts and cumulus cells were cultured at passage 5 or 6 in 10% FBS DMEM at 37°C in 5% CO2 humidified air, and used as nuclear donors. After donor cell transfer, somatic cell-cytoplast pairs were then fused by applying two direct current pulses at 2.0kVcm−1 for a duration of 10μs/pulse. Fused embryos were activated in 10mgmL−1 cycloheximide in M199+7.5% FBS for 4h. Embryos were cultured in CR1aa plus 3mgmL−1 BSA for 2 days (initiation of activation=Day 0) at 39°C, 5% CO2, 5% O2 and 90% N2, and then cultured on bovine cumulus monolayers in CR1aa medium supplemented with 7.5% FBS for 5 successive days. Expanding and hatching blastocysts on Day 7 were selected for cyropreservation via liquid nitrogen surface vitrification (LNSV). Vitrification solution contained HEPES-buffered TCM199 supplemented with 20% FBS, ethylene glycol and dimethylsulphoxide. A droplet of 1–2μL vitrification solution containing two blastocysts was directly dropped onto a cooled surface within 30s after 3min incubation in equilibration solution. Vitrified NT embryos were kept at −150°C vapor phase until recipients were synchronized to Day 7 for ET. Embryos are warmed and subsequently washed several times in rehydration solutions and M199+7.5% FBS medium. The warmed embryos from initial trials were cultured for 2h to evaluate their viability after cryopreservation. Non-surgical transfer was carried out to transfer two embryos to a synchronous recipient, and pregnancy was determined by palpation via rectum around Day 70 of transfer. After warming of vitrified embryos, similar high survival rates were achieved in NT embryos derived from either cumulus (93.8%, n=16), or fibroblast cells (95.8%, n=48) as nuclear donors, respectively (P>0.05). The pregnancy data (Table 1) on Day 70 of gestation indicated that there were no significant differences among ET groups with fresh NT blastocysts, vitrified cumulus NT and fibroblast NT embryos (P>0.05). This study demonstrates that cryopreserved bovine NT embryos via LNSV vitrification can maintain an in vivo developmental viability comparable to freshly produced NT counterparts. Further developmental potential of vitrified NT embryos to term is under investigation. Table 1 Pregnancy outcomes of vitrifield bovine NT embryos after embryo transfer

The Phosphatase MKP1 Is a Transcriptional Target of p53 Involved in Cell Cycle Regulation

The tumor suppressor p53 protein suppresses cell growth by inducing cell cycle arrest or apoptosis. Despite the fact that p53-dependent p21-mediated G1 arrest induced by DNA damage is well defined, the role of p53 in the cell cycle in response to the MAKP signaling remains to be determined. Here we show that MKP1, a member of the dual specificity protein phosphatase family capable of inactivating MAPKs, is a transcriptional target of p53. MKP1 mRNA and protein levels were increased upon p53 activation in several well defined p53-regulated cell systems. p53 bound to a consensus p53 binding site located in the second intron of the MKP1 gene and transactivated MKP1 in reporter gene assays. Inhibition of phosphatase activity impaired p53-mediated G1 arrest in arrested human glioblastoma GM cells in response to growth factor stimuli. Importantly conditional expression of MKP1 prevented arrested human cancer cells from entering into the cell cycle. Thus, these results provide a novel mechanism by which p53 controls the cell cycle in response to the MAPK signaling in the absence of DNA damage and suggest that p53 may negatively control the MAKP pathway via MKP1.

Electrophile tocopheryl quinones in apoptosis and mutagenesis: thermochemolysis of thiol adducts with proteins and in cells.

Electrophile tocopheryl quinones from the phenolic antioxidants gamma-tocopherol and delta-tocopherol form Michael adducts with the thiol nucleophile glutathione. These tocopheryl quinones are involved in cytotoxicity, apoptosis, and mutagenesis, and their biologic properties are associated with the depletion of intracellular thiols. We now show that both proteins and tissues treated with the electrophile gamma-tocopheryl quinone (gamma-TQ) form thiol adducts. The monoglutathion-S-yl derivative of gamma-TQ was subjected to thermochemolysis with the strong methylating base tetramethylammonium hydroxide. GC/MS showed four signature peaks and a fragmentation pattern characteristic of the thiol adduct. Similarly, pure monoglutathion-S-yl and diglutathion-S-yl derivatives of delta-TQ were subjected to thermochemolysis, and GC/MS showed characteristic fragmentation patterns for thiol adducts. The four signature peaks were identified when pure proteins with accessible thiol groups (hemoglobin and histone), FBS, and tissue culture medium and cell preparations were treated with gamma-TQ. Signature peaks in both complete medium and washed cells showed the presence of both soluble and insoluble thiol adducts. The effective or free arylating electrophile concentration in complete medium should always be evaluated in tissue culture studies. gamma-TQ is a mutagen but not a genotoxin; therefore, the histone adduct may be a previously unrecognized histone modification involved in chromatin dynamics leading to mutagenesis.

Edaravone, a radical scavenger, may enhance or produce antiproliferative effects of fluvastatin, amlodipine, ozagrel, GF109203X and Y27632 on cultured basilar artery smooth muscle cells.

Proliferation of vascular smooth muscle cells stimulated by reactive oxygen species (ROS) may play a pivotal role in the pathogenesis of atherosclerosis. To clarify mechanisms by which ROS promote vascular atherogenesis, effects of fluvastatin, amlodipine, ozagrel (thromboxane synthetase inhibitor), GF109203X (a protein kinase C inhibitor) and Y27632 (a ROCK inhibitor) on the proliferation of guinea-pig basilar artery smooth muscle cells (GBa-SM3) in a 5% FBS culture medium were studied over 3 d in the presence or absence of a free radical scavenger, edaravone. Viability of cells at the end of incubation was measured by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. Results demonstrated that fluvastatin and amlodipine by themselves possess antiproliferative effects on the GBa-SM3 cells at 10-100 microM and 0.1-1 microM, respectively. While edaravone possessed no antiproliferative effect by itself at 100 microM, it significantly (p<0.05) augmented the antiproliferative effects of fluvastatin and amlodipine. In addition, ozagrel, GF109203X and Y27632 possessed no appreciable effects on the cell growth by themselves. However, coincubation of edaravone at 100 microM with these agents elicited significant antiproliferative effects for ozagrel, GF109203X and Y27632 at 10-100 microM, 1-10 microM and 0.1-1 microM, respectively. In conclusion, edaravone may have clinically beneficial interactions with fluvastatin, amlodipine and ozagrel regarding the prevention of vascular atherosclerosis. The interactions between edaravone and the inhibitors of protein kinase C and ROCK were suggestive of possible contributions of ROS-triggered intracellular signals associated with these enzymes to vascular atherogenesis, but further studies are required for confirmation.

Differential responsiveness of cord and adult blood monocytes to hepatocyte growth factor.

Monocytes as antigen-presenting cells play an important role in host defence. There are several cytokines affecting monocyte function. We demonstrate that both adult and cord blood monocytes constitutively express hepatocyte growth factor (HGF) receptor, MET. HGF significantly down-regulated MET expression of adult blood monocytes, compared with cord blood monocytes, when cultured either in RPMI-1640 containing 10% FBS or serum-free medium. Surface levels of MET correlated with c-met mRNA levels both in adult and cord blood when cultured. MET expression was down-regulated by treating with actinomycin D or cycloheximide. HGF stimulated DNA synthesis of adult monocytes, but not cord blood. HGF enhanced antigen-presenting capacity of adult blood monocytes but not cord blood monocytes. HGF up-regulated HLA class I expression in adult monocytes but not in cord blood monocytes. The current results suggest that the failure of cord blood monocytes to respond to HGF may be responsible, in large part, for their functional immaturity.

Identification of link protein during follicle development and cumulus cell cultures in rats.

Cumulus oocyte complex (COC) expansion is induced through hyaluronic acid production and accumulation of proteins of the inter-alpha-trypsin inhibitor family in the gonadotropin-stimulated cumulus cells. Link protein, a glycoprotein found in cartilage, interacts specifically with hyaluronic acid and stabilizes the binding of proteoglycan monomers to hyaluronic acid to form aggregates. The aim of this study was to investigate the expression of immunoreactive link protein during follicle development in rats and in cumulus cells in culture by immunohistochemistry and Western blot as well as by specific enzyme-linked immunosorbent assay. Immunohistochemical analysis revealed that the extracellular matrix of cumulus cells that were morphologically at a stage of COC expansion were markedly stained for link protein, whereas granulosa cells from immature follicles were not stained. Cumulus cells deposited link protein into the extracellular matrix in an in vitro culture system. The staining intensity was negated by the treatment with hyaluronidase, suggesting that the link protein is bound to hyaluronic acid. We have identified a 42-kDa immunoreactive link protein in rat ovary during the preovulatory period and in COC extracts. Addition of FSH to the medium of cumulus cells in culture supplemented with 10% FBS and oocyte-conditioned medium resulted in an increased rate of link protein synthesis. This work suggests that the cumulus cells synthesize the link protein that may stabilize the binding of inter-alpha-trypsin inhibitor or dermatan sulfate proteoglycan to hyaluronic acid to make up hyaluronic acid-rich matrix aggregate.

Integrins and the cytoskeleton: focal adhesion kinase and paxillin.

Increases in intraglomerular pressure are known to organization leading to stress fibre formation, and predispose for the development of glomerular sclerosis. focal adhesion assembly accompany tyrosine phosMesangial cells are exposed to high glomerular capilphorylation of FAK and paxillin. Thus, these structures lary pressures and appear to be a potential primary play important roles in the intracellular signal transtarget within the glomerulus for increased mechanical duction from extracellular stimuli. Because of the stress. We have studied the effect of cyclic stretch on possible involvement of cell to substratum interactions the mesangial cellular function to mimic the condition in the mechanical deformation-induced mesangial cell of mesangial cells in glomerular hypertension, and function alterations, we examined the effect of stretch previously demonstrated that subjecting cultured rat on the integrin aggregation, stress fibre formation, and mesangial cells to the continuous cycles of stretch/ tyrosine phosphorylation of FAK and paxillin. relaxation stimulates, cell proliferation, prostaglandin, Rat cultured mesangial cells were plated on six-well TGF-b, and extracellular matrix (ECM) components plates with a flexible bottom coated with type I collagen production. Translocation of protein kinase C (PKC) (Flexcell Corp., PA). Cells were grown to subconfluactivity to the membrane and increase in calcium ence and rendered quiescent by incubation for 48 h in uptake and total calcium content, expression of several 0.5% FBS medium and exposed to cycles of stretching immediate early genes including c-fos and egr-1, were and relaxation using a computer-driven, vacuumalso observed in the early time course of the stretch. operated, stress-providing instrument (Flexercell Strain The mechanisms by which these cells sense passive Unit 100B; Flexcell Corp.). The condition of stretch/ stretch and transduce it into signals leading to specific relaxation was 9 to 12% elongation and 30 cycles per gene expression have not yet been determined. min. F-actin or b1 integrin was stained using FITCIntegrins are a family of a,b-heterodimeric transphalloidin or anti-b1-integrin antibody. Tyrosine phosmembrane proteins that connect the cytoskeleton to phorylation of FAK and paxillin were studied by the ECM. Recent studies have shown that integrinWestern blotting after immunoprecipitation of cell mediated adhesion regulates a variety of intracellular lysates using phosphotyrosine antibody. After probing events, including induction of cell spreading, cytoskeleFAK, the membrane was stripped and reprobed with tal organization, and activation of phosphoinsitide paxillin. synthesis. Thus integrins mediate a variety of signalling When mesangial cells were subjected to stretch, cascades. The focal adhesions are localized at the sites F-actin staining by FITC-phalloidin showed stress fibre of cell attachment to the ECM and the most concenformation at 3 min reaching a peak at 10 min. This trated sites of tyrosine phosphorylation. The focal stress fibre formation disappeared 30 min after subadhesion consists of a complex of proteins such as jecting to stretch. The staining pattern of b1-integrin p125 focal adhesion kinase (FAK) and paxillin. FAK after 10 min of stretch also changed from diffuse is the focal adhesion tyrosine kinase protein and autostaining before stretch becoming more condensed at phosphorylated with the focal adhesion complex the periphery of the cell, this indicates clustering of assembly. Paxillin, which interacts with other focal b1-integrin by stretching. Angiotensin II, ionomycin, adhesion proteins and the cytoskeleton, such as vincuand activation of PKC with phorbol dibutyrate lin and FAK, is thought to be a substrate of FAK and induced stress fibre formation, but the degree and also phosphorylated with the focal adhesion complex duration of stress fibre formation were weaker and assembly. The focal adhesion complex connects inteshorter than those induced by stretch. We also examgrin with cytoskeletal filaments and thus acts both as ined the effect of a longer time course of stretch on a signalling device and a connection to the cytostress fibre formation. After 24 and 48 h of stretch, skeleton. Many stimuli, such as integrin occupancy condensed stress fibre was observed and the stress with a ligand, induce integrin aggregation, F-actin fibres were aligned with their long axis perpendicular to the direction of stretch. As the mechanisms of this Correspondence and offprint requests to: Takashi Yasada, Department stress fibre formation after a longer stretch time seemed of Medicine 1, St Marianna University School of Medicine, 2-16-1 Sugao Miyamae-ku, Kawasaki-shi, 216-8511, Japan. to be different from those observed in the early time

Stromal factors support the expansion of the whole hemopoietic spectrum from bone marrow CD34+DR- cells and of some hemopoietic subsets from CD34+ and CD34+DR+ cells.

Ex vivo expanded bone marrow CD34+DR- cells could offer a graft devoid of malignant cells able to promptly reconstitute hemopoiesis after transplant. We investigated the specific expansion requirements of this subpopulation compared to the more mature CD34+ and CD34+DR+ populations. The role of stromal factors was assessed by comparing the expansion obtained when the cells were cultured in (1) long-term bone marrow culture (LTBMC) medium conditioned by an irradiated human BM stroma (CM), (2) medium supplemented with 15% FBS (FBSM) and (3) non-conditioned LTBMC medium (LTM) for 21 days. The effect of the addition of G-CSF (G) and/or of MIP-1alpha (M) to a combination of IL-3, SCF, IL-6 and IL-11 (3, S, 6, 11) was analyzed. Compared to CD34+DR- cells, CD34+ and CD34+DR+ cells gave rise to a similar number of viable cells and to a lower progenitor expansion. The expansion potential of CD34+ and CD34+DR+ cells was equivalent in CM and in FBSM except for both the emergence of CD61 + megakaryocytic cells and LTC-IC maintenance which were improved by culture in CM. In contrast, expansion from CD34+DR- cells was enhanced by CM for all the parameters tested. Compared to FBSM, CM induced a higher level of CFU-GM and BFU-E expansion and allowed the emergence of CD61+ cells. HPP-CFC were maintained or expanded in CM but decreased in FBSM. Compared to input, the number of LTC-IC remaining after 21 days of CD34+DR- expansion culture was strongly decreased in FBSM and variably maintained or expanded in CM. Comparison with LTM indicated that stroma conditioning is responsible for this effect. G-CSF significantly improved CFU-GM and HPP-CFC expansion from CD34+DR- cells without being detrimental to the LTC-IC pool. The growth of CD61+ cells was significantly enhanced by G-CSF in CM. Addition of MIP-1alpha had no significant effect either on progenitor expansion or on LTC-IC, regardless of culture medium. We conclude that factors present in stroma- conditioned medium are necessary to support the expansion of the whole spectrum of hematopoietic cells from CD34+DR- cells and to support the expansion of cell subsets from CD34+ and CD34+DR+.