Cytoplasmic Fragments Research Articles

Abstract 3787: D, L-Sulforaphane inhibits constitutive and interleukin 6-induced activation of signal transducer and activator of transcription 3 in human prostate cancer cells

Abstract D, L-Sulforaphane (SFN), a synthetic analogue of broccoli-derived L-isomer, inhibits viability of human prostate cancer cells in culture and in vivo. We also showed recently that oral administration of SFN prevents development of prostate cancer and pulmonary metastasis in a transgenic mouse model (TRAMP mice) by reducing cell proliferation and augmenting activity of natural killer cells. However, the mechanism underlying anticancer effect of SFN is not fully understood. We now demonstrate that SFN inhibits constitutive and interleukin 6 (IL6)-induced activation of signal transducer and activator of transcription 3 (STAT3), which is implicated in development and progression of prostate cancer. Growth suppressive concentrations of SFN (20 and 40 μmol/L) inhibited constitutive (DU145 cells) and IL6-induced (LNCaP cells) phosphorylation of STAT3 (Tyr705) as well as its upstream regulator Janus-activated kinase 2 (JAK2; Tyr1007/1008). Treatment of DU145 and LNCaP cells with SFN resulted in suppression of IL6-induced transcriptional activity of STAT3 as revealed by luciferase reporter assay and nuclear translocation of STAT3 as judged by immunofluorescence microscopy. The SFN-mediated inhibition of STAT3 activation was due to down-regulation of JAK2 mRNA expression in both cell lines. Levels of many STAT3-regulated gene products including Bcl-2, cyclin D1, and survivin were also down-regulated in SFN-treated cells. The IL6-mediated activation of STAT3 conferred partial but marked protection against SFN-induced apoptosis in LNCaP cells as evidenced by cleavage of poly-(ADP-ribose)-polymerase and caspase-3 and cytoplasmic histone-associated DNA fragmentation. In addition, siRNA knockdown of STAT3 in DU145 cells resulted in a modest yet statistically significant increase in SFN-induced apoptotic DNA fragmentation. The results of the present study demonstrate SFN-mediated inhibition of STAT3 activation in human prostate cancer cells. This investigation was supported by the National Cancer Institute grant CA115498-05. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3787.

P66Shc Is Indispensable for Phenethyl Isothiocyanate–Induced Apoptosis in Human Prostate Cancer Cells

Naturally occurring phenethyl isothiocyanate (PEITC) selectively inhibits growth of cancer cells by causing apoptosis, but the mechanism of cell death induction is not fully understood. We now show, for the first time, that growth factor adapter protein p66(Shc) is indispensable for PEITC-induced apoptosis. Mouse embryonic fibroblasts derived from p66(Shc) knockout mice were significantly more resistant to PEITC-mediated growth inhibition, cytoplasmic histone-associated apoptotic DNA fragmentation, and caspase-3 activation compared with wild-type fibroblasts. The PEITC treatment resulted in induction as well as increased Ser(36) phosphorylation of p66(Shc) in PC-3 and LNCaP human prostate cancer cells. Knockdown of p66(Shc) protein conferred significant protection against PEITC-mediated cytoplasmic histone-associated DNA fragmentation as well as production of reactive oxygen species in both PC-3 and LNCaP cells. The PEITC-treated PC-3 and LNCaP cells exhibited increased binding of p66(Shc) with prolyl isomerase Pin1, a protein implicated in translocation of p66(Shc) to mitochondria. Consistent with these results, treatment of PC-3 cells with PEITC resulted in translocation of p66(Shc) to the mitochondria as judged by immunoblotting using cytosolic and mitochondrial fractions and immunofluorescence microscopy. Growth suppression and apoptosis induction in tumor xenografts in vivo by oral administration of PEITC to the PC-3 tumor-bearing male athymic mice were accompanied by statistically significant increase in the level of Ser(36)-phosphorylated p66(Shc). Collectively, these results provide novel insight into the critical role of p66(Shc) in regulation of PEITC-induced apoptotic cell death in human prostate cancer cells.

Exclusion of cytoplasmic fragments in flow cytometric analysis of lymphoma samples using DyeCycle Violet (144.1)

Abstract Cytoplasmic fragments that originate from fragile lymphoid cells and are commonly seen in lymphoma fine needle aspirate samples. These cytoplasmic fragments can interfere with accurate gating of target cells and quantification protocols used for flow cytometry because of their variable size and expression of lymphoid cell surface antigens on their membrane. The aim of this study was to develop a method to efficiently exclude cytoplasmic fragments in lymphoma samples analyzed by flow cytometry. Tumor cells prepared from lymph node biopsy and fine needle aspirate samples of dogs with lymphoma were stained using DyeCycle Violet (DCV, a membrane-permeable DNA-binding fluorescent dye), antibodies against cell surface antigens, and 7-amino-actinomycin D (7-AAD, a membrane impermeable, DNA-binding fluorescent dye). Multi-parameter flow cytometry was used to exclude antigen-positive cytoplasmic fragments for analysis based on the selective uptake and fluorescence of these dyes. Both cytoplasmic fragments, which are DCV-negative and CD45-positive, and dead cells, which are 7-AAD-positive, could be efficiently separated from tumor cells in lymphoma samples. DCV staining is a useful method to improve the gate setting and quantitative analyses of lymphoma samples by flow cytometry. DI and JFM contributed equally to this work

Sulforaphane Inhibits Constitutive and Interleukin-6–Induced Activation of Signal Transducer and Activator of Transcription 3 in Prostate Cancer Cells

D,L-sulforaphane (SFN), a synthetic analogue of broccoli-derived L-isomer, inhibits viability of human prostate cancer cells and prevents development of prostate cancer and distant site metastasis in a transgenic mouse model. However, the mechanism underlying the anticancer effect of SFN is not fully understood. We now show that SFN inhibits constitutive and interleukin-6 (IL-6)-inducible activation of signal transducer and activator of transcription 3 (STAT3), which is an oncogenic transcription factor activated in many human malignancies, including prostate cancer. Growth-suppressive concentrations of SFN (20 and 40 micromol/L) decreased constitutive (DU145 cells) and IL-6-induced (DU145 and LNCaP cells) phosphorylation of STAT3 (Tyr(705)) as well as its upstream regulator Janus-activated kinase 2 (Tyr(1007/1008)). Exposure of DU145 and LNCaP cells to SFN resulted in suppression of (a) IL-6-induced transcriptional activity of STAT3 as judged by luciferase reporter assay and (b) nuclear translocation of phospho-STAT3 as revealed by immunofluorescence microscopy. Levels of many STAT3-regulated gene products, including Bcl-2, cyclin D1, and survivin, were also reduced in SFN-treated cells. The IL-6-mediated activation of STAT3 conferred partial but marked protection against SFN-induced apoptosis as evidenced by cytoplasmic histone-associated DNA fragmentation and cleavage of poly(ADP-ribose) polymerase and procaspase-3. Furthermore, knockdown of STAT3 protein using small interfering RNA resulted in a modest yet statistically significant increase in SFN-induced apoptotic DNA fragmentation in DU145 cells. Suppression of STAT3 activation was also observed in cells treated with naturally occurring analogues of SFN. In conclusion, the present study indicates that inhibition of STAT3 partially contributes to the proapoptotic effect of SFN.

Altered Transcript Reveals an Orf507 Sterility-Related Gene in Chili Pepper (Capsicum annuum L.)

A previously reported cytoplasmic male sterility-associated fragment was detected in chili pepper (Capsicum annuum L.) and sequenced in the mitochondria of a sterile plant. This fragment, 16 kb in size, contains two common mitochondrial genes, coxII and atp6-2, and the newly detected gene orf456, which appears to be a strong candidate gene for the male sterile phenotype. The data show that one nucleotide (Cytosine) at the +449 position is missing compared with the original sequence data of orf456. By translation to the amino acid sequence, the termination codon was found 49 nt downstream from the original site. This hypothetical extension was confirmed by RT-PCR, which showed a sequence size of 507 bp. Since pollen fertility can be restored by a nuclear restorer of fertility gene (Rf), the transcription of orf507 was checked in four stages of flower development. The RT-PCR resulted in a fragment in stage III in the restorer line, indicating a possible role in microspore development.

Kinase-active Signaling Complexes of Bacterial Chemoreceptors Do Not Contain Proposed Receptor−Receptor Contacts Observed in Crystal Structures

The receptor dimers that mediate bacterial chemotaxis form high-order signaling complexes with CheW and the kinase CheA. From the packing arrangement in two crystal structures of different receptor cytoplasmic fragments, two different models have been proposed for receptor signaling arrays: the trimers-of-dimers and hedgerow models. Here we identified an interdimer distance that differs substantially in the two models, labeled the atoms defining this distance through isotopic enrichment, and measured it with (19)F-(13)C REDOR. This was done in two types of receptor samples: isolated bacterial membranes containing overexpressed, intact receptor and soluble receptor fragments reconstituted into kinase-active signaling complexes. In both cases, the distance found was not compatible with the receptor dimer-dimer contacts observed in the trimers-of-dimers or in the hedgerow models. Comparisons of simulated and observed REDOR dephasing were used to deduce a closest approach distance at this interface, which provides a constraint for the possible arrangements of receptor assemblies.

Cytoskeletal coherence requires myosin-IIA contractility

Maintaining a physical connection across cytoplasm is crucial for many biological processes such as matrix force generation, cell motility, cell shape and tissue development. However, in the absence of stress fibers, the coherent structure that transmits force across the cytoplasm is not understood. We find that nonmuscle myosin-II (NMII) contraction of cytoplasmic actin filaments establishes a coherent cytoskeletal network irrespective of the nature of adhesive contacts. When NMII activity is inhibited during cell spreading by Rho kinase inhibition, blebbistatin, caldesmon overexpression or NMIIA RNAi, the symmetric traction forces are lost and cell spreading persists, causing cytoplasm fragmentation by membrane tension that results in 'C' or dendritic shapes. Moreover, local inactivation of NMII by chromophore-assisted laser inactivation causes local loss of coherence. Actin filament polymerization is also required for cytoplasmic coherence, but microtubules and intermediate filaments are dispensable. Loss of cytoplasmic coherence is accompanied by loss of circumferential actin bundles. We suggest that NMIIA creates a coherent actin network through the formation of circumferential actin bundles that mechanically link elements of the peripheral actin cytoskeleton where much of the force is generated during spreading.

Staurosporine-induced programmed cell death in Blastocystis occurs independently of caspases and cathepsins and is augmented by calpain inhibition

Previous studies have shown that the protozoan parasite Blastocystis exhibits apoptotic features with caspase-like activity upon exposure to a cytotoxic monoclonal antibody or the anti-parasitic drug metronidazole. The present study reports that staurosporine (STS), a common apoptosis inducer in mammalian cells, also induces cytoplasmic and nuclear features of apoptosis in Blastocystis, including cell shrinkage, phosphatidylserine (PS) externalization, maintenance of plasma membrane integrity, extensive cytoplasmic vacuolation, nuclear condensation and DNA fragmentation. STS-induced PS exposure and DNA fragmentation were abolished by the mitochondrial transition pore blocker cyclosporine A and significantly inhibited by the broad-range cysteine protease inhibitor iodoacetamide. Interestingly, the apoptosis phenotype was insensitive to inhibitors of caspases and cathepsins B and L, while calpain-specific inhibitors augmented the STS-induced apoptosis response. While the identities of the proteases responsible for STS-induced apoptosis warrant further investigation, these findings demonstrate that programmed cell death in Blastocystis is complex and regulated by multiple mediators.

A novel testis-specific Na H exchanger is involved in sperm motility and fertility

Sodium-hydrogen exchanger as a channel for regulation of intracellular pH might be a crucial modulator of sperm capacitation and motility. Three members of this family have been identified in spermatozoa. A novel protein testis-specific sodium-hydrogen exchanger named mtsNHE was cloned in the present study. The mtsNHE localizing on principle piece of sperm flagellum contained 12 predicted transmembrane regions without cytoplasmic fragment at carboxyl terminus. Hydrophilic region was common in the sodium-hydrogen exchanger family members. Polyclonal antibodies to trans-membrane region significantly reduced sperm motility, acrosome reaction and ratio of in vitro fertilization. By in-pouring the antibodies in sperm solution, intracellular pH and calcium concentration were decreased. Muscle injection of female mice with the specific gene vaccine of mtsNHE, significantly stepped down fertility rate. Considering its specific expression and involvement in the regulation of fertility, the mtsNHE might be a potential target molecule for developing a new male contraceptive.

Slug suppression induces apoptosis via Puma transactivation in rheumatoid arthritis fibroblast-like synoviocytes treated with hydrogen peroxide.

Inadequate apoptosis contributes to synovial hyperplasia in rheumatoid arthritis (RA). Recent study shows that low expression of Puma might be partially responsible for the decreased apoptosis of fibroblast-like synoviocytes (FLS). Slug, a highly conserved zinc finger transcriptional repressor, is known to antagonize apoptosis of hematopoietic progenitor cells by repressing Puma transactivation. In this study, we examined the expression and function of Slug in RA FLS. Slug mRNA expression was measured in the synovial tissue (ST) and FLS obtained from RA and osteoarthritis patients. Slug and Puma mRNA expression in FLS by apoptotic stimuli were measured by real-time PCR analysis. FLS were transfected with control siRNA or Slug siRNA. Apoptosis was quantified by trypan blue exclusion, DNA fragmentation and caspase-3 assay. RA ST expressed higher level of Slug mRNA compared with osteoarthritis ST. Slug was significantly induced by hydrogen peroxide (H2O2) but not by exogenous p53 in RA FLS. Puma induction by H2O2 stimulation was significantly higher in Slug siRNA-transfected FLS compared with control siRNA-transfected FLS. After H2O2 stimulation, viable cell number was significantly lower in Slug siRNA-transfected FLS compared with control siRNA-transfected FLS. Apoptosis enhancing effect of Slug siRNA was further confirmed by ELISA that detects cytoplasmic histone-associated DNA fragments and caspase-3 assay. These data demonstrate that Slug is overexpressed in RA ST and that suppression of Slug gene facilitates apoptosis of FLS by increasing Puma transactivation. Slug may therefore represent a potential therapeutic target in RA.

Solid-State NMR Demonstrates that Active Signaling Complexes of Bacterial Chemoreceptors Do Not Adopt the Proposed Trimer-Of-Dimers Structure

The receptor dimers that mediate bacterial chemotaxis form signaling complexes with CheW and the kinase CheA. Based on the packing arrangement observed in two different crystal structures of two different receptor cytoplasmic fragments, two different models have been proposed for receptor signaling arrays: the trimers-of-dimers and hedgerow models. We have identified an inter-dimer distance predicted to be substantially different by the two models, labeled the atoms defining this distance through isotopic enrichment, and measured it with 19F-13C REDOR. This was done in two types of receptor samples: isolated bacterial membranes containing overexpressed, intact receptor, and soluble receptor fragments reconstituted into kinase-active signaling complexes. In both cases, the distance found was incompatible with both the trimers-of-dimers and the hedgerow models. Comparisons of simulated and observed REDOR dephasing were used to deduce a closest-approach distance at this interface, which provides a constraint for the possible arrangements of receptor assemblies in the kinase-active signaling state.This research was supported by U.S. Public Health Service Grant GM47601; DJF was partially supported by National Research Service Award T32 GM08515.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Evolving a robust signal transduction pathway from weak cross‐talk

We have evolved a robust two-component signal transduction pathway from a sensor kinase (SK) and non-partner response regulator (RR) that show weak cross-talk in vitro and no detectable cross-talk in vivo in wild-type strains. The SK, CpxA, is bifunctional, with both kinase and phosphatase activities for its partner RR. We show that by combining a small number of mutations in CpxA that individually increase phosphorylation of the non-partner RR OmpR, phosphatase activity against phospho-OmpR emerges. The resulting circuit also becomes responsive to input signal to CpxA. The effects of combining these mutations in CpxA appear to reflect complex intragenic interactions between multiple sites in the protein. However, by analyzing a simple model of two-component signaling, we show that the behavior can be explained by a monotonic change in a single parameter controlling protein–protein interaction strength. The results suggest one possible mode of evolution for two-component systems with bifunctional SKs whereby the remarkable properties and competing reactions that characterize these systems can emerge by combining mutations of the same effect.

Clinical Benefit of INCB7839, a Potent and Selective Inhibitor of ADAM10 and ADAM17, in Combination with Trastuzumab in Metastatic HER2 Positive Breast Cancer Patients.

Abstract Background: In HER2 over-expressing breast cancer cells, the HER2 protein can be cleaved by a metalloproteinase, ADAM10. While the extracellular domain (ECD) of HER2 is released into the serum, the truncated HER2 receptor fragment, termed p95, remains in the tumor cell membrane as a constitutively active receptor tyrosine kinase. Previous studies have shown that the presence of p95 in tumor cells is associated with poor clinical outcomes in patients with metastatic HER2 positive breast cancer and it was recently shown that patients with p95+ HER2 positive breast cancer are resistant to trastuzumab-based therapy. Therefore, inhibition of HER2 cleavage by the ADAM10/ADAM17 inhibitor, INCB7839, which reduces the formation of soluble HER2 ECD as well as p95 levels in the tumor, may enhance the clinical efficacy of trastuzumab in HER2+ breast cancer patients. Materials and Methods: This study is a single arm modified dose escalation open label trial of INCB7839 + trastuzumab in women with HER2+ metastatic breast cancer, naïve to chemotherapy. Three doses of INCB7839 were studied (100 mg, 200 mg, 300 mg BID) with 6 patients/cohort and an expansion arm at the 300mg BID dose. Herceptin was administered on a Q3 week schedule. Pharmacokinetics, plasma HER2 ECD levels and p95 expression in tumor tissue were assessed in addition to clinical response and safety. Results: 39 patients have been enrolled in the study and assessment of HER2 ECD levels, p95 status and best clinical response completed on 30 patients. INCB7839 administration results in a dose-dependent reduction in the elevated levels of circulating HER2 ECD with a mean of ∼80% inhibition achieved at the highest dose tested (300 mg BID). At the 300mg BID dose, the current overall response rate is 40% (6/15 evaluable patients) with a higher response rate (6/11 or 55%) observed in patients with average plasma concentrations of INCB7839 above the IC50 for reduction of ECD levels, 320nM. Importantly, INCB7839 increases the clinical response rate in p95+ patients, with equivalent responses observed in the p95+ and p95- patients treated with INCB7839 + trastuzumab. The combination has been generally safe and well tolerated. Discussion: Proteolytic cleavage of the HER2 receptor by ADAM proteases results in the formation of a cytoplasmic fragment (p95) that possesses constitutive kinase activity with the release of ECD. Importantly, elevated levels of ECD and/or p95 have been associated with poor prognosis and clinical data suggest that p95 affords resistance to trastuzumab. Biomarker and clinical data from this trial demonstrate that INCB7839 can markedly reduce HER2 cleavage in patients with HER2+ breast cancer, and suggests that INCB7839, by inhibiting the HER2 shedding process, can render a subpopulation of HER2+ patients (as defined by the presence of p95) that would be predicted to be trastuzumab resistant clinically responsive to trastuzumab therapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5056.

Ligand‐Stimulated VEGFR2 Signaling is Regulated by Co‐Ordinated Trafficking and Proteolysis

Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration. Ligand-stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT-0 subunit caused differential effects on ligand-stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF-A-induced intracellular signaling to c-Akt and endothelial nitric oxide synthase (eNOS). VEGF-A-stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF-A-mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C-dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF-stimulated endothelial signaling and cell migration.

Induction of apoptosis in intestine following ethanol intoxication and burn injury

Disruption of intestinal barrier integrity and the subsequent translocation of bacteria or their products are often implicated in lethal sepsis and multiple organ failure following major trauma and in ICU patients. We evaluated the effect of ethanol intoxication and burn injury on intestinal cell apoptosis. Male rats (|250 g) were gavaged with ethanol to achieve a blood ethanol level of |100 mg/dL prior to |12.5% total body surface area burn or sham injury. One day after injury, rats were sacrificed and small intestinal specimens were collected. The samples were sonicated and centrifuged. The clear supernatant was analyzed for phospho-p38 and various apoptotic markers using western blot and specific antibodies. We found that ethanol intoxication combined with burn injury resulted in a significant up-regulation of phospho-p38 in comparison to sham animals irrespective of ethanol treatment. Our results indicated a significant increase in proapoptotic proteins Bad and Bax in the homogenates prepared from rats receiving a combined insult of ethanol and burn injury compared to either ethanol or burn injury alone. The level of cleaved caspase 3 was also significantly up-regulated following ethanol and burn injury compared to the sham animals irrespective of ethanol intoxication. Anti-apoptotic protein Mcl-1 was significantly down regulated while the level of Bcl-2 remained unchanged after ethanol and burn injury compared to sham animals. Increase in intestinal apoptosis following ethanol and burn injury was further confirmed by measuring the cytoplasmic histone-associated DNA fragments by cell death detection ELISA. These findings indicate that ethanol combined with burn injury causes an increase in apoptosis in the intestine. Such an increase in apoptosis may impair the intestinal epithelial barrier and thus enhance the possibility of translocation of intestinal bacteria and their products following ethanol and burn injury. (Supported by R01AA015731 & R21AA015979 and the Dr. Ralph and Marian C. Falk Medical Research Trust).

Infusing Mature Megakaryocytes Into Mice Yields Functional Platelets: Biological and Clinical Implications.

Abstract 225Thrombopoiesis, the process by which circulating platelets (plts) arise from megakaryocytes (Megs) remains incompletely understood. Two models had been proposed: (1) release of plts produced from Megs within the marrow space and (2) release of plts by circulating Megs within the pulmonary vasculature. Recently, murine two-photon electron microscopy suggested that the actual process may involve an intermediate model, wherein Megs adjacent to vascular spaces within the marrow shed large cytoplasmic fragments into the circulation, which presumably go on to form plts in the vascular space. We have now tested whether infused cultured Megs can form circulating, functional plts. Cultured Megs from the fetal livers of Day 14 wildtype (WT) C57Bl6 murine embryos grown in the presence of thrombopoietin for 10 days were separated using a bovine serum albumin gradient into two cell fractions: (1) large, mature CD41+/CD42+ Megs (average ploidy = 16–32N) and (2) an admixture of proplatelets and small CD41+/CD42+ Megs (average ploidy = 2-4N). These fractions were separately infused into hαIIb mice that expressed human, but not mouse, αIIb on their platelets. Donor-derived circulating plts were detected using species-specific antibodies to distinguish them from recipient hαIIb+ plts. Infusions of isolated WT plts as a positive control resulted in immediately detectable donor plts, and these plts lasted 24–36 hrs. Infused large Megs produced plts with delayed kinetics. Few donor plts were detectable at 5 mins, followed by a peak at 30–60 min. These plts were normal in size as determined by side-scatter and lasted for ∼24 hrs. The peak yield was ∼50-100 plts/infused Meg. In these studies, these derived plt levels reached as high as 10% of the total circulating plts in these mice who had plt counts of >8×105/μl. Maximal tolerated dose of infused large Megs was not tested. Infusion of the proplatelets/small Megs fraction generated an immediate peak of circulating plts and these lasted <24 hrs. The yield of plts was approximately equal to the number of infused proplatelets/platelets in this admixture. Half-lives of the derived plts were unaffected by the inclusion of GM6001 or TAPI-1, both of which have been reported to block metalloproteinase MMP9/ADAM17 and lengthen the half-life of culture-derived plts. Functionality of the derived plts from both cell fractions was compared to infused plts in situ using the laser-injury cremaster system. Qualitatively, derived plts were incorporated as well as infused plts into growing thrombi. Remarkably, we also detected large CD41+ cells, which circulated and occasionally became incorporated into thrombi. These cells were much more common in the proplatelet/small Meg infusions than in the large Megs infusion. Thus, our studies show that whole, mature Megs shed a significant number of plts in circulation within mice. Plts level achievable in the recipient mouse approximate a clinically relevant level. These plts are normal in size and appear to be functional. Further studies to demonstrate where plts are released from these Megs and the basis for their shortened half-life remain to be performed; however, these studies suggest an alternative source of circulating platelets and a novel strategy for generating sufficient plts from cultured Megs for clinical use. Disclosures:Poncz:Diagnostica Stago: Patents & Royalties.

Interaction of TorsinA with Its Major Binding Partners Is Impaired by the Dystonia-associated ΔGAG Deletion

Early onset (DYT1) torsion dystonia is a dominantly inherited movement disorder associated with a three-base pair (DeltaGAG) deletion that removes a glutamic acid residue from the protein torsinA. TorsinA is an essential AAA(+) (ATPases associated with a variety of cellular activities) ATPase found in the endoplasmic reticulum and nuclear envelope of higher eukaryotes, but what it does and how changes caused by the DeltaGAG deletion lead to dystonia are not known. Here, we asked how the DYT1 mutation affects association of torsinA with interacting proteins. Using immunoprecipitation and mass spectrometry, we first established that the related transmembrane proteins LULL1 and LAP1 are prominent binding partners for torsinA in U2OS cells. Comparative analysis demonstrates that these two proteins are targeted to the endoplasmic reticulum or nuclear envelope by their divergent N-terminal domains. Binding of torsinA to their C-terminal lumenal domains is stabilized when residues in any one of three motifs implicated in ATP hydrolysis (Walker B, sensor 1, and sensor 2) are mutated. Importantly, the DeltaGAG deletion does not stabilize this binding. Indeed, deleting the DeltaGAG encoded glutamic acid residue from any of the three ATP hydrolysis mutants destabilizes their association with LULL1 and LAP1C, suggesting a possible basis for loss of torsinA function. Impaired interaction of torsinA with LULL1 and/or LAP1 may thus contribute to the development of dystonia.

The use of mitochondrial nutrients to improve the outcome of infertility treatment in older patients

We present a hypothesis emphasizing the role of mitochondrial dysfunction in reproductive senescence and suggesting the use of mitochondrial nutrients as an adjuvant treatment in older patients with infertility.

Apoptosis Induced by Duck Reovirus p10.8 Protein in Primary Duck Embryonated Fibroblast and Vero E6 Cells

Muscovy duck reovirus (MDRV) is an important poultry pathogen that causes high morbidity and mortality in ducklings. The mechanisms by which viruses kill susceptible cells, and ultimately produce diseases, in Muscovy duck remain poorly understood. In this study, we focused on the biologic functions of the MDRV p10.8 protein in vitro. The p10.8 protein is a small protein of MDRV that is encoded by the first open reading frame of the S4 segment. In our study, the p10.8-encoding gene was individually cloned and expressed in bacterial and eukaryotic cells. The p10.8 protein had no potential transmembrane domain; it shared no sequence similarity to other known fusion-associated small transmembrane proteins encoded by the avian reovirus, Nelson Bay virus or baboon reovirus; and it did not show any syncytium formation activity. The p10.8 protein induced apoptosis when expressed by itself in transfected primary Muscovy duck embryonic fibroblasts or in Vero E6 cells. Four assays were used to analyze the apoptosis induced by p10.8: DNA ladder formation; terminal deoxynucleotidyl transferase dUTP nick end labeling; enzyme-linked immunosorbent assay detection of cytoplasmic histone-associated DNA fragments; and nuclear staining with propidium iodide. Two deletion products, p10.8delta1 (1-63aa; amino acid position) and p10.8delta2 (64-96aa), were constructed. Deletion analysis suggests that p10.8delta1 (1-63aa) is important in mediating p10.8-induced apoptosis because its deletion abolishes induction of apoptosis.

Defragmentation of low grade day 3 embryos resulted in a sustained reduction in fragmentation, but did not improve compaction, or blastulation rates

OBJECTIVE: Anuclear cytoplasmic fragments formed in early embryos are associated with diminished implantation. We have reported that defragmentation results in IVF implantation rates equivalent to unfragmented embryos. This study assesses whether day 3 defragmentation results in improved day 5 embryo fragmentation, compaction and blastulation rates.DESIGN: Prospective blinded randomized trial of day 3 embryo defragmentation.MATERIALS AND METHODS: Following IRB approval, 50 fragmented low grade embryos, unlikely to be transferred, of equal fragmentation and cell number were randomized in sets of two from the same patient to undergo hatching and defragmentation or hatching alone. The embryos were then evaluated under 400X inverted microscopy by a blinded embryologist on days 4 and 5, for percent fragmentation and stage. Assisted hatching was performed on all day 3 embryos with a micro-manipulator, laser ablating 5% of the zona pellucida. Defragmentation was performed on “treatment” embryos using oil filled injectors to suction out fragments from around and between the blastomeres. Embryos were isolated and cultured in Cook medium to day 5. T-test and chi square or fisher exact test were performed with Systat 12.1 software.Tabled 1Day 5 Post-Defragmentation ResultsResultsNon-DefragmentedDefragmentedP ValuePercent Fragmentation18.8%6.4%0.002Compaction80%72%0.74Blastula Formation32%32%1.00 Open table in a new tab CONCLUSIONS: Defragmentation of low grade day 3 embryos resulted in a sustained reduction in percent fragmentation through day 5, but did not increase the rate of compaction or blastula formation. OBJECTIVE: Anuclear cytoplasmic fragments formed in early embryos are associated with diminished implantation. We have reported that defragmentation results in IVF implantation rates equivalent to unfragmented embryos. This study assesses whether day 3 defragmentation results in improved day 5 embryo fragmentation, compaction and blastulation rates. DESIGN: Prospective blinded randomized trial of day 3 embryo defragmentation. MATERIALS AND METHODS: Following IRB approval, 50 fragmented low grade embryos, unlikely to be transferred, of equal fragmentation and cell number were randomized in sets of two from the same patient to undergo hatching and defragmentation or hatching alone. The embryos were then evaluated under 400X inverted microscopy by a blinded embryologist on days 4 and 5, for percent fragmentation and stage. Assisted hatching was performed on all day 3 embryos with a micro-manipulator, laser ablating 5% of the zona pellucida. Defragmentation was performed on “treatment” embryos using oil filled injectors to suction out fragments from around and between the blastomeres. Embryos were isolated and cultured in Cook medium to day 5. T-test and chi square or fisher exact test were performed with Systat 12.1 software. CONCLUSIONS: Defragmentation of low grade day 3 embryos resulted in a sustained reduction in percent fragmentation through day 5, but did not increase the rate of compaction or blastula formation.