Gradual Cell Death Research Articles

Temporal Progression of Excitotoxic Calcium Following Distal Middle Cerebral Artery Occlusion in Freely Moving Mice.

Ischemic stroke is recognized as one of the leading causes of adult disability, morbidity, and death worldwide. Following stroke, acute neuronal excitotoxicity can lead to many deleterious consequences, one of which is the dysregulation of intracellular calcium ultimately culminating in cell death. However, to develop neuroprotective treatments that target neuronal excitotoxicity, it is essential to know the therapeutic time window for intervention following an ischemic event. To address this question, the current study aimed to characterize the magnitude and temporal progression of neuronal intracellular calcium observed following distal middle cerebral artery occlusion (dMCAO) in mice. Using the calcium fluorescence indicator, GCaMP, we tracked neuronal population response in freely moving animals immediately following dMCAO in both the core infarct and peri-infarct regions. Our results demonstrate that calcium excitotoxicity following artery occlusion can be generally characterized by two phases: a transient increase in activity that lasts tens of minutes, followed by a long, slow sustained increase in fluorescence signal. The first phase is primarily thought to represent neuronal hyperexcitability, defining our therapeutic window, while the second may represent gradual cell death. Importantly, we show that the level of intracellular calcium following artery occlusion correlated with the infarct size at 24 h demonstrating a direct connection between excitotoxicity and cell death in our stroke model. In addition, we show that administration of the NMDA antagonist MK-801 resulted in both a decrease in calcium signal and a subsequent reduction in the infarct size. Altogether, this study represents the first demonstration in freely moving animals characterizing the temporal progression of toxic calcium signaling following artery occlusion. In addition, these results define a critical time window for neuroprotective therapeutic intervention in mice.

Assessment of Potential Cytotoxicity During Vital Observation at the BioStation CT

In recent decades, the methods based on the cell technologies have become more relevant for medical and pharmaceutical research. In this paper, weconsidered the BioStation CT using for continuous in vivo observation of Ehrlich ascitic carcinoma cell culture under the influence of two active substances in different concentrations (from 1.25 to 20 mg/l). As a result of the carcinoma cells condition monitoring for 4.5 days it was shown that substance No 2 does not affect the cell viability while substance No 1 causes the carcinoma cell death, and this active substance effect is dose-dependent. Photodocumentation at two-hour intervals allowed us to research differences in the rate of cell destruction (intensive disintegration in the first day with further stabilization of the living cells number or gradual cell death throughout the experiment), as well as differences in the time of 50 % mortality reaching. Thus in the study it was demonstrated that due to the fact that the BioStation CT combines the properties of a CO2 -incubator and a microscope this device is promising for toxicological studies and significantly expands the detection possibilities of processes occurring with living cells for a sufficiently long time period making possible further analysis of the cell behavior characteristics throughout the experiment, and that is fundamentally different from the systems allowing only the final result fixation of long-term active substance exposure.

Abstract B78: Irreversible electroporation (IRE) acts as an “in situ vaccine” and induces antitumor immune responses in pancreatic cancer

Abstract Introduction: Cancer immunotherapy has made waves of progress in recent years with the advent of checkpoint inhibitor therapies against several cancers. However, pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded immunotherapy approaches. PC hosts a notoriously immunosuppressive microenvironment comprised of an abundance of immunosuppressive tumor associated macrophages, myeloid derived suppressor cells (MDSCs) and regulatory T-cells with a scarcity of effector CD8+ T-cells. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures. Unlike thermal ablation techniques, IRE results in a gradual apoptotic cell death and is currently in clinical use for selected patients with locally advanced PC. We hypothesize that the abundant release of antigens by IRE can induce an “in situ vaccination” effect that can elicit adaptive T-cell-mediated anti-tumor immune responses. Methods: We have developed a robust syngeneic immunocompetent mouse model of PC using a cell line established from a tumor arising in a LSL-KrasG12D/+; LSL-Trp53R172H/+; PDX1Cre/+; LSL-ROSA26 Luc/+ mouse (KPC). We have utilized the ECM 830 square wave pulse generator to deliver IRE (100 μsec pulses of electricity at 1500 V/cm using a two-needle array probe) to subcutaneous tumors measuring 5-7 mm in diameter. The effects of IRE on primary tumor growth and secondary tumor challenge were observed. Flow cytometry-based immunoprofiling studies of tumors at one week post-IRE were performed in triplicate and presented as mean difference between IRE and no treatment (standard error). *P<0.01 by student’s t-test was considered statistically significant. Results: Vaccination studies with irradiated KPC cells confirmed that they were immunogenic in C57BL/6 mice. In preliminary studies, we identified the minimal IRE dose to induce tumor regression to be 150 pulses, with complete regression in 3 of 9 tumors (33%). Survival was prolonged in the IRE groups, particularly in mice with complete regression. This outcome was confirmed to be immune-mediated, as IRE of tumor-bearing immunodeficient Rag-1-/- mice, which lack functional T and B cell components, results in progressive outgrowth with similar kinetics as untreated mice. When immunocompetent mice with tumor regression were re-challenged two weeks later with 105 cells injected into the contralateral flank, we observed no growth in the secondary tumors, confirming adaptive immune activation post-IRE. Tumor immunoprofiling revealed a 43±2%* decrease in the number of tumor-infiltrating MDSCs (CD11b+/CD11c+/Gr-1hi) post-IRE, with a similar decrease in the respective tumor draining inguinal lymph nodes. An increase in total tumor infiltrating lymphocytes (TILs) was also observed, including an 18±2%* increase in CD8+ TILs and a concomitant large increase (2.5 fold*) in PD-1+/CD8+ double positive T-cells. Conclusion: These results suggest that IRE is capable of triggering an antitumor immune response that contributes to the local effects of IRE and inhibits growth of secondary tumor re-challenge. IRE affects the composition of immune cells in the tumor microenvironment, including a large increase in PD-1+/CD8+ TILs. These effects may be augmented by combining IRE with checkpoint blockade, which is a focus of our ongoing studies. Citation Format: Jayanth Shankara Narayanan, Tomoko Hayashi, Aaron M. Miller, Stephen P. Schoenberger, Rebekah R. White. Irreversible electroporation (IRE) acts as an “in situ vaccine” and induces antitumor immune responses in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B78.

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation.

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Iron-Chelating Drugs Enhance Cone Photoreceptor Survival in a Mouse Model of Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a group of hereditary retinal degeneration in which mutations commonly result in the initial phase of rod cell death followed by gradual cone cell death. The mechanisms by which the mutations lead to photoreceptor cell death in RP have not been clearly elucidated. There is currently no effective treatment for RP. The purpose of this work was to explore iron chelation therapy for improving cone survival and function in the rd10 mouse model of RP. Two iron-chelating drugs, 5-(4-(2-hydroxyethyl) piperazin-1-yl (methyl)-8-hydroxyquinoline (VK28) and its chimeric derivative 5-(N-methyl-N-propargyaminomethyl)-quinoline-8-oldihydrochloride (VAR10303), were injected intraperitoneally to rd10 mice every other day starting from postnatal day 14. We investigate the effects of the two compounds on cone rescue at three time points, using a combination of immunocytochemistry, RT-PCR, Western blot analysis, and a series of visual function tests. VK28 and VAR10303 treatments partially rescued cones, and significantly improved visual function in rd10 mice. Moreover, we showed that the neuroprotective effects of VK28 and VAR10303 were correlated to inhibition of neuroinflammation, oxidative stress, and apoptosis. Furthermore, we demonstrated that downregulation of NF-kB and p53 is likely to be the mechanisms by which proinflammatory mediators and apoptosis are reduced in the rd10 retina, respectively. VK28 and VAR10303 provided partial histologic and functional rescue of cones in RD10 mice. Our study demonstrated that iron chelation therapy might represent an effective therapeutic strategy for RP patients.

Abstract 499: IgH enhancers hs3b/hs4 are dispensable for c-Myc deregulation in mouse plasmacytomas with T(12;15) translocations

Abstract c-Myc deregulating t(12;15) chromosomal translocation is the hallmark cytogenetic abnormality of murine plasmacytomas (PCTs). In the great majority of PCTs, the immunoglobulin heavy chain (IgH) locus is broken between Eμ enhancer and the 3’ regulatory region (3’RR), making the latter the major candidate for orchestrating c-Myc deregulation. To elucidate the role of 3’RR in tumorigenesis, we induced PCTs in mice deficient for the major 3’RR enhancer elements, namely hs3b and hs4 (3’KO). Unexpectedly, and contrary to the previous observations made in a mouse lymphoma model, Hs3b/Hs4 3’KO mice did develop t(12;15)-positive PCTs, although with a lower incidence than wild-type controls. In heterozygous mice, there was no allelic bias in targeting IgH for the t(12;15). Molecular analysis of IgH/Myc junctions revealed dominance of Sμ region breakpoints compared to the prevalence of hits into Sγ or Sα in the controls. Analysis of c-Myc expression as well as Ig secretion in 3’KO PCT cell lines revealed no significant differences from the controls. Our current findings highlight the complexity of the 3’RR and the potential of its components to compensate for each other in the context of differentiation from B-lymphocyte to plasma cell. We next extended our observations to include a PCT model where Cre-mediated deletion can remove all four 3'IgH enhancers: hs3a, hs1,2, hs3b and hs4. In PCT cell lines where c-Myc is translocated to an IgH BAC transgene with a floxed 3’IgH enhancer region, induction of retrovirally-expressed Cre-recombinase causes gradual Myc downregulation and complete cell death within 96 hours. The later observation confirms the requirement of the full 3’RR for c-Myc deregulation by T(12;15). Citation Format: Alexander L. Kovalchuk, Herbert C. Morse. IgH enhancers hs3b/hs4 are dispensable for c-Myc deregulation in mouse plasmacytomas with T(12;15) translocations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 499. doi:10.1158/1538-7445.AM2017-499

Mild MPP+ exposure-induced glucose starvation enhances autophagosome synthesis and impairs its degradation

Parkinson’s disease (PD) is a prevalent neurodegenerative disorder, mainly characterised by the progressive loss of dopaminergic neurons. MPP+ has been widely used as a PD-related neurotoxin, and their reports suggested the several hypotheses for neuronal cell death. However, most of these hypotheses come from the studies about the acute MPP+ exposure. We previously revealed that mild MPP+ exposure (10 and 200 μM), which induces gradual cell death, impairs autophagosome degradation at 48 h. In the present study, we further investigated the specific events of mild MPP+ exposure and revealed that mild MPP+ exposure causes the cell death through glucose starvation, but not acute toxic model (2.5 and 5 mM). At 36 h after mild MPP+ exposure, autophagosome synthesis was enhanced owing to glucose starvation and continued to enhance until 48 h, despite impaired autophagosome degradation. Inhibition of autophagosome synthesis reduced mild MPP+-induced cell death. In conclusion, we clarified that glucose starvation-enhanced autophagosome synthesis occurs at an earlier stage than impaired autophagosome degradation and is important in mild MPP+ toxicity.

Cold acclimation improves chill tolerance in the migratory locust through preservation of ion balance and membrane potential.

Most insects have the ability to alter their cold tolerance in response to temporal temperature fluctuations, and recent studies have shown that insect cold tolerance is closely tied to the ability to maintain transmembrane ion gradients that are important for the maintenance of cell membrane potential (Vm). Several studies have therefore suggested a link between preservation of Vm and cellular survival after cold stress, but none has measured Vm in this context. We tested this hypothesis by acclimating locusts (Locusta migratoria) to high (31°C) and low temperature (11°C) for 4 days before exposing them to cold stress (0°C) for up to 48 h and subsequently measuring ion balance, cell survival, muscle Vm, and whole animal performance. Cold stress caused gradual muscle cell death, which coincided with a loss of ion balance and depolarization of muscle Vm The loss of ion balance and cell polarization were, however, dampened markedly in cold-acclimated locusts such that the development of chill injury was reduced. To further examine the association between cellular injury and Vm we exposed in vitro muscle preparations to cold buffers with low, intermediate, or high [K+]. These experiments revealed that cellular injury during cold exposure occurs when Vm becomes severely depolarized. Interestingly, we found that cellular sensitivity to hypothermic hyperkalaemia was lower in cold-acclimated locusts that were better able to defend Vm whilst exposed to high extracellular [K+]. Together these results demonstrate a mechanism of cold acclimation in locusts that improves survival after cold stress: increased cold tolerance is accomplished by preservation of Vm through maintenance of ion homeostasis and decreased K+ sensitivity.

Expression of DNMT1 in neural stem/precursor cells is critical for survival of newly generated neurons in the adult hippocampus

Adult neurogenesis persists throughout life in the dentate gyrus (DG) of the hippocampus, and its importance has been highlighted in hippocampus-dependent learning and memory. Adult neurogenesis consists of multiple processes: maintenance and neuronal differentiation of neural stem/precursor cells (NS/PCs), followed by survival and maturation of newborn neurons and their integration into existing neuronal circuitry. However, the mechanisms that govern these processes remain largely unclear. Here we show that DNA methyltransferase 1 (DNMT1), an enzyme responsible for the maintenance of DNA methylation, is highly expressed in proliferative cells in the adult DG and plays an important role in the survival of newly generated neurons. Deletion of Dnmt1 in adult NS/PCs (aNS/PCs) did not affect the proliferation and differentiation of aNS/PCs per se. However, it resulted in a decrease of newly generated mature neurons, probably due to gradual cell death after aNS/PCs differentiated into neurons in the hippocampus. Interestingly, loss of DNMT1 in post-mitotic neurons did not influence their survival. Taken together, these findings suggest that the presence of DNMT1 in aNS/PCs is crucial for the survival of newly generated neurons, but is dispensable once they accomplish neuronal differentiation in the adult hippocampus.

Pretreatment of epithelial cells with live streptococcus pneumoniae had no detectable effect on influenza A virus replication in vitro (MPF1P.774)

Abstract Influenza A virus (IAV) and Streptococcus pneumoniae (pneumococcus) are two major upper respiratory tract pathogens responsible for exacerbated disease in coinfected individuals. Information on the direct effect of S. pneumoniae on IAV replication in humans is unknown. Such an investigation is important because S. pneumoniae is a commensal of the human upper respiratory tract. The aim of this study was to determine whether treatment of epithelial cells with S. pneumoniae affects IAV replication, determined with the help of a standard immunofluorescence assay (IFA). For this study, four IAV permissive epithelial cell lines including two human-derived cell lines, 12 pneumococcal strains including recent human clinical isolates which represent different genetic backgrounds and serotypes, and six IAV strains of varying genetic nature and pathogenic potential including the pandemic 2009 H1N1 virus were used. Our results suggested that pretreatment of MDCK cells with 7.5×10^6 colony-forming unit (CFU) of live S. pneumoniae resulted in gradual cell-death in a time-dependent manner (1/2 hr to 4 hr). However, pretreatment of cells with 7.5×10^5 and lower CFU of S. pneumoniae had no detectable effect on either the morphology of epithelial cells or on the IAV replication. However, the effect of pneumococci on IAV replication may be different during a coinfection in vivo in the human upper respiratory tract.

Pretreatment of Epithelial Cells with Live Streptococcus pneumoniae Has No Detectable Effect on Influenza A Virus Replication In Vitro

Influenza A virus (IAV) and Streptococcus pneumoniae (pneumococcus) are two major upper respiratory tract pathogens responsible for exacerbated disease in coinfected individuals. Despite several studies showing increased susceptibility to secondary bacterial infections following IAV infection, information on the direct effect of S. pneumoniae on IAV in vitro is unknown. This is an important area of investigation as S. pneumoniae is a common commensal of the human upper respiratory tract, present as an important coinfecting pathogen with IAV infection. A recent study showed that S. pneumoniae enhances human metapneumovirus infection in polarized bronchial epithelial cells in vitro. The aim of the current study was to determine whether treatment of epithelial cells with S. pneumoniae affects IAV replication using a standard immunofluorescence assay (IFA). For this study we used four IAV permissive epithelial cell lines including two human-derived cell lines, 12 pneumococcal strains including recent human clinical isolates which represent different genetic backgrounds and serotypes, and six IAV strains of varying genetic nature and pathogenic potential including the pandemic 2009 H1N1 virus. Our results suggested that pretreatment of MDCK cells with 7.5×106 colony-forming units (CFUs) of live S. pneumoniae resulted in gradual cell-death in a time-dependent manner (0.5 to 4 hr). But, pretreatment of cell lines with 7.5×105 and lower CFUs of S. pneumoniae had no detectable effect on either the morphology of cells or on the IAV replication. However, unlike in epithelial cell lines, due to influence of secreted host factors the effect of pneumococci on IAV replication may be different during coinfections in vivo in the human upper respiratory tract, and in vitro with primary human polarized bronchial epithelial cells.

Comparative Proteomic Analysis of Wheat (Triticum aestivum L.) Hybrid Necrosis

Hybrid necrosis is the gradual premature death of leaves or plants in certain F1 hybrids of wheat (Triticum aestivum L.). Comparison of protein expression in necrotic and normal wheat leaves showed that the abundance of 33 proteins was changed significantly, and 24 of these proteins were identified. These proteins were involved in plant growth and development, antioxidation, photosynthesis and carbon assimilation, amino acid and protein biosynthesis, cytological signal transduction, DNA and RNA modification, protein transport, folding and assembly according to their functions. The down-regulation of uroporphyrinogen decarboxylase and the up-regulation of lipoxygenases in necrotic leaves may be related to the oxidative stress in the necrotic cells. The heat shock proteins may play the cytoprotective role. The differential expression of photosynthesis and carbon assimilation related proteins indicated chlorophyll biosynthesis and chloroplast development were inhibited and might finally cause the gradual chlorosis and cell death in necrotic leaves. The results of this study give a comprehensive picture of the post-transcriptional response to necrosis in hybrid wheat leaves and serve as a platform for further characterization of gene function and regulation in wheat hybrid necrosis.

Size-dependent in vitro cytotoxicity assay of gold nanoparticles

Gold nanoparticles (AuNps) may serve as a promising model to address the size-dependent biological response of cell lines. Their size can be controlled with great precision during chemical synthesis. AuNps have potential applications in drug delivery, cancer diagnosis, and therapy, in the food industry, and for environmental remediation. However, some of the recent literature contains conflicting data regarding the cytotoxicity of gold nanoparticles. Against this background, a systematic study of water soluble gold nanoparticles stabilized by citrate ranging in size from 3 nm to 45 nm were synthesized. The cytotoxicity of these particles were tested by employing the (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction assay), the neutral red cellular uptake assay, and lactate dehydrogenase release assay. Noticeable differences in the cytotoxic effects depending on the assay, and the nanoparticle size have been found. Citrate stabilized gold nanoparticles with sizes of 3 nm, 8 nm, and 30 nm were more sensitive to the cell lines and caused gradual cell death within 24 h at higher concentrations. This results in IC50 values ranging from 57 to 78 μgmL−1 depending on the particle size, and cell line combinations. In contrast, AuNps with diameters of 5 nm, 6 nm, 10 nm, 17 nm, and 45 nm were nontoxic up to three to four fold higher concentrations, and at long-term exposure.

Microencapsulation of small intestinal neuroendocrine neoplasm cells for tumor model studies

Basic cancer research is dependent on reliable in vitro and in vivo tumor models. The serotonin (5-HT) producing small intestinal neuroendocrine tumor cell line KRJ-1 has been used in in vitro proliferation and secretion studies, but its use in in vivo models has been hampered by problems related to the xeno-barrier and tumor formation. This may be overcome by the encapsulation of tumor cells into alginate microspheres, which can function as bioreactors and protect against the host immune system. We used alginate encapsulation of KRJ-1 cells to achieve long-term functionality, growth and survival. Different conditions, including capsule size, variations in M/G content, gelling ions (Ca(2+) /Ba(2+)) and microcapsule core properties, and variations in KRJ-1 cell condition (single cells/spheroids) were tested. Viability and cell growth was evaluated with MTT, and confocal laser scanner microscopy combined with LIVE/DEAD viability stains. 5-HT secretion was measured to determine functionality. Under all conditions, single cell encapsulation proved unfavorable due to gradual cell death, while encapsulation of aggregates/spheroids resulted in surviving, functional bioreactors. The most ideal spheroids for encapsulation were 200-350 μm. Long-term survival (>30 days) was seen with solid Ca(2+) /Ba(2+) microbeads and hollow microcapsules. Basal 5-HT secretion was increased (sixfold) after hollow microcapsule encapsulation, while Ca(2+) /Ba(2+) microbeads was associated with normal basal secretion and responsiveness to cAMP/PKA activation. In conclusion, encapsulation of KRJ-1 cells into hollow microcapsules produces a bioreactor with a high constitutively activate basal 5-HT secretion, while Ca(2+) /Ba(2+) microbeads provide a more stable bioreactor similar to non-encapsulated cells. Alginate microspheres technology can thus be used to tailor different functional bioreactors for both in vitro and in vivo studies.

Cardiomyocyte death: mechanisms and translational implications

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Although treatments have improved, development of novel therapies for patients with CVD remains a major research goal. Apoptosis, necrosis, and autophagy occur in cardiac myocytes, and both gradual and acute cell death are hallmarks of cardiac pathology, including heart failure, myocardial infarction, and ischemia/reperfusion. Pharmacological and genetic inhibition of autophagy, apoptosis, or necrosis diminishes infarct size and improves cardiac function in these disorders. Here, we review recent progress in the fields of autophagy, apoptosis, and necrosis. In addition, we highlight the involvement of these mechanisms in cardiac pathology and discuss potential translational implications.

Evidence of protandry in Aa Rchb.f. (Orchidaceae, Cranichideae)

Protandry has been described in orchids since Darwin (1862) as a strategy to promote cross-pollination. Three different forms of protandry have been reported in tribe Cranichideae: downward movement of the labellum in Spiranthes species, upward movement of the column in Sauroglossum elatum Lindl. and Manniella spp., and downward movement of the column in Prescottia stachyodes (Sw.) Lindl. In the present work, through longitudinal and transverse sections of flowers of different developmental stages, we reported column movement of Aa erosa (Rchb.f.) Schltr. and the mechanism responsible for the movement. Our result shows that in an early flower stage (male phase), the column of Aa erosa is straight. The gradual cell death of the dorsal side of the column and size increase of the cells of the ventral side cause the column to bend downward to almost 90o representing the female stage. Some authors have suggested self-pollination in Aa. But flies exploring inflorescences of Aa species have been observed in the field by the author. This observation plus the evidence of protandry in Aa erosa could discard self- pollination as the only strategy of pollination in this genus.

High-dose γ-irradiation enhances the expression of a transgene controlled by the immediate-early CMV promoter in stably transfected tumor cells

γ-irradiation is commonly used to inactivate whole-cell anticancer vaccines containing viable tumor cells. To evaluate the effect of γ-irradiation on transgene expression in tumor cells, human and mouse cell lines were stably transfected with constructs expressing the granulocyte macrophage colony-stimulating factor (GM-CSF) or green fluorescent protein (GFP) gene under the control of the immediate-early CMV promoter. Irradiation of cells at 20–100 Gy caused a loss of proliferation capacity and gradual cell death, with the survival depending on the irradiation dose. G2/M cells accumulated in irradiated cultures, while the portion of S-phase cells was reduced. Surviving cells displayed activation of β-galactosidase and morphological changes associated with cell senescence. Mitochondrial dehydrogenase activity did not change with the irradiation dose. Irradiated cells retained transgene expression. Moreover, the amount of secreted GM-CSF and GFP production significantly increased after γ-irradiation, up to tenfold in cells exposed to 100 Gy. Transgene expression increased gradually and positively correlated with the total irradiation dose. The results demonstrate that γ-irradiation at 100 Gy is optimal for whole-cell anticancer vaccine inactivation.

Antioxidants reduce cone cell death in a model of retinitis pigmentosa

Retinitis pigmentosa (RP) is a label for a group of diseases caused by a large number of mutations that result in rod photoreceptor cell death followed by gradual death of cones. The mechanism of cone cell death is uncertain. Rods are a major source of oxygen utilization in the retina and, after rods die, the level of oxygen in the outer retina is increased. In this study, we used the rd1 mouse model of RP to test the hypothesis that cones die from oxidative damage. A mixture of antioxidants was selected to try to maximize protection against oxidative damage achievable by exogenous supplements; alpha-tocopherol (200 mg/kg), ascorbic acid (250 mg/kg), Mn(III)tetrakis (4-benzoic acid) porphyrin (10 mg/kg), and alpha-lipoic acid (100 mg/kg). Mice were treated with daily injections of the mixture or each component alone between postnatal day (P)18 and P35. Between P18 and P35, there was an increase in two biomarkers of oxidative damage, carbonyl adducts measured by ELISA and immunohistochemical staining for acrolein, in the retinas of rd1 mice. The staining for acrolein in remaining cones at P35 was eliminated in antioxidant-treated rd1 mice, confirming that the treatment markedly reduced oxidative damage in cones; this was accompanied by a 2-fold increase in cone cell density and a 50% increase in medium-wavelength cone opsin mRNA. Antioxidants also caused some preservation of cone function based upon photopic electroretinograms. These data support the hypothesis that gradual cone cell death after rod cell death in RP is due to oxidative damage, and that antioxidant therapy may provide benefit.

2b Protein is essential to induce a novel gradual cell death in Zucchini yellow mosaic virus-inoculated cucumber cotyledon co-infected with Cucumber mosaic virus

Cucumber cotyledons inoculated with Cucumber mosaic virus (CMV, Pepo strain) or Zucchini yellow mosaic virus (ZYMV, Z5-1 isolate) developed either mild chlorotic spots or no symptoms. Cotyledons treated with CMV plus ZYMV also developed mild chlorotic spots. However, plants ZYMV-inoculated cotyledons had veinal yellowing and gradual cell death by 20 days postinoculation (dpi) when co-inoculated with CMV on the other cotyledon. When analyzing this synergism, an enzyme-linked immunosorbent assay showed that CMV gradually increased in CMV-inoculated cotyledons of plants, with the other cotyledon mock- or ZYMV-inoculated. However, CMV significantly increased at 9 to 14 dpi in the ZYMV-inoculated cotyledons of plants co-infected with CMV. ZYMV similarly increased in cotyledon pairs of both co-infected and singly infected plants. Inoculation with PepoΔ2b, a modified Pepo-CMV that lacks translation of the 2b protein, revealed that PepoΔ2b without the 2b protein systemically infected cucumber but induced no symptoms on cotyledons or true leaves. Plants with a ZYMV-inoculated cotyledon and co-infected with PepoΔ2b did not undergo cell death; nevertheless, PepoΔ2b was at high levels comparable to levels of CMV in the ZYMV-inoculated cotyledon. The 2b protein thus seems essential for induction of the novel gradual cell death in ZYMV-inoculated cotyledons of cucumbers co-infected with CMV.