Necrosis and necroptosis in germ cell depletion from mammalian ovary

The maximum number of germ cells is present during the fetal life in mammals. Follicular atresia results in rapid depletion of germ cells from the cohort of the ovary. At the time of puberty, only a few hundred (<1%) germ cells are either culminated into oocytes or further get eliminated during the reproductive life. Although apoptosis plays a major role, necrosis as well as necroptosis, might also be involved in germ cell elimination from the mammalian ovary. Both necrosis and necroptosis show similar morphological features and are characterized by an increase in cell volume, cell membrane permeabilization, and rupture that lead to cellular demise. Necroptosis is initiated by tumor necrosis factor and operated through receptor interacting protein kinase as well as mixed lineage kinase domain-like protein. The acetylcholinesterase, cytokines, starvation, and oxidative stress play important roles in necroptosis-mediated granulosa cell death. The granulosa cell necroptosis directly or indirectly induces susceptibility toward necroptotic or apoptotic cell death in oocytes. Indeed, prevention of necrosis and necroptosis pathways using their specific inhibitors could enhance growth/differentiation factor-9 expression, improve survivability as well as the meiotic competency of oocytes, and prevent decline of reproductive potential in several mammalian species and early onset of menopause in women. This study updates the information and focuses on the possible involvement of necrosis and necroptosis in germ cell depletion from the mammalian ovary.     

Proteasomal inhibition sensitizes cervical cancer cells to mitomycin C-induced bystander effect: the role of tumor microenvironment

Inaccessibility of drugs to poorly vascularized strata of tumor is one of the limiting factors in cancer therapy. With the advent of bystander effect (BE), it is possible to perpetuate the cellular damage from drug-exposed cells to the unexposed ones. However, the role of infiltrating tumor-associated macrophages (TAMs), an integral part of the tumor microenvironment, in further intensifying BE remains obscure. In the present study, we evaluated the effect of mitomycin C (MMC), a chemotherapeutic drug, to induce BE in cervical carcinoma. By using cervical cancer cells and differentiated macrophages, we demonstrate that MMC induces the expression of FasL via upregulation of PPARγ in both cell types (effector cells) in vitro, but it failed to induce bystander killing in cervical cancer cells. This effect was primarily owing to the proteasomal degradation of death receptors in the cervical cancer cells. Pre-treatment of cervical cancer cells with MG132, a proteasomal inhibitor, facilitates MMC-mediated bystander killing in co-culture and condition medium transfer experiments. In NOD/SCID mice bearing xenografted HeLa tumors administered with the combination of MMC and MG132, tumor progression was significantly reduced in comparison with those treated with either agent alone. FasL expression was increased in TAMs, and the enhanced level of Fas was observed in these tumor sections, thereby causing increased apoptosis. These findings suggest that restoration of death receptor-mediated apoptotic pathway in tumor cells with concomitant activation of TAMs could effectively restrict tumor growth.Owing to the heterogeneous nature and scanty vascularization, the access of anticancer regimen to all strata of the tumor is one of the major challenges in cancer therapy. Current response rate to chemotherapy is far from desirable and warrants formulating the strategies to enhance specificity and efficacy of the anticancer regimens. Of late, the phenomenon of bystander effect (BE), which refers to transmission of death signals from the drug-exposed cells to the unexposed cells, is being explored to improve the therapeutic response. Although BE has been well documented in radiotherapy^{1, 2} and experimental approaches of gene therapy,^{3, 4} very limited information is available with respect to conventional chemotherapeutic drugs. We have previously demonstrated the chemotherapy-induced bystander killing in breast cancer cells⁵ and hepatocellular carcinoma cells.⁶ Recently, other groups also have demonstrated the occurrence of chemotherapy-induced BE in breast cancer⁷ and lung cancer,^{8, 9} which is in agreement with our studies. BE has been shown to be dependent on cell type and class of drugs,⁶ and the role of tumor microenvironment in response to chemotherapeutic drug-induced BE is poorly understood.Cervical cancer is one of the most common solid tumors. Mitomycin C (MMC), a DNA alkylating agent, has been widely used in this malignancy as a constituent of combination therapy.¹⁰ From the pharmacological point of view, MMC is effective at relatively low dose with minimal organ-associated toxicity¹¹ and it has been shown to activate innate immunity.¹² However, therapeutic efficacy of MMC principally depends on other drug types in combination therapy.¹³ Therefore, a well-designed strategy that could enhance the efficacy of MMC is desirable. MMC has been demonstrated to induce BE in hepatocellular carcinoma, but not in cervical cancer cells.⁶ Although the precise mechanisms of bystander killing remain elusive, we have previously reported the involvement of death ligands,^{5, 6} which was later supported by other studies.^{7, 8, 9} The ability of cancer cells to escape programmed cell death has a critical role in the survival of cancer cells and tumor progression. Despite the presence of cellular apoptotic factors, cancer cells reprogram their molecular events and signaling to evade apoptosis.¹⁴ Moreover, it has been reported that exposure to proteasomal inhibitor inhibits the growth of various cancer cells and sensitize them to death ligand-mediated death by stabilizing death receptors.^{15, 16, 17} Considering these notions, we speculated that non-functionality of death receptors could be one of the possible factors associated with defective BE in cervical cancer. We, therefore, hypothesized that treating cervical cancer cells with combination of MMC and proteasomal inhibitor could elicit BE, and thereby may significantly improve the therapeutic outcome.Till date, studies explicate cancer cells exposed to chemotherapy as the effector cells in inducing bystander-mediated killing. However, owing to the heterogeneous nature of cellular population in tumor, other cellular components are also likely to have a key role in inducing BE. Tumor microenvironment consists of a heterogeneous mass of malignant as well as nonmalignant cells. The nonmalignant cells include endothelial, fibroblast and immune cells that establish multitude of interactions among themselves and also with malignant cells.⁹ Macrophages are the most abundant immune cells present in tumors, also termed as tumor-associated macrophages (TAMs).¹⁸ TAMs are differentiated monocytes that infiltrate the tumor microenvironment, and are exposed to chemotherapeutic regimen. Studies have demonstrated that TAMs could account for approximately more than 60% of tumor mass in some cancers.^{19, 20, 21} TAMs exposed to radiations² and chemotherapy²² have been shown to have a significant role in inducing BE. Studies support the notion that targeting TAMs could improve the therapeutic index of various drugs.^{10, 23} Increased sensitivity to cyclophosphamide¹⁴ and cisplatin²⁴ has been shown in co-culture system involving cancer cells and macrophages. Under chemotherapy, increased recruitment of macrophages with enhanced expression of tumoricidal factors like perforin and granzyme,²² death ligands¹⁰ or ROS ²⁵ has been reported in tumors. Therefore, we speculated that BE could further be amplified by infiltrating macrophages resulting in enhanced therapeutic efficacy of anticancer regimens. In the present study, we evaluated combination effect of MMC and MG132 in enhancing bystander killing of cancer cells in vitro and in vivo, in part, through the involvement of cancer cells and TAMs. Herein, we demonstrate that stabilization of Fas on cervical cancer cells facilitates dramatic reduction in tumor progression as a consequence of increase in apoptosis. This study could be helpful in designing novel therapeutic strategies in treating cancer by involving proteasomal inhibitors in combination with chemotherapeutic drugs that specifically activate death receptor-mediated killing.     

A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces

We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.     

在韩国境内Potentilla fragarioides var.sprengeliana的遗传多样 …

根据２２个等位酶位点遗传变异,探讨了韩国境内委陵菜（Ｐｏｔｅｎｔｉｌｌａ ｆｒａｇａｒｉｏｉｄｅｓ Ｌ．ｖａｒ．ｓｐｒｅｎｇｅｌｉａｎａ）的遗传多样性和种群结构。酶位点的多态位点百分比为５９．１％。种和种群水平上的遗传多样性比较高,分别为Ｈｅｓ＝０．２１０,Ｈｅｐ＝０．１９９;而种群的分化水平则相对较低（ＧＳＴ＝０．０７４）。１９个种群中随机交配的偏差为ＦＩＳ＝０．３３１。每代迁移数的间接估计     

Endoplasmic reticulum stress-mediated inhibition of NSMase2 elevates plasma membrane cholesterol and attenuates NO production in endothelial cells

Chronic exposure of blood vessels to cardiovascular risk factors such as free fatty acids, LDL-cholesterol, homocysteine and hyperglycemia can give rise to endothelial dysfunction, partially due to decreased synthesis and bioavailability of nitric oxide (NO). Many of these same risk factors have been shown to induce endoplasmic reticulum (ER) stress in endothelial cells. The objective of this study was to examine the mechanisms responsible for endothelial dysfunction mediated by ER stress. ER stress elevated both intracellular and plasma membrane (PM) cholesterols in BAEC by ~ 3-fold, indicated by epifluorescence and cholesterol oxidase methods. Increases in cholesterol levels inversely correlated with neutral sphingomyelinase 2 (NSMase2) activity, endothelial nitric oxide synthase (eNOS) phospho-activation and NO-production. To confirm that ER stress-induced effects on PM cholesterol were a direct consequence of decreased NSMase2 activity, enzyme expression was either enhanced or knocked down in BAEC. NSMase2 over-expression did not significantly affect cholesterol levels or NO-production, but increased eNOS phosphorylation by ~ 1.7-fold. Molecular knock down of NSMase2 decreased eNOS phosphorylation and NO-production by 50% and 40%, respectively while increasing PM cholesterol by 1.7-fold and intracellular cholesterol by 2.7-fold. Furthermore, over-expression of NSMase2 in ER-stressed BAEC lowered cholesterol levels to within control levels as well as nearly doubled the NO production, restoring it to ~ 74% and 68% of controls using tunicamycin and palmitate, respectively. This study establishes NSMase2 as a pivotal enzyme in the onset of endothelial ER stress-mediated vascular dysfunction as its inactivation leads to the attenuation of NO production and the elevation of cellular cholesterol.     

Toxicity of catechol to alternaria spp

In slide-germination tests concentration of 50 p.p.m. or more of catechol were inhibitory to spore germination ofAlternaria tomato, A. solani, A. melongenae, A. longipes, A. brassicicola. A. brassicae, A. raphani, A. triticina and anAlternaria sp. isolated from onion. More than 70 % inhibition of conidial germination was caused by 100 p.p.m. of the compound. Nearly complete or complete inhibition occurred in 200 and 500 p.p.m. Elongation of germ tubes was similarly affected. A 3-minute dip in 500 p.p.m. catechol solution did not affect the germinability of seeds of radish, cabbage, cauliflower and knol kohl. Similar treatment of radish seeds reduced the seed-borne inoculum ofA. raphani from 42 % to 1 %. In one preliminary experiment, radish seedlings raised from treated seeds, appeared to be resistant to artificial inoculation withA. raphani.Journal Series Paper no. 100 from U.P. Agricultural University Experiment Station.     

Nox4-dependent ROS modulation by amino endoperoxides to induce apoptosis in cancer cells

Tumor metastasis is the main cause of death in cancer patients. Anoikis resistance is one critical malefactor of metastatic cancer cells to resist current clinical chemotherapeutic treatments. Although endoperoxide-containing compounds have long been suggested as anticancer drugs, few have been clinically employed due to their instability, complex synthesis procedure or low tumor cell selectivity. Herein, we describe a one-pot strategy to synthesize novel amino endoperoxides and their derivatives with good yields and stabilities. In vitro cell-based assays revealed that 4 out of the 14 amino endoperoxides selectively induce metastatic breast carcinoma cells but not normal breast cells to undergo apoptosis, in a dose-dependent manner. Mechanistic studies showed that the most potent amino endoperoxide, 4-Me, is selective for cancer cells expressing a high level of Nox4. The anticancer effects are further shown to be associated with reduced O₂⁻:H₂O₂ ratio and increased ·OH level in the cancerous cells. Animal study showed that 4-Me impairs orthotopic breast tumor growth as well as tumor cell metastasis to lymph nodes. Altogether, our study suggests that anticancer strategies that focus on redox-based apoptosis induction in tumors are clinically viable.     

Evolutionary divergence times in the Annonaceae: evidence of a late Miocene origin of Pseuduvaria in Sundaland with subsequent diversification in New Guinea

Background  

Phylogenetic analyses of the Annonaceae consistently identify four clades: a basal clade consisting of Anaxagorea, and a small 'ambavioid' clade that is sister to two main clades, the 'long branch clade' (LBC) and 'short branch clade' (SBC). Divergence times in the family have previously been estimated using non-parametric rate smoothing (NPRS) and penalized likelihood (PL). Here we use an uncorrelated lognormal (UCLD) relaxed molecular clock in BEAST to estimate diversification times of the main clades within the family with a focus on the Asian genus Pseuduvaria within the SBC. Two fossil calibration points are applied, including the first use of the recently discovered Annonaceae fossil Futabanthus. The taxonomy and morphology of Pseuduvaria have been well documented, although no previous dating or biogeographical studies have been undertaken. Ancestral areas at internal nodes within Pseuduvaria are determined using dispersal-vicariance analysis (DIVA) and weighted ancestral area analysis (WAAA).