Apoptosis-like Programmed Cell Death Research Articles

Fungal Apoptosis-Related Proteins

Programmed cell death (PCD) plays a crucial role in the development and homeostasis maintenance of multicellular organisms. Apoptosis is a form of PCD that prevents pathological development by eliminating damaged or useless cells. Despite the complexity of fungal apoptosis mechanisms being similar to those of plants and metazoans, fungal apoptosis lacks the core regulatory elements of animal apoptosis. Apoptosis-like PCD in fungi can be triggered by a variety of internal and external factors, participating in biological processes such as growth, development, and stress response. Although the core regulatory elements are not fully understood, apoptosis-inducing factor and metacaspase have been found to be involved. This article summarizes various proteins closely related to fungal apoptosis, such as apoptosis-inducing factor, metacaspase, and inhibitors of apoptosis proteins, as well as their structures and functions. This research provides new strategies and ideas for the development of natural drugs targeting fungal apoptosis and the control of fungal diseases.

Iris setosa Pall. ex Link Extract Reveals Amoebicidal Activity against Acanthamoeba castellanii and Acanthamoeba polyphaga with Low Toxicity to Human Corneal Cells.

Acanthamoeba keratitis (AK) is a sight-threatening and difficult-to-treat ocular infection. The significant side effects of current AK treatments highlight the urgent need to develop a safe and effective AK medication. In this study, the amoebicidal activity of Iris setosa Pall. ex Link extract (ISE) against Acanthamoeba was examined and its specific amoebicidal mechanism was explored. ISE induced significant morphological changes in Acanthamoeba trophozoites and exhibited amoebicidal activity against A. castellanii and A. polyphaga. ISE was further fractionated into five subfractions by sequential extraction with n-hexane, chloroform, ethyl acetate, n-butanol, and water, and their amoebicidal activities and underlying amoebicidal mechanisms were investigated. The n-butanol subfraction of ISE (ISE-BuOH) displayed selective amoebicidal activity against the Acanthamoeba species with minimal cytotoxicity in human corneal cells (HCE-2). ISE-BuOH triggered apoptosis-like programmed cell death (PCD) in amoebae, characterized by DNA fragmentation, increased ROS production, and caspase-3 activity elevation. ISE-BuOH also demonstrated a partial cysticidal effect against the amoeba species. ISE-BuOH could be a promising candidate in the development of therapeutic drugs for AK.

Botrytis cinerea type II inhibitor of apoptosis BcBIR1 enhances the biocontrol capacity of Coniothyrium minitans.

Apoptosis-like programmed cell death is associated with fungal development, ageing, pathogenicity and stress responses. Here, to explore the potential of Botrytis cinerea type II inhibitor of apoptosis (IAP) BcBIR1 in elevating the biocontrol efficacy of Coniothyrium minitans, the BcBIR1 gene was heterologously expressed in C. minitans. Results indicated that the strains expressing BcBIR1 had higher rates of conidiation, mycelial growth and biomass growth than the wild-type strain. Moreover, BcBIR1 was found to inhibit apoptosis, indicating its role as an IAP in C. minitans. Under various abiotic stresses, the growth rates of BcBIR1-expressing strains were significantly higher than that of the wild-type strain. Moreover, the conidial survival rate of the BcBIR1-expressing strains treated with ultraviolet irradiation was enhanced. In antifungal activity assay, the culture filtrates of BcBIR1-expressing strains displayed a stronger inhibitory effect on B. cinerea and Sclerotinia sclerotiorum than the wild-type strain. The study also found that BcBIR1 expression increased the mycoparasitism against the sclerotia, but not the hyphae of S. sclerotiorum. Taken together, these results suggest that BcBIR1 enhances vegetative growth, conidiation, anti-apoptosis activity, abiotic stress resistance, antifungal activity and mycoparasitism in C. minitans. As an IAP, BcBIR1 may improve the control capacity of C. minitans against S. sclerotiorum.

Identification of algicidal monoterpenoids from four chemotypes of Cinnamomum camphora and their algicidal mechanisms on Microcystis aeruginosa

Cyanobacterial blooms cause serious environmental issues, and plant secondary metabolites are considered as new algaecide for controlling them. Cinnamomum camphora produces a wide spectrum of terpenoids and has 4 main chemotypes, including linalool, camphor, eucalyptol and borneol chemotype. To develop the new cyanobacterial algaecide by using suitable chemotype of Cinnamomum camphora and the main terpenoids, we analyzed the terpenoid composition in the 4 chemotype extracts, evaluated the algicidal effects of the extracts and their typical monoterpenoids on Microcystis aeruginosa, and investigated the algicidal mechanism of the stronger algicidal agents. Among the 4 chemotypes, eucalyptol and borneol chemotype extracts exhibited stronger algicidal effects. In the 4 chemotype extracts, monoterpenoids were the main compounds, of which linalool, camphor, eucalyptol and borneol were the typical components. Among the 4 typical monoterpenoids, eucalyptol and borneol showed stronger algicidal effects, which killed 78.8% and 100% M. aeruginosa cells, respectively, at 1.2 mM after 48 h. In 1.2 mM eucalyptol and borneol treatments, the reactive oxygen species levels markedly increased, and the caspase-3-like activity also raised. With prolonging the treatment time, M. aeruginosa cells gradually shrank and wrinkled, and the cell TUNEL fluorescence intensity and DNA degradation gradually enhanced, indicating that the lethal mechanism is causing apoptosis-like programmed cell death (PCD). Therefore, eucalyptol and borneol chemotype extracts and their typical monoterpenoids have the potential for developing as algaecides to control cyanobacteria through triggering apoptosis-like PCD.

Vibrio parahaemolyticus thermostable direct haemolysin induces non-classical programmed cell death despite caspase activation.

Thermostable direct haemolysin (TDH) is the key virulence factor secreted by the human gastroenteric bacterial pathogen Vibrio parahaemolyticus. TDH is a membrane-damaging pore-forming toxin. It evokes potent cytotoxicity, the mechanism of which still remains under-explored. Here, we have elucidated the mechanistic details of cell death response elicited by TDH. Employing Caco-2 intestinal epithelial cells and THP-1 monocytic cells, we show that TDH induces some of the hallmark features of apoptosis-like programmed cell death. TDH triggers caspase-3 and 7 activations in the THP-1 cells, while caspase-7 activation is observed in the Caco-2 cells. Interestingly, TDH appears to induce caspase-independent cell death. Higher XIAP level and lower Smac/Diablo level upon TDH intoxication provide plausible explanation for the functional inability of caspases in the THP-1 cells, in particular. Further exploration reveals that mitochondria play a central role in the TDH-induced cell death. TDH triggers mitochondrial damage, resulting in the release of AIF and endonuclease G, responsible for the execution of caspase-independent cell death. Among the other critical mediators of cell death, ROS is found to play an important role in the THP-1 cells, while PARP-1 appears to play a critical role in the Caco-2 cells. Altogether, our work provides critical new insights into the mechanism of cell death induction by TDH, showing a common central theme of non-classical programmed cell death. Our study also unravels the interplay of crucial molecules in the underlying signalling processes. Our findings add valuable insights into the role of TDH in the context of the host-pathogen interaction processes.

Programmed cell death in Xanthomonas axonopodis pv. glycines is associated with modulation of gene expression resulting in altered states of motility, biofilm and virulence

One of the foremost report of apoptosis-like programmed cell death (PCD) came from Xanthomonas axonopodis pv. glycines (Xag), which displayed rapid post-exponential cell death in PCD inducing media (PIM) but not in a non-inducing media (PNIM). The current study aims to decipher for the first time, the advantages of the existence of PCD in this phytopathogenic microorganism. Analysis of RNA-seq under inducing and non-inducing conditions, revealed differential expression of a number of genes related to key physiology of Xag, such as, motility, xanthan biosynthesis and export as well as virulence. A PCD negative mutant Xag M42 displayed diminished virulence and a contrasting transcriptome pattern. In vitro experiments revealed that under PCD inducing condition, Xag produced negligible xanthan gum as well as extracellular amylase, displayed enhanced swarming motility, released copious e-DNA and formed scanty biofilm. Lack of ‘diffusible signalling factor’ production was eliminated as possible reason for PCD-induction. Altogether, it appears that, in planta existence of the pathogen metabolically resembles PNIM, and on being transferred to PIM, the cells experience oxidative stress and circumvents it by adopting PCD as an altruistic response. Survival of the remaining population is encouraged by upregulating motility, detachment from the fragile biofilm to achieve dispersal.

Phragmites australis (Cav.) Trin. ex Steud. Extract Induces Apoptosis-like Programmed Cell Death in Acanthamoeba castellanii Trophozoites.

Acanthamoeba keratitis (AK) is an infectious ocular disease which is difficult to diagnose correctly and cure. Development of an effective and safe therapeutic drug for AK is needed. Our preliminary screening of more than 200 extracts from wild plants collected in Korea suggested the potential amoebicidal activity of Phragmites australis (Cav.) Trin. ex Steud. extract (PAE) against Acanthamoeba species. Here, we aimed to analyze the amoebicidal activity of PAE on Acanthamoeba and its underlying amoebicidal mechanism. PAE induced amoebicidal activity against both A. castellanii and A. polyphaga trophozoites, while it showed low cytotoxicity in human corneal epithelial cells (HCE-2) and human retinal pigment epithelial cells (ARPE-19). Transmission electron microscopy analysis showed subcellular morphological changes, such as increased granules, abnormal mitochondria, and atypical cyst wall formation, in the PAE-treated A. castellanii. Fluorometric apoptosis assay and TUNEL assay revealed apoptosis-like programmed cell death (PCD) in the PAE-treated A. castellanii. The PAE treatment increased reactive oxygen species production and reduced mitochondrial membrane potential in the amoeba. The enhanced expression of autophagy-associated genes was also detected. These results suggested that PAE exerted a promising amoebicidal effect on A. castellanii trophozoites via the PCD pathway. PAE could be a potential candidate for developing a therapeutic drug for AK.

Apoptosis is not conserved in plants as revealed by critical examination of a model for plant apoptosis-like cell death

BackgroundAnimals and plants diverged over one billion years ago and evolved unique mechanisms for many cellular processes, including cell death. One of the most well-studied cell death programmes in animals, apoptosis, involves gradual cell dismantling and engulfment of cellular fragments, apoptotic bodies, through phagocytosis. However, rigid cell walls prevent plant cell fragmentation and thus apoptosis is not applicable for executing cell death in plants. Furthermore, plants are devoid of the key components of apoptotic machinery, including phagocytosis as well as caspases and Bcl-2 family proteins. Nevertheless, the concept of plant “apoptosis-like programmed cell death” (AL-PCD) is widespread. This is largely due to superficial morphological resemblances between plant cell death and apoptosis, and in particular between protoplast shrinkage in plant cells killed by various stimuli and animal cell volume decrease preceding fragmentation into apoptotic bodies.ResultsHere, we provide a comprehensive spatio-temporal analysis of cytological and biochemical events occurring in plant cells subjected to heat shock at 40–55 °C and 85 °C, the experimental conditions typically used to trigger AL-PCD and necrotic cell death, respectively. We show that cell death under both conditions was not accompanied by membrane blebbing or formation of apoptotic bodies, as would be expected during apoptosis. Instead, we observed instant and irreversible permeabilization of the plasma membrane and ATP depletion. These processes did not depend on mitochondrial functionality or the presence of Ca2+ and could not be prevented by an inhibitor of ferroptosis. We further reveal that the lack of protoplast shrinkage at 85 °C, the only striking morphological difference between cell deaths induced by 40–55 °C or 85 °C heat shock, is a consequence of the fixative effect of the high temperature on intracellular contents.ConclusionsWe conclude that heat shock-induced cell death is an energy-independent process best matching definition of necrosis. Although the initial steps of this necrotic cell death could be genetically regulated, classifying it as apoptosis or AL-PCD is a terminological misnomer. Our work supports the viewpoint that apoptosis is not conserved across animal and plant kingdoms and demonstrates the importance of focusing on plant-specific aspects of cell death pathways.

Manipulation of Tropomysin 1 in iPSCs to Enhance in Vitro Blood Cell Production

Donor-derived blood transfusions are critical to our healthcare system, but do not fully meet the needs of patients with multiple alloantibodies, rare blood types, or HLA-sensitization. These needs have fueled the concept of ex vivo blood cell production, which might address issues related to demographic aging, infectious outbreaks transmitted by transfusions, and rare blood types. Blood cells produced in vitro could be used for transfusions, and could also be used as blood bank testing reagents (Coleman et al, Transfusion 2019). One major challenge in deploying this system is efficiently scaling up cell production. We used machine learning and genome-edited induced pluripotent stem cell (iPSC) models to determine that Tropomyosin 1 (TPM1) normally inhibits in vitro hematopoiesis. TPM1 knockout (TPM1KO) iPSCs produced 2-fold more hematopoietic progenitor cells (HPCs) than controls, thereby increasing production of mature blood cells that were functionally normal (Thom et al, BMC Biol 2020). During human hematopoiesis, HPCs arise from specialized vascular 'hemogenic endothelial' cells (HE) with distinct surface markers that can be used for identification and isolation. To define molecular mechanisms by which TPM1 regulates in vitro primitive hematopoiesis, we performed RNA sequencing analysis on sorted KDR+CD31+ endothelial cells and CD43+ HPCs from TPM1KO and control cultures. TPM1KO endothelial cells and HPCs had altered expression of genes and pathways known to regulate HE biology, including cell adhesion, integrin expression, and integrin-mediated signaling (p<0.05). 'Anoikis' is an apoptosis-like programmed cell death that occurs after extracellular matrix detachment. This process may limit nascent non-adherent HPC production in vitro, but has not been previously studied. TPM1KO cells showed increased expression of N-cadherin and RAP1-activating genes; increased N-cadherin and activated RAP1 limit anoikis in other biological contexts. In sum, these results suggested that TPM1KO cultures increased HE production and/or survival. To analyze HE production at the single cell level, we sorted KDR+CD31+CD43- endothelial cells and plated them in limiting dilution. We cultured sorted cells in hematopoietic cytokines for 7 days, and analyzed the number of wells in which CD43+ HPCs arose from sorted TPM1KO and control cells. Using limiting dilution analysis (Hu & Smyth,J. Immunol. Methods 2009), we found that TPM1KO cultures produced 2-fold more HE than controls. These results show that TPM1KO enhances in vitro hematopoiesis by increasing HE and subsequent HPC production, perhaps by limiting anoikis in nascent HPCs. TPM1-mediated regulation at the HE stage represents a novel mechanism that may be genetically or pharmacologically exploited to augment in vitro hematopoiesis. These findings will help boost in vitro HPC and blood cell production to clinically relevant scales, supporting efforts to produce blood cells for direct transfusion and/or to be used as clinical screening reagents. Disclosures No relevant conflicts of interest to declare.

5-Aminouracil and other inhibitors of DNA replication induce biphasic interphase\u2013mitotic cells in apical root meristems of Allium cepa

Key messageInduction of biphasic interphase–mitotic cells and PCC is connected with an increased level of metabolism in root meristem cells of Allium cepa.Previous experiments using primary roots of Allium cepa exposed to low concentrations of hydroxyurea have shown that long-term DNA replication stress (DRS) disrupts essential links of the S–M checkpoint mechanism, leading meristem cells either to premature chromosome condensation (PCC) or to a specific form of chromatin condensation, establishing biphasic organization of cell nuclei with both interphase and mitotic domains (IM cells). The present study supplements and extends these observations by describing general conditions under which both abnormal types of M-phase cells may occur. The analysis of root apical meristem (RAM) cell proliferation after prolonged mild DRS indicates that a broad spectrum of inhibitors is capable of generating PCC and IM organization of cell nuclei. These included: 5-aminouracil (5-AU, a thymine antagonist), characterized by the highest efficiency in creating cells with the IM phenotype, aphidicolin (APH), an inhibitor of DNA polymerase α, 5-fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase, methotrexate (MTX), a folic acid analog that inhibits purine and pyrimidine synthesis, and cytosine arabinoside (Ara-C), which inhibits DNA replication by forming cleavage complexes with topoisomerase I. As evidenced using fluorescence-based click chemistry assays, continuous treatment of onion RAM cells with 5-AU is associated with an accelerated dynamics of the DNA replication machinery and significantly enhanced levels of transcription and translation. Furthermore, DRS conditions bring about an intensified production of hydrogen peroxide (H2O2), depletion of reduced glutathione (GSH), and some increase in DNA fragmentation, associated with only a slight increase in apoptosis-like programmed cell death events.

The ROS-associated programmed cell death causes the decline of pollen viability recovered from cryopreservation in Paeonia lactiflora.

After cryopreservation, the occurrence of apoptosis-like programmed cell death events induced by the accumulation of ROS reduces pollen viability. Cryopreservation, as a biotechnological means for long-term preservation of pollen, has been applied to many species. However, after cryopreservation, the viability of pollen significantly decreases via a mechanism that is not completely clear. In this study, the pollen of Paeonia lactiflora 'Zi Feng Chao Yang', which exhibits significantly reduced viability after liquid nitrogen (LN2) storage, was used to study the relationship among pollen viability, programmed cell death (PCD) and reactive oxygen species (ROS). The apoptosis rate was increased significantly in pollen with decreased viability after cryopreservation, and the changes in ROS generation and hydrogen peroxide (H2O2) were consistent with the apoptosis rate. Correlation analysis results showed that the apoptosis rate is positively correlated with ROS generation and H2O2 content. In addition, ascorbic acid (AsA), glutathione (GSH) and ascorbic acid reductase (APX) levels were significantly correlated with ROS and H2O2. After LN2 preservation for 8months, the exogenous antioxidants AsA and GSH at appropriate concentrations significantly decreased H2O2 content, inhibited PCD indicator levels, and increased cryopreserved pollen viability. These observations suggest that PCD occurred in pollen during LN2 preservation for 1-8months and was induced by the accumulation of ROS in pollen after cryopreservation, thus explaining the main reasons for the reduction in pollen viability after cryopreservation in LN2.

Studying the roles of calcium and magnesium in cell death in the serpentine native plant Alyssum inflatum NYÁRÁDY through cell suspension culture technique

Calcium is an essential element for plants’ survival and ability to deal with environmental stresses. However, it can cause cell death due to cellular disequilibrium. Serpentine plants are sensitive to high concentrations of Ca2+, which induces lethal symptoms, especially under environmental stress. In this study, the direct effects of Ca2+ on cell death were investigated in cell cultures of Alyssum inflatum, a serpentine plant native to Western Iran, and results were compared to a non-serpentinitic congeneric species A. saxatile. The results were also compared to the effects of Mg2+ treatments in both species, as another determinative factor in serpentinite soil is high Mg2+ content. Plasma membrane permeability, reactive oxygen species (ROS), and malondialdehyde (MDA) production were measured as physiological cell injury indices. In A. inflatum higher levels of ROS and MDA were observed in Ca2+-treated cells (5 mM or more), while in A. saxatile they were measured in Mg2+-treated cells (5 mM or more). In serpentine species, results indicated that cell death by Ca2+ was more intensive than the cell death by Mg2+, which were observed with less intensity in non-serpentine plants. Microscopic studies showed that cell death occurred via apoptosis-like programmed cell death (AL-PCD). Therefore, Ca2+ sensitivity and AL-PCD as mechanistic reasons for their non-serpentine intolerance would be a crucial consideration in cellular researches concerning serpentine plants, which could be employed in green technologies such as phytoremediation.

Laterals take it better \u2013 Emerging and young lateral roots survive lethal salinity longer than the primary root in Arabidopsis

Plant responses to salinity have been extensively studied over the last decades. Despite the vast accumulated knowledge, the ways Arabidopsis lateral roots (LR) cope with lethal salinity has not been fully resolved. Here we compared the primary root (PR) and the LR responses during events leading to lethal salinity (NaCl 200 mM) in Arabidopsis. We found that the PR and young LR responded differently to lethal salinity: While the PR died, emerging and young LR’s remained strikingly viable. Moreover, “age acquired salt tolerance” (AAST) was observed in the PR. During the 2 days after germination (DAG) the PR was highly sensitive, but at 8 DAG there was a significant increase in the PR cell survival. Nevertheless, the young LR exhibited an opposite pattern and completely lost its salinity tolerance, as it elongated beyond 400 µm. Examination of several cell death signatures investigated in the young LR showed no signs of an active programmed cell death (PCD) during lethal salinity. However, Autophagic PCD (A-PCD) but not apoptosis-like PCD (AL-PCD) was found to be activated in the PR during the high salinity conditions. We further found that salinity induced NADPH oxidase activated ROS, which were more highly distributed in the young LR compared to the PR, is required for the improved viability of the LR during lethal salinity conditions. Our data demonstrated a position-dependent resistance of Arabidopsis young LR to high salinity. This response can lead to identification of novel salt stress coping mechanisms needed by agriculture during the soil salinization challenge.

Suppression of water-bloom cyanobacterium Microcystis aeruginosa by algaecide hydrogen peroxide maximized through programmed cell death

The global expansion and intensification of toxic cyanobacterial blooms require effective algaecides. Algaecides should be selective, effective, fast-acting, and ideally suppress cyanotoxin production. In this study, whether both maximum growth suppression and minimal toxin production can be simultaneously achieved was tested with a selective algaecide H2O2, through its ability to induce apoptosis-like programmed cell death (AL PCD) in a common bloom species Microcystis aeruginosa. Under doses of 1−15 mg L−1, non-monotonic dose-response suppression of H2O2 on M. aeruginosa were observed, where maximal cell death and minimal microcystin production both occurred at a moderate dose of 10 mg L−1 H2O2. Maximal cell death was indeed achieved through AL PCD, as revealed by integrated biochemical, structural, physiological and transcriptional evidence; transcriptional profile suggested AL PCD was mediated by mazEF and lexA systems. Higher H2O2 doses directly led to necrosis in M. aeruginosa, while lower doses only caused recoverable stress. The integrated data showed the choice between the two modes of cell death is determined by the intracellular energy state under stress. A model was proposed for suppressing M. aeruginosa with AL PCD or necrosis. H2O2 was demonstrated to simultaneously maximize the suppression of both growth and microcystin production through triggering AL PCD.

Changes in apoptosis-like programmed cell death and viability during the cryopreservation of pollen from Paeonia suffruticosa

Pollen after cryopreservation has a variety of change trends in viability, with most pollens showing decreased viability. The role of apoptosis-like programmed cell death (AL-PCD) in changing viability is not exactly clear. AL-PCD is an important biological activity of cells that plays a key role in the normal development of plants, maintenance of homeostasis and resistance to interference from various environmental factors. However, a relationship between the diverse changes in pollen viability after liquid nitrogen (LN) preservation and AL-PCD has rarely been reported. The results of this study show that after cryopreservation, the viability of pollen from six cultivars of Paeonia suffruticosa presents different changes, and the occurrence of AL-PCD in pollen of these six cultivars varies. When the viability of the cultivars was significantly higher than that of fresh pollen or the viability showed no significant difference from that of fresh pollen, intracellular reactive oxygen species (ROS) generation was not significantly changed and the activity of the caspase-3-like enzyme was relatively low. When the viability of the cultivars after cryopreservation was significantly lower than that of fresh pollen, ROS generation significantly increased and the caspase-3-like enzymatic activity was higher than the control level. The pollen apoptosis rate increased for all three viability change types, and correlation analysis results demonstrated a significant negative correlation between the apoptosis rate and pollen viability (P < 0.05). However, the DNA fragmentation of AL-PCD late indicators was not characterized in the pollen of these six cultivars. Based on these findings, we concluded that AL-PCD occurs during pollen cryopreservation and is related to the differences in pollen viability after cryopreservation. After cryopreservation, six cultivars of P. suffruticosa pollen has a different change trends in viability, and the difference of pollen viability is related to apoptosis-like programmed cell death.

HSP70 improves the viability of cryopreserved Paeonia lactiflora pollen by regulating oxidative stress and apoptosis-like programmed cell death events

Heat shock proteins (HSPs) have many positive stabilizing effects on the proteins of stressed cells. This study investigated the effect of HSP70 on cryopreserved pollen. HSP70 was differentially expressed in Paeonia lactiflora and Magnolia denudate pollen before and after exposure to liquid nitrogen (LN). In this study, cryopreserved pollen samples of P. lactiflora ‘Fen Yu Nu’, ‘Zi Feng Chao Yang’ and ‘Hong Pan Tuo Jing’ were incubated with HSP70 at six concentrations, and the pollen germination, oxidative stress and apoptosis-like programmed cell death indicators were measured after LN storage. HSP70 significantly improved pollen viability after LN storage, and the concentration of HSP70 that was required varied from cultivar to cultivar, ranging from 0.5 to 10 μg/mL. Compared to the activity of the control treatment without HSP70, the activity of superoxide dismutase (SOD) increased, the contents of reactive oxygen species (ROS) and malondialdehyde (MDA) decreased, and the Ca2+ level, apoptosis rate and caspase-3-like activity decreased depending on the appropriate concentration of HSP70 added. Pollen germination was negatively correlated with MDA content, Ca2+ level, and caspase-3-like activity and positively correlated with SOD activity. In conclusion, HSP70 improved the viability of pollen after cryopreservation by improving the activity of antioxidant enzymes, reducing the contents of ROS and MDA, and affecting the Ca2+ signal to inhibit apoptosis-like programmed cell death events. This report is the first time to describe the application of HSP70 for improving cryopreserved pollen viability by regulating oxidative and apoptosis-like programmed cell death events, and provide a novel insight into the mechanisms of HSP70 action in cryopreservation. After the pollen was rewarmed and then incubated with appropriate concentrations of exogenous HSP70, it significantly increasing the post-freezing germination of the pollen. Which by affecting oxidative stress and apoptosis-like events, the ROS level, MDA content, intracellular Ca2+ concentration, apoptosis rate and caspase-3-like enzyme activity were reduced, and SOD activity was increased, thereby significantly increasing the post-freezing germination of the pollen.

(+)-Spectaline and Iso-6-Spectaline Induce a Possible Cross-Talk between Autophagy and Apoptosis in Trypanosoma brucei rhodesiense.

In our previous study, two known piperidine alkaloids (+)-spectaline (1) and iso-6-spectaline (2) were isolated from the leaves of Senna spectabilis and showed no toxic effect on L6 cells. In view of the potential use of piperidine alkaloids in S. spectabilis for the treatment of sleeping sickness, further investigation on the cell death actions of the parasite after treatment with compound 1 and 2 suggested that the treated parasites died by a process of autophagy based on the characteristic morphological alterations observed in intracellular T. b. rhodesiense. In search for apoptosis, interestingly, trypanosomes treated with high concentration of compound 1 and 2 after 72 h significantly induced an early apoptosis-like programmed cell death (PCD) such as phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and caspases activation. No DNA laddering discriminated late apoptosis event. Taken together, these findings demonstrated the potential of compound 1 and 2 as a natural chemotherapeutic capable of inducing a possible cross-talk between autophagy and apoptosis in T. b. rhodesiense.

Antifungal activity and mechanism of action of Ou-gon (Scutellaria root extract) components against pathogenic fungi

Ou-gon, an extract from Scutellaria baicalensis Georgi root, has been shown to exhibit pronounced antifungal activity. The present study aimed to identify antifungal components of Ou-gon and to determine their mechanism of action against pathogenic fungi. Antifungal activity was assessed by the microbroth dilution method using four common human pathogenic fungi, Trichophyton rubrum, Trichophyton mentagrophytes, Aspergillus fumigatus, and Candida albicans. Components of crude Ou-gon extract were separated by reversed-phase high-performance liquid chromatography. Active antifungal components were identified by liquid chromatography-electrospray ionization tandem mass spectrometry. Terminal deoxynucleotidyl transferase dUTP nick end-labelling assay, SYTOX® green uptake assay, determination of intracellular reactive oxygen species and mitochondrial membrane potential as well as microscopy (confocal laser microscopy, scanning and transmission electron microscopy) were used to probe the mode of action. Two components with potent antifungal activity, baicalein and wogonin, were identified in Ou-gon. Baicalein showed potent antifungal activity against the four fungi tested. Wogonin displayed antifungal activity against all four fungi except C. albicans. The components are considered to induce apoptosis-like programmed cell death via hyperproduction of reactive oxygen species. This study enhances our understanding of the antifungal activity of Kampo medicine, and may contribute to the development of new and safe antifungal therapeutics.

Disassembly of dying cells in diverse organisms.

Programmed cell death (PCD) is a conserved phenomenon in multicellular organisms required to maintain homeostasis. Among the regulated cell death pathways, apoptosis is a well-described form of PCD in mammalian cells. One of the characteristic features of apoptosis is the change in cellular morphology, often leading to the fragmentation of the cell into smaller membrane-bound vesicles through a process called apoptotic cell disassembly. Interestingly, some of these morphological changes and cell disassembly are also noted in cells of other organisms including plants, fungi and protists while undergoing 'apoptosis-like PCD'. This review will describe morphologic features leading to apoptotic cell disassembly, as well as its regulation and function in mammalian cells. The occurrence of cell disassembly during cell death in other organisms namely zebrafish, fly and worm, as well as in other eukaryotic cells will also be discussed.

Do Fungi Undergo Apoptosis-Like Programmed Cell Death?

This question of whether fungi undergo apoptosis-like programmed cell death can be separated into two questions. One question is about applying the term "apoptosis" to fungi, and the other is a more challenging question of whether fungi have evolved mechanisms that inflict self-injury. The answers to both questions depend on the definitions applied to "apoptosis" and "programmed cell death." Considering how these and other cell death terms originated and are currently defined for animals, some confusion arises when the terms are applied to fungi. While it is difficult to defend the concept of fungal apoptosis, the more interesting issue is whether fungi will eventually be found to encode programmed or extemporaneous self-destructive processes, as suggested by intriguing new findings.