The role of apoptosis-induced proliferation for regeneration and cancer

Genes dedicated to killing cells must have evolved because of their positive effects on organismal survival. Positive functions of apoptotic genes have been well established in a large number of biological contexts, including their role in eliminating damaged and potentially cancerous cells. More recently, evidence has suggested that proapoptotic proteins-mostly caspases-can induce proliferation of neighboring surviving cells to replace dying cells. This process, that we will refer to as "apoptosis-induced proliferation," may be critical for stem cell activity and tissue regeneration. Depending on the caspases involved, at least two distinct types of apoptosis-induced proliferation can be distinguished. One of these types have been studied using a model in which cells have initiated cell death, but are prevented from executing it because of effector caspase inhibition, thereby generating "undead" cells that emit persistent mitogen signaling and overgrowth. Such conditions are likely to contribute to certain forms of cancer. In this review, we summarize the current knowledge of apoptosis-induced proliferation and discuss its relevance for tissue regeneration and cancer.     

Regional TMPRSS2 V197M Allele Frequencies Are Correlated with COVID-19 Case Fatality Rates

Coronavirus disease, COVID-19 (coronavirus disease 2019), caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), has a higher case fatality rate in European countries than in others, especially East Asian ones. One potential explanation for this regional difference is the diversity of the viral infection efficiency. Here, we analyzed the allele frequencies of a nonsynonymous variant rs12329760 (V197M) in the TMPRSS2 gene, a key enzyme essential for viral infection and found a significant association between the COVID-19 case fatality rate and the V197M allele frequencies, using over 200,000 present-day and ancient genomic samples. East Asian countries have higher V197M allele frequencies than other regions, including European countries which correlates to their lower case fatality rates. Structural and energy calculation analysis of the V197M amino acid change showed that it destabilizes the TMPRSS2 protein, possibly negatively affecting its ACE2 and viral spike protein processing.     

Rhizosphere Competitiveness of Trichloroethylene-Degrading, Poplar-Colonizing Recombinant Bacteria

Indigenous bacteria from poplar tree (Populus canadensis var. eugenei ‘Imperial Carolina’) and southern California shrub rhizospheres, as well as two tree-colonizing Rhizobium strains (ATCC 10320 and ATCC 35645), were engineered to express constitutively and stably toluene o-monooxygenase (TOM) from Burkholderia cepacia G4 by integrating the tom locus into the chromosome. The poplar and Rhizobium recombinant bacteria degraded trichloroethylene at a rate of 0.8 to 2.1 nmol/min/mg of protein and were competitive against the unengineered hosts in wheat and barley rhizospheres for 1 month (colonization occurred at a level of 1.0 × 10⁵ to 23 × 10⁵ CFU/cm of root). In addition, six of these recombinants colonized poplar roots stably and competitively with populations as large as 79% ± 12% of all rhizosphere bacteria after 28 days (0.2 × 10⁵ to 31 × 10⁵ CFU/cm of root). Furthermore, five of the most competitive poplar recombinants (e.g., Pb3-1 and Pb5-1, which were identified as Pseudomonas sp. strain PsK recombinants) retained the ability to express TOM for 29 days as 100% ± 0% of the recombinants detected in the poplar rhizosphere expressed TOM constitutively.     

Role of the Rice Hexokinases OsHXK5 and OsHXK6 as Glucose Sensors

The Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) is recognized as an important glucose (Glc) sensor. However, the function of hexokinases as Glc sensors has not been clearly demonstrated in other plant species, including rice (Oryza sativa). To investigate the functions of rice hexokinase isoforms, we characterized OsHXK5 and OsHXK6, which are evolutionarily related to AtHXK1. Transient expression analyses using GFP fusion constructs revealed that OsHXK5 and OsHXK6 are associated with mitochondria. Interestingly, the OsHXK5ΔmTP-GFP and OsHXK6ΔmTP-GFP fusion proteins, which lack N-terminal mitochondrial targeting peptides, were present mainly in the nucleus with a small amount of the proteins seen in the cytosol. In addition, the OsHXK5NLS-GFP and OsHXK6NLS-GFP fusion proteins harboring nuclear localization signals were targeted predominantly in the nucleus, suggesting that these OsHXKs retain a dual-targeting ability to mitochondria and nuclei. In transient expression assays using promoter∷luciferase fusion constructs, these two OsHXKs and their catalytically inactive alleles dramatically enhanced the Glc-dependent repression of the maize (Zea mays) Rubisco small subunit (RbcS) and rice α-amylase genes in mesophyll protoplasts of maize and rice. Notably, the expression of OsHXK5, OsHXK6, or their mutant alleles complemented the Arabidopsis glucose insensitive2-1 mutant, thereby resulting in wild-type characteristics in seedling development, Glc-dependent gene expression, and plant growth. Furthermore, transgenic rice plants overexpressing OsHXK5 or OsHXK6 exhibited hypersensitive plant growth retardation and enhanced repression of the photosynthetic gene RbcS in response to Glc treatment. These results provide evidence that rice OsHXK5 and OsHXK6 can function as Glc sensors.In higher plants, sugars are known to function as signaling molecules in addition to being a fundamental source of fuel for carbon and energy metabolism. Indeed, sugars have been shown to regulate physiological processes during the entire plant life cycle, from germination to flowering and senescence, and to function during defense responses to biotic and abiotic stresses (Jang and Sheen, 1994; Jang et al., 1997; Perata et al., 1997; Smeekens and Rook, 1997; Smeekens, 1998; Wingler et al., 1998; Rolland et al., 2001, 2006; Leon and Sheen, 2003; Gibson, 2005; Biemelt and Sonnewald, 2006; Seo et al., 2007). Therefore, to sustain normal plant growth and development, rigorous sugar sensing and signaling systems are important for coordinating and modulating many essential metabolic pathways.Glc, one of the main products of photosynthesis, is the most widely recognized sugar molecule that regulates plant signaling pathways (Koch, 1996; Yu et al., 1996; Ho et al., 2001; Chen, 2007). Yeast (Saccharomyces cerevisiae) has several Glc sensors, including the hexokinase ScHXK2, Glc transporter-like proteins Sucrose nonfermenting 3 (Snf3) and Restores glucose transport 2 (Rgt2), and G protein-coupled receptor Gpr1. These sensors have been reported to sense the internal and external Glc status as part of mechanisms controlling cell growth and gene expression (Rolland et al., 2001; Lemaire et al., 2004; Santangelo, 2006). Similarly, recent studies in plants have unveiled sugar sensing and signaling systems mediated by hexokinase as a Glc sensor or G protein-coupled receptors in a hexokinase-independent way (Rolland et al., 2001, 2002, 2006; Chen et al., 2003; Moore et al., 2003; Holsbeeks et al., 2004; Cho et al., 2006b; Huang et al., 2006). In addition, plant Snf1-related protein kinase 1 (SnRK1), which is an ortholog of the yeast Snf1, plays important roles linking sugar signal, as well as stress and developmental signals, for the global regulation of plant metabolism, energy balance, growth, and survival (Baena-González et al., 2007; Lu et al., 2007; Baena-González and Sheen, 2008).In addition to the catalytic role of hexokinase in plants, which is to facilitate hexose phosphorylation to form hexose-6-P, the role of hexokinase as an evolutionarily conserved Glc sensor was first recognized from biochemical, genetic, and molecular studies of Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) transgenic plants and glucose insensitive2 (gin2) mutants (Jang et al., 1997; Rolland et al., 2002; Harrington and Bush, 2003; Moore et al., 2003; Cho et al., 2006b). Transgenic plants expressing catalytically inactive AtHXK1 mutant alleles in the gin2 mutant background have provided compelling evidence that the catalytic and sensory functions of AtHXK1 are uncoupled in the Arabidopsis plant (Moore et al., 2003). Furthermore, proteomics and yeast two-hybrid interaction experiments have revealed that in the nucleus, AtHXK1 interacts with two partners, the vacuolar H⁺-ATPase B1 and the 19S regulatory particle of proteasome subunit, to directly control the expression of specific photosynthetic genes (Cho et al., 2006b; Chen, 2007). In these studies, the interactions between AtHXK1 and vacuolar H⁺-ATPase B1 or 19S regulatory particle of proteasome subunit appeared not to require the enzymatic activity of AtHXK1. In the tomato (Solanum lycopersicum) plant, AtHXK1 expression causes a reduction in photosynthesis, growth inhibition, and the induction of rapid senescence (Dai et al., 1999), which are all characteristics of sugar sensing and signaling in photosynthetic tissues. With the exception of Arabidopsis HXK1, the role of hexokinases as Glc sensors has yet to be demonstrated in other plant species (Halford et al., 1999; Veramendi et al., 2002; Rolland et al., 2006).Hexokinases have been shown to associate with various subcellular compartments, including mitochondria, chloroplasts, Golgi complexes, endoplasmic reticula, plasma membranes, and cytosols, suggesting numerous distinct intracellular functions (Schleucher et al., 1998; Wiese et al., 1999; Frommer et al., 2003; Olsson et al., 2003; Giese et al., 2005; Cho et al., 2006a; Kandel-Kfir et al., 2006; Rezende et al., 2006; Damari-Weissler et al., 2007). In yeast, the Glc sensor ScHXK2 has a nuclear localization signal (NLS) within its N-terminal domain and resides partly in the nucleus in addition to the cytosol (Herrero et al., 1998; Randez-Gil et al., 1998). Furthermore, the nuclear localization of ScHXK2 is required for Glc repression of several genes, such as SUC2, HXK1, and GLK1 (Herrero et al., 1998; Rodríguez et al., 2001). A portion of cellular AtHXK1, which is predominantly associated with mitochondria, was also found to reside in the nucleus (Yanagisawa et al., 2003; Cho et al., 2006b). Under conditions of Glc excess, it has thus been hypothesized that nuclear AtHXK1 binds its substrate Glc, resulting in the suppression of target gene expression (Cho et al., 2006b; Chen, 2007).We have previously isolated 10 rice (Oryza sativa) hexokinases, OsHXK1 through OsHXK10, and demonstrated that all of these subtypes possess hexokinase activity (Cho et al., 2006a). The results of this previous study showed that OsHXK4 and OsHXK7 reside in the chloroplast stroma and cytosol, respectively. Based on sequence similarity and subcellular localization, we have identified two rice hexokinases homologous to AtHXK1, OsHXK5 and OsHXK6. The subcellular localization of OsHXK5 and OsHXK6, observed with GFP fusion constructs, suggested that OsHXK5 and OsHXK6 retain a dual-targeting ability to mitochondria and nuclei. This finding prompted us to examine whether these homologues play a role in Glc sensing and signaling in rice. To address this question, we observed the function of OsHXK5 and OsHXK6 in mesophyll protoplasts of maize (Zea mays) and rice and in transgenic rice plants. In addition, we transformed the Arabidopsis gin2-1 mutant with either wild-type or catalytically inactive alleles of OsHXK5 and OsHXK6 and analyzed their sugar sensing and signaling characteristics. Finally, the conserved role of hexokinase as a Glc sensor in Arabidopsis and rice plants is discussed.     

Aerobic degradation of mixtures of tetrachloroethylene, trichloroethylene, dichloroethylenes, and vinyl chloride by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1

A recombinant strain of Escherichia coli (JM109/pBZ1260) expressing constitutively toluene-o-xylene monooxygenase (ToMO) of Pseudomonas stutzeri OX1 degraded binary mixtures (100 microM each) of tetrachloroethylene (PCE) with either trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), cis-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), or vinyl chloride (VC). PCE degradation was 8-20% for these binary mixtures, while TCE and trans-DCE with PCE were degraded at 19%, 1,1-DCE at 37%, cis-DCE at 97%, and VC at 27%. The host P. stutzeri OXI was also found to degrade binary mixtures of PCE/TCE, PCE/cis-DCE, and PCE/VC when induced with toluene. Degradation of quaternary mixtures of PCE/TCE/trans-DCE/VC and PCE/TCE/cis-DCE/VC by JM109/pBZ1260 were also investigated as well as mixtures of PCE/TCE/trans-DCE/1,1-DCE/cis-DCE/VC; when all the chlorinated compounds were present, the best degradation occurred with 24-51% removal of each. For these degradation reactions, 39-85% of the stoichiometric chloride expected from complete degradation of the chlorinated ethenes was detected. The time course of PCE/TCE/1,1-DCE degradation was also measured for a mixture of 8, 17, and 6 microM, respectively; initial degradation rates were 0.015, 0.023. and 0.029 nmol/min x mg protein, respectively. This indicates that for the first time an aerobic enzyme can degrade mixtures of all chlorinated ethenes, including the once--so it was believed-completely recalcitrant PCE.     

The 18-kDa Translocator Protein Inhibits Vascular Cell Adhesion Molecule-1 Expression via Inhibition of Mitochondrial Reactive Oxygen Species

Translocator protein 18 kDa (TSPO) is a mitochondrial outer membrane protein and is abundantly expressed in a variety of organ and tissues. To date, the functional role of TSPO on vascular endothelial cell activation has yet to be fully elucidated. In the present study, the phorbol 12-myristate 13-acetate (PMA, 250 nM), an activator of protein kinase C (PKC), was used to induce vascular endothelial activation. Adenoviral TSPO overexpression (10–100 MOI) inhibited PMA-induced vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) expression in a dose dependent manner. PMA-induced VCAM-1 expressions were inhibited by Mito-TEMPO (0.1–0.5 μM), a specific mitochondrial antioxidants, and cyclosporin A (1–5 μM), a mitochondrial permeability transition pore inhibitor, implying on an important role of mitochondrial reactive oxygen species (ROS) on the endothelial activation. Moreover, adenoviral TSPO overexpression inhibited mitochondrial ROS production and manganese superoxide dismutase expression. On contrasts, gene silencing of TSPO with siRNA increased PMA-induced VCAM-1 expression and mitochondrial ROS production. Midazolam (1–50 μM), TSPO ligands, inhibited PMA-induced VCAM-1 and mitochondrial ROS production in endothelial cells. These results suggest that mitochondrial TSPO can inhibit PMA-induced endothelial inflammation via suppression of VCAM-1 and mitochondrial ROS production in endothelial cells.     

The parthenogenetic activation of canine oocytes with Ca-EDTA by various culture periods and concentrations

In the present study, canine oocytes were exposed to various concentrations of and durations of exposure to EDTA saturated with Ca(2+) (Ca-EDTA), a cell membrane-impermeable metal ion chelator, to determine if parthenogenetic activation could be induced. When oocytes were cultured for 48 or 72 h in parthenogenetic activation medium (PAM) without Ca-EDTA (control) or PAM supplemented with 1 or 5mM Ca-EDTA, the highest rate of pronuclear formation (PN) was obtained in oocytes cultured in 1mM Ca-EDTA for 48 h (8.0%; P<0.05). There was no pronuclear formation in the control group (PAM without Ca-EDTA). Oocytes treated with 5mM Ca-EDTA for 48 h or 1mM Ca-EDTA for 72 h formed a parthenogenetic pronucleus (3.1 and 4.5, respectively). However, there was no pronuclear formation in oocytes treated with 5mM Ca-EDTA for 72 h. In summary, exposure to Ca-EDTA can induce pronuclear formation in canine oocytes.     

Selenium acts as an insulin-like molecule for the down-regulation of diabetic symptoms via endoplasmic reticulum stress and insulin signalling proteins in diabetes-induced non-obese diabetic mice

To investigate whether selenium (Sel) treatment would impact on the onset of diabetes, we examined serum biochemical components including glucose and insulin, endoplasmic reticulum (ER) stress and insulin signalling proteins, hepatic C/EBP-homologous protein (CHOP) expression and DNA fragmentation in diabetic and non-diabetic conditions of non-obese diabetic (NOD) mice. We conclude that (i) Sel treatment induced insulin-like effects in lowering serum glucose level in Sel-treated NOD mice, (ii) Sel-treated mice had significantly decreased serum biochemical components associated with liver damage and lipid metabolism, (iii) Sel treatment led to the activation of the ER stress signal through the phosphorylation of JNK and eIF2 protein and insulin signal mechanisms through the phosphorylation of Akt and PI3 kinase, and (iv) Sel-treated mice were significantly relieved apoptosis of liver tissues indicated by DNA fragmentation assay in the diabetic NOD group. These results suggest that Sel compounds not only serve as insulin-like molecules for the downregulation of glucose level and the incidence of liver damage, but may also have the potential for the development of new drugs for the relief of diabetes by activating the ER stress and insulin signalling pathways.     

In vivo label‐free functional photoacoustic monitoring of ischemic reperfusion

Pressure ulcer formation is a common problem among patients confined to bed or restricted to wheelchairs. The ulcer forms when the affected skin and underlying tissues go through repeated cycles of ischemia and reperfusion, leading to inflammation. This theory is evident by intravital imaging studies performed in immune cell–specific, fluorescent reporter mouse skin with induced ischemia‐reperfusion (I‐R) injuries. However, traditional confocal or multiphoton microscopy cannot accurately monitor the progression of vascular reperfusion by contrast agents, which leaks into the interstitium under inflammatory conditions. Here, we develop a dual‐wavelength micro electro mechanical system (MEMS) scanning–based optical resolution photoacoustic microscopy (OR‐PAM) system for continuous label‐free functional imaging of vascular reperfusion in an IR mouse model. This MEMS‐OR‐PAM system provides fast scanning speed for concurrent dual‐wavelength imaging, which enables continuous monitoring of the reperfusion process. During reperfusion, the revascularization of blood vessels and the oxygen saturation (sO₂) changes in both arteries and veins are recorded, from which the local oxygen extraction ratios of the ischemic tissue and the unaffected tissue can be quantified. Our MEMS‐OR‐PAM system provides novel perspectives to understand the I‐R injuries. It solves the problem of dynamic label‐free functional monitoring of the vascular reperfusion at high spatial resolution.