Differential susceptibility to hydrogen sulfide-induced apoptosis between PHLDA1-overexpressing oral cancer cell lines and oral keratinocytes: Role of PHLDA1 as an apoptosis suppressor

Hydrogen sulfide (H₂S) is a novel gasotransmitter that plays multiple biological roles in various body systems. In addition to its endogenous production, H₂S is produced by bacteria colonizing digestive organs, including the oral cavity. H₂S was previously shown to enhance pro-apoptotic effects in cancer cell lines, although the mechanisms involved remain unclear. To properly assess the anti-cancer effects of H₂S, however, investigations of apoptotic effects in normal cells are also necessary. The aims of this study were (1) to compare the susceptibility to H₂S-induced apoptosis between the oral cancer cell line Ca9-22 and oral keratinocytes that were derived from healthy gingiva, and (2) to identify candidate genes involved in the induction of apoptosis by H₂S. The susceptibility to H₂S-induced apoptosis in Ca9-22 cells was significantly higher than that in keratinocytes. H₂S exposure in Ca9-22 cells, but not keratinocytes, enhanced the expression of pleckstrin homology-like domain, family A, member 1 (PHLDA1), which was identified through a differential display method. In addition, PHLDA1 expression increased during actinomycin D-induced apoptosis in Ca9-22 cells. Knockdown of PHLDA1 expression by small interfering RNA in Ca9-22 cells led to expression of active caspase 3, thus indicating apoptosis induction. The tongue cancer cell line SCC-25, which expresses PHLDA1 at a high level, showed similar effects. Our data indicate that H₂S is an anti-cancer compound that may contribute to the low incidence of oral cancer. Furthermore, we demonstrated the role of PHLDA1 as an apoptosis suppressor.     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Bioisostere of valtrate,anti-HIV principle by inhibition for nuclear export of Rev

Rational design by the MO calculation disclosed 5,6-dihydrovaltrate (2) as the bioisostere of valtrate (1), the Rev-export inhibitor with anti-HIV activity. The synthesis of 2 was accomplished by ingenious use of asymmetric Diels–Alder reaction and stereoselective epoxidation associated with the adjacent hydroxyl group. Because of similar biological potency to 1, the analog 2 should be recognized as a promising scaffold for new anti-HIV agents with an unprecedented mechanism of action, inhibition for nuclear export of Rev protein, in the conventional remedy.     

Adsorption of mutagens by refined corn bran

Refined corn bran (RCB), a dietary fiber derived from the mechanical refining of corn hulls, effectively adsorbed various environmental mutagens. When RCB was added at a concentration of 10 mg/ml to an aqueous solution of dinitropyrene (DNP), 91.6% of the mutagenicity towards Salmonella tester strain TA98 disappeared. Under similar conditions decreases in mutagenicity of DNP using wheat bran and cellulose powder were 58.4% and 43.0%, respectively. The adsorption of DNP to the fibers appeared irreversible since little mutagenicity was recovered by washing the treated fibers with aqueous buffer solutions of various pHs. Even with an organic solvent (methanol: ammonium hydroxide 50:1), only 2/3 of the mutagenicity of DNP was recovered. RCB could similarly adsorb mutagenic heterocyclic amines such as IQ, Trp-P-1, Trp-P-2, Glu-P-1, and Glu-P-2.     

Expression level of enzymes related to in situ estrogen synthesis and clinicopathological parameters in breast cancer patients

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17β-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor β, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.     

Direct Comparisons of 2D and 3D Dental Microwear Proxies in Extant Herbivorous and Carnivorous Mammals

The analysis of dental microwear is commonly used by paleontologists and anthropologists to clarify the diets of extinct species, including herbivorous and carnivorous mammals. Currently, there are numerous methods employed to quantify dental microwear, varying in the types of microscopes used, magnifications, and the characterization of wear in both two dimensions and three dimensions. Results from dental microwear studies utilizing different methods are not directly comparable and human quantification of wear features (e.g., pits and scratches) introduces interobserver error, with higher error being produced by less experienced individuals. Dental microwear texture analysis (DMTA), which analyzes microwear features in three dimensions, alleviates some of the problems surrounding two-dimensional microwear methods by reducing observer bias. Here, we assess the accuracy and comparability within and between 2D and 3D dental microwear analyses in herbivorous and carnivorous mammals at the same magnification. Specifically, we compare observer-generated 2D microwear data from photosimulations of the identical scanned areas of DMTA in extant African bovids and carnivorans using a scanning white light confocal microscope at 100x magnification. Using this magnification, dental microwear features quantified in 2D were able to separate grazing and frugivorous bovids using scratch frequency; however, DMTA variables were better able to discriminate between disparate dietary niches in both carnivorous and herbivorous mammals. Further, results demonstrate significant interobserver differences in 2D microwear data, with the microwear index remaining the least variable between experienced observers, consistent with prior research. Overall, our results highlight the importance of reducing observer error and analyzing dental microwear in three dimensions in order to consistently interpret diets accurately.     

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.     

Autotomy-induced life history plasticity in band-legged ground cricket Dianemobius nigrofasciatus

Crickets can autotomize their limbs when attacked by predators. This enables them to escape death, but imposes a short-term cost on their escape speed and a long-term cost on their future mating ability. Therefore, adaptive response compensated for the cost of autotomy might be advantageous for autotomized individuals. In the present study, we examined whether autotomy induced life history plasticities compensating for the future cost in the band-legged ground cricket Dianemobius nigrofasciatus . Life history traits of D. nigrofasciatus were compared between autotomized and intact individuals. The developmental time and head width of the individuals that were autotomized as fourth instar nymphs were significantly shorter and smaller, respectively, than those of intact individuals. However, the adult longevity, number of eggs laid and oviposition schedule did not vary between autotomized and intact individuals. In addition, there was no difference between individuals autotomized at the fourth instar and adult stages in these three traits. Early maturation in the autotomized individuals might be advantageous through reducing the risk of predation owing to the shorter period in nymphal stages. The cost of small body size in the autotomized females might not be so great because of no significant difference in fecundity between autotomized and intact individuals. However, the cost of small body size was unclear in the autotomized males because in general larger males were preferred by females. These results indicated autotomy-induced life history that might reduce the cost of autotomy.