alpha-Tomatine, the major saponin in tomato, induces programmed cell death mediated by reactive oxygen species in the fungal pathogen Fusarium oxysporum

The tomato saponin alpha-tomatine has been proposed to kill sensitive cells by binding to cell membranes followed by leakage of cell components. However, details of the modes of action of the compound on fungal cells are poorly understood. In the present study, mechanisms involved in alpha-tomatine-induced cell death of fungi were examined using a filamentous pathogenic fungus Fusarium oxysporum. alpha-Tomatine-induced cell death of F. oxysporum (TICDF) occurred only under aerobic conditions and was blocked by the mitochondrial F(0)F(1)-ATPase inhibitor oligomycin, the caspase inhibitor D-VAD-fmk, and protein synthesis inhibitor cycloheximide. Fungal cells exposed to alpha-tomatine showed TUNEL-positive nuclei, depolarization of transmembrane potential of mitochondria, and reactive oxygen species (ROS) accumulation. These results suggest that TICDF occurs through a programmed cell death process in which mitochondria play a pivotal role. Pharmacological studies using inhibitors suggest that alpha-tomatine activates phosphotyrosine kinase and monomeric G-protein signaling pathways leading to Ca(2+) elevation and ROS burst in F. oxysporum cells.     

Pathogenic mitochondrial DNA-induced respiration defects in hematopoietic cells result in anemia by suppressing erythroid differentiation

Anemia is a symptom in patients with Pearson syndrome caused by the accumulation of mutated mitochondrial DNA (mtDNA). Such mutated mtDNAs have been detected in patients with anemia. This suggested that respiration defects due to mutated mtDNA are responsible for the anemia. However, there has been no convincing experimental evidence to confirm the pathophysiological relation between respiration defects in hematopoietic cells and expression of anemia. We address this issue by transplanting bone marrow cells carrying pathogenic mtDNA with a large-scale deletion (ΔmtDNA) into normal mice. The bone marrow-transplanted mice carried high proportion of ΔmtDNA only in hematopoietic cells, and resultant the mice suffered from macrocytic anemia. They show abnormalities of erythroid differentiation and weak erythropoietic response to a stressful condition. These observations suggest that hematopoietic cell-specific respiration defects caused by mtDNAs with pathogenic mutations are responsible for anemia by inducing abnormalities in erythropoiesis.     

Sex‐specific aging in animals: Perspective and future directions

Sex differences in aging occur in many animal species, and they include sex differences in lifespan, in the onset and progression of age‐associated decline, and in physiological and molecular markers of aging. Sex differences in aging vary greatly across the animal kingdom. For example, there are species with longer‐lived females, species where males live longer, and species lacking sex differences in lifespan. The underlying causes of sex differences in aging remain mostly unknown. Currently, we do not understand the molecular drivers of sex differences in aging, or whether they are related to the accepted hallmarks or pillars of aging or linked to other well‐characterized processes. In particular, understanding the role of sex‐determination mechanisms and sex differences in aging is relatively understudied. Here, we take a comparative, interdisciplinary approach to explore various hypotheses about how sex differences in aging arise. We discuss genomic, morphological, and environmental differences between the sexes and how these relate to sex differences in aging. Finally, we present some suggestions for future research in this area and provide recommendations for promising experimental designs.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Study of color variation in the solitary ascidian Halocynthia roretzi, collected in the Inland Sea of Japan

In the vicinity of Yashiro Island in the Inland Sea of Japan, the solitary ascidian (tunicate) Halocynthia roretzi with tunics of various colors were collected. Samples of these animals were sorted into three groups on the basis of visual observation of tunic color. The red group includes animals with dark-red, light-red, or orange tunics. The pink group includes animals with tunic colors ranging between red and white. The white group includes only animals with completely white tunics. Animals in the white group lacked color internally, with the exception of the hepatopancreas and the gonads in breeding season; the epidermis and gill basket were white. In contrast, animals of both the red group and the pink group were colored internally, with red-orange epidermis and yellow gill basket. Alloreactivity was tested by mixed-hemocyte incubation between different animals belonging to the same color group and between animals belonging to different color groups. Alloreactivity between animals of the white group was 56.3%, between animals of the pink group was 60.0%, and between animals of the red group was 69.3%. The relatively high frequency of compatible combinations among the white animals is discussed.     

Changes in the thermal dissipation and the electron flow in the water-water cycle in rice grown under conditions of physiologically low temperature

Effects of low temperature on chlorophyll (Chl) fluorescence, gas exchange rate, the amounts of xanthophyll cycle pigments (Xp) and the activities of several antioxidant enzymes were examined in the 8th leaf of two rice (Oryza sativa L.) cultivars (japonica and indica types) and rbcS antisense rice. All plants were grown hydroponically at 25/20 degrees C (day/night), and then exposed to 20/17 degrees C (day/night) after full expansion of the 8th leaf, or exposed to either 20/17 degrees C or 15/13 degrees C (day/night) during the expansion of the 8th leaf. All plants exposed to low temperatures showed a decrease in CO(2) assimilation rate without photoinhibition, and increases in the fraction of thermal dissipation in PSII, and in the electron flux through the water-water cycle (WWC) were observed. Although the increase of thermal dissipation was associated with increases in the ratio of carotenoids to Chl, the ratio of Xp to carotenoids and the de-epoxidation state of Xp, the increase of the electron flux of WWC was not accompanied by an increase in the activities of antioxidant enzymes. Such photoprotective responses did not differ between during and after full expansion of the leaf, and did not differ among the three genotypes. Quantitative analyses on the dissipation of excess light energy showed that thermal dissipation makes a larger contribution than WWC. Thus, although low temperature led to a decrease in CO(2) assimilation, rice potentially coped with the excess light energy by increasing the thermal dissipation and the electron flux of WWC under low temperature irrespective of leaf development and genotypes.     

Identification of two novel mutations in adenine phosphoribosyltransferase gene in patients with 2,8-dihydroxyadenine urolithiasis

Five mutations in the adenine phosphoribosyltransferase (APRT) gene have been described in Japanese patients with APRT deficiency. We investigated the APRT gene from three patients with APRT deficiency and two novel mutations, G133D and V84M, were determined.     

Structural and functional analysis of the gpsA gene product of Archaeoglobus fulgidus: a glycerol-3-phosphate dehydrogenase with an unusual NADP+ preference

NAD(+)-dependent glycerol-3-phosphate dehydrogenase (G3PDH) is generally absent in archaea, because archaea, unlike eukaryotes and eubacteria, utilize glycerol-1-phosphate instead of glycerol-3-phosphate for the biosynthesis of membrane lipids. Surprisingly, the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus comprises a G3PDH ortholog, gpsA, most likely due to horizontal gene transfer from a eubacterial organism. Biochemical characterization proved G3PDH-like activity of the recombinant gpsA gene product. However, unlike other G3PDHs, the up to 85 degrees C thermostable A. fulgidus G3PDH exerted a 15-fold preference for NADPH over NADH. The A. fulgidus G3PDH bears the hallmarks of adaptation to halotolerance and thermophilicity, because its 1.7-A crystal structure showed a high surface density for negative charges and 10 additional intramolecular salt bridges compared to a mesophilic G3PDH structure. Whereas all amino acid residues required for dihydroxyacetone phosphate binding and reductive catalysis are highly conserved, the binding site for the adenine moiety of the NAD(P) cosubstrate shows a structural variation that reflects the observed NADPH preference, for example, by a putative salt bridge between R49 and the 2'-phosphate.     

Effect of irradiance on the partitioning of assimilated carbon during the early phase of grain filling in rice

Low irradiance in the early phase of grain filling in rice often results in a low grain yield, but its effects on the partitioning of previously or recently assimilated carbon within the plant or panicle have not been seriously examined. The objective of this study was to demonstrate the effect of shading during the different stages in the early phase of grain filling on the partitioning of previously or recently assimilated carbon among constituent organs and into superior and inferior spikelets of the panicle in rice (Oryza sativa L. 'Sasanishiki') plants using 13C as a tracer. Plants were grown either under low (shading) or moderate (non-shading) irradiance (120 and 800 micromol quantum m(-2) s(-1)) for 3 or 4 d before or after the 13CO2 feeding at heading, full-heading or milky stages during the early phase of grain filling. Four days after the 13CO2 feeding, the proportion of labelled (previously assimilated) carbon partitioned into the panicle was 17% higher in plants grown under low irradiance compared with plants grown under moderate irradiance at the full-heading stage (7-11 d after heading), while the proportion partitioned into the culm was 13% lower. The light treatments for 3 d were conducted before the 13CO2 feeding and partitioning of the labelled (recently-assimilated) carbon into spikelets was examined 6 h after feeding. The amount of labelled carbon partitioned into the spikelets of the secondary branch (inferior grains) in the plants grown under low irradiance was only 31% when compared with plants grown under moderate irradiance at the full-heading stage, although the partitioning of labelled carbon into the apical spikelets of the primary branch (superior grains) was not affected by the light treatments. These results clearly indicate that preferential partitioning of assimilated carbon into the panicle occurs under low irradiance at around 7-11 d after heading and that the priority of superior spikelets for assimilated carbon intensifies. This phenomenon is thought to be an important strategy for such rice cultivars as used in this study to achieve a certain proportion of ripened grains even under light limited conditions.     

Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have studied the biochemical diversity of several GH 12 homologs. The H. schweinitzii Cel12A enzyme differs from the T. reesei Cel12A enzyme by only 14 amino acids (93% sequence identity), but is much less thermally stable. The bacterial Cel12A enzyme from S. sp. 11AG8 shares only 28% sequence identity to the T. reesei enzyme, and is much more thermally stable. Each of the 14 sequence differences from H. schweinitzii Cel12A were introduced in T. reesei Cel12A to determine the effect of these amino acid substitutions on enzyme stability. Several of the T. reesei Cel12A variants were found to have increased stability, and the differences in apparent midpoint of thermal denaturation (T(m)) ranged from a 2.5 degrees C increase to a 4.0 degrees C decrease. The least stable recruitment from H. schweinitzii Cel12A was A35S. Consequently, the A35V substitution was recruited from the more stable S. sp. 11AG8 Cel12A and this T. reesei Cel12A variant was found to have a T(m) 7.7 degrees C higher than wild type. Thus, the buried residue at position 35 was shown to be of critical importance for thermal stability in this structural family. There was a ninefold range in the specific activities of the Cel12 homologs on o-NPC. The most and least stable T. reesei Cel12A variants, A35V and A35S, respectively, were fully active. Because of their thermal tolerance, S. sp. 11AG8 Cel12A and T. reesei Cel12A variant A35V showed a continual increase in activity over the temperature range of 25 degrees C to 60 degrees C, whereas the less stable enzymes T. reesei Cel12A wild type and the destabilized A35S variant, and H. schweinitzii Cel12A showed a decrease in activity at the highest temperatures. The crystal structures of the H. schweinitzii, S. sp. 11AG8, and T. reesei A35V Cel12A enzymes have been determined and compared with the wild-type T. reesei Cel12A enzyme. All of the structures have similar Calpha traces, but provide detailed insight into the nature of the stability differences. These results are an example of the power of homolog recruitment as a method for identifying residues important for stability.