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.     

Hydrogen production by termite gut protists: characterization of iron hydrogenases of Parabasalian symbionts of the termite Coptotermes formosanus

Cellulolytic flagellated protists in the guts of termites produce molecular hydrogen (H(2)) that is emitted by the termites; however, little is known about the physiology and biochemistry of H(2) production from cellulose in the gut symbiotic protists due to their formidable unculturability. In order to understand the molecular basis for H(2) production, we here identified two genes encoding proteins homologous to iron-only hydrogenases (Fe hydrogenases) in Pseudotrichonympha grassii, a large cellulolytic symbiont in the phylum Parabasalia, in the gut of the termite Coptotermes formosanus. The two Fe hydrogenases were phylogenetically distinct and had different N-terminal accessory domains. The long-form protein represented a phylogenetic lineage unique among eukaryotic Fe hydrogenases, whereas the short form was monophyletic with those of other parabasalids. Active recombinant enzyme forms of these two Fe hydrogenases were successfully obtained without the specific auxiliary maturases. Although they differed in their extent of specific activity and optimal pH, both enzymes preferentially catalyzed H(2) evolution rather than H(2) uptake. H(2) evolution, at least that associated with the short-form enzyme, was still active even under high hydrogen partial pressure. H(2) evolution activity was detected in the hydrogenosomal fraction of P. grassii cells; however, the vigorous H(2) uptake activity of the endosymbiotic bacteria compensated for the strong H(2) evolution activity of the host protists. The results suggest that termite gut symbionts are a rich reservoir of novel Fe hydrogenases whose properties are adapted to the gut environment and that the potential of H(2) production in termite guts has been largely underestimated.     

Comparison Study of Allelochemicals and Bispyribac-Sodium on the Germination and Growth Response of Echinochloa crus-galli L.

The phytotoxic effects of two allelochemicals (trans-cinnamic acid and syringaldehyde) at different concentrations (1000, 100, 10, and 1 µM) on seed germination, seedling growth, and physiological and biochemical changes of Echinochloa crus-galli L. were tested by comparison to a commercial herbicide ‘Nominee’ (that is, 100 g/L bispyribac-sodium). trans-Cinnamic acid and the herbicide inhibited seed germination completely at 100 µM, whereas for syringaldehyde, complete inhibition required 1000 µM. However, with 100 µM syringaldehyde, the seed germination of the test species was 53% of the control. Allelochemicals and the herbicide delayed seed germination and significantly affected the speed of germination index (S), speed of cumulative germination index (AS), and coefficient of germination rate (CRG). The roots were more affected when nutrients were not added to the growth bioassay. In general, with the increasing concentration of allelochemicals from 100 to 1000 µM, the inhibitory effects increased. Via microscopy analysis, we found leaf blade wilting and necrosis at concentrations above 100 µM in allelochemical-treated plants. Roots of E. crus-galli treated with 1000 µM allelochemicals had black points on root nodes but had no root hairs. The anatomy of roots treated with allelochemicals (1000 µM) showed contraction or reduction of root pith cells as well as fewer and larger vacuoles compared to the control. The allelochemicals also showed remarkable effects on seedling growth, SPAD index, chlorophyll content, and free proline content in a pot culture bioassay, indicating that trans-cinnamic acid and syringaldehyde are potent inhibitors of E. crus-galli growth and can be developed as herbicides for future weed management strategies.

     

Co-culture of fibroblasts and hepatic parenchymal cells induces metabolic changes and formation of a three-dimensional structure

Co-culturing of tendon fibroblasts and liver parenchymal cells in Williams' medium E supplemented with fetal bovine serum, hormones, and L-ascorbic acid 2-phosphate, a long acting vitamin C derivative, resulted in formation of three-dimensional structure. Both growth of fibroblasts and their production of collagen were inhibited, however production of albumin by the hepatocytes was much better preserved than when individual cells were cultured separately, indicating epithelial-mesenchymal interactions stimulate reorganization of the liver-like tissue from isolated cells.     

Downregulation of the voltage-dependent calcium channel (VDCC) beta-subunit mRNAs in pancreatic islets of type 2 diabetic rats

The present study was undertaken to determine whether altered expression of the VDCC beta-subunits in pancreatic beta-cells could play a role in the changes in beta-cell sensitivity to glucose that occur with diabetes. Application of competitive RT-PCR procedure revealed that in normal Wistar rats, LETO and prediabetic OLETF rats, the beta(2)-subunit mRNA levels were 60-200-fold greater than the levels for the beta(3)-subunit. These findings suggest that the beta(2)-subunit as well as the beta-cell type VDCC1 alpha(1)-subunit may be the predominant form of the VDCC expressed in pancreatic beta-cells. The levels of mRNA encoding the beta-subunits and the beta-cell type alpha(1)-subunit as well as insulin were significantly reduced in diabetic rats. Perfusion experiments revealed that diabetic rats showed the higher basal insulin secretion and profoundly impaired insulin secretory responses to glucose compared with non-diabetic rats. Alternatively, impaired insulin secretory responses to glucose in high dose glucose-infused rats were recovered partly with the elevation of mRNA levels of the VDCC beta(2)- and beta(3)-subunits as well as the alpha(1)-subunit by the treatment with diazoxide. Thus, considering the possibility that the most striking effect of the VDCC alpha(1) beta-subunit coexpression in pancreatic beta-cells might occur on activation kinetics like the skeletal muscle, the impairment of further activation of the VDCCs to acute glucose challenge caused by the reduced expressions of the alpha(1) beta-subunits mRNAs in type 2 diabetic animals might be at least partly associated with the alterations in beta-cell sensitivity to glucose.     

Theoretical studies of the ATP hydrolysis mechanism of myosin

The ATP hydrolysis mechanism of myosin was studied using quantum chemical (QM) and molecular dynamics calculations. The initial model compound for QM calculations was constructed on the basis of the energy-minimized structure of the myosin(S1dc)-ATP complex, which was determined by molecular mechanics calculations. The result of QM calculations suggested that the ATP hydrolysis mechanism of myosin consists of a single elementary reaction in which a water molecule nucleophilically attacked gamma-phosphorus of ATP. In addition, we performed molecular dynamics simulations of the initial and final states of the ATP hydrolysis reaction, that is, the myosin-ATP and myosin-ADP.Pi complexes. These calculations revealed roles of several amino acid residues (Lys185, Thr186, Ser237, Arg238, and Glu459) in the ATPase pocket. Lys185 maintains the conformation of beta- and gamma-phosphate groups of ATP by forming the hydrogen bonds. Thr186 and Ser237 are coordinated to a Mg(2+) ion, which interacts with the phosphates of ATP and therefore contributes to the stabilization of the ATP structure. Arg238 and Glu459, which consisted of the gate of the ATPase pocket, retain the water molecule acting on the hydrolysis at the appropriate position for initiating the hydrolysis.     

Epigenetic mark sequence of the H19 gene in human sperm

We have investigated the epigenetic mark in the human H19 gene. The H19 promoter is methylation-free in human sperm, but it is methylated in the paternally derived allele of most adult tissues. Consequently, the H19 gene is exclusively transcribed from the maternal allele. It was demonstrated that the differentially methylated region (DMR) located 2 kb upstream from mouse H19 is essential for the imprinting of H19. A 39 bp sequence in DMR has a high degree of similarity between humans, mice and rats. The highly conserved 15 bp core region of the consensus sequence contains four methylatable sites, and thus has been proposed as a potential imprinting mark region. In this study, fine epigenetic sequencing analysis was performed on the sperm DNA in comparison with other adult organs. Interestingly, the conserved sequence of the potential mark region was methylated in almost all the sperm genomes analyzed. Furthermore, the single dinucleotide CpG, whose methylation affects the accessibility of the element to CTCF, was methylated in the conserved core in the human sperm. These results suggest that the human core sequences may act as an imprinting center in the reciprocal monoallelic expression of H19.     

Reversibility of n-3 fatty acid deficiency-induced alterations of learning behavior in the rat: level of n-6 fatty acids as another critical factor

Rats fed a semipurified diet supplemented with 3% (w/w) safflower oil [Saf, n-3 fatty acid deficient, high linoleic acid (18:2n-6)] through two generations exhibit decreased correct response ratios in a brightness-discrimination learning test compared with rats fed 3% perilla oil [Per, high alpha-linolenic acid (18:3n-3)]. This is associated with a decreased DHA (22:6n-3)-to-arachidonic acid (20:4n-6) ratio in brain lipids. In the first set of experiments, dietary oil was shifted from Saf to a mixture of 2.4% safflower oil plus 0.6% DHA after weaning (Saf-DHA), but all parameters measured in the learning test were essentially unchanged. Brain 22:6n-3 content of the Saf-DHA group reached that of the Per group but the levels of 20:4n-6 and docosatetraenoic acid (22:4n-6) did not decrease to those of the Per group at the start of the test. In the second set of experiments, dietary oil was shifted to a mixture of 0.6% safflower oil plus 1.2% oleic acid (OA) plus 1.2% DHA (Saf-OA-DHA group) with 18:2n-6 content comparable to that of the Per group. The Saf-OA-DHA group exhibited a learning performance similar to that of the Per group; brain 22:6n-3, 20:4n-6, and 22:4n-6 contents were also comparable to those of the Per group. These results indicate that the altered learning behavior associated with a long-term n-3 fatty acid deficiency is reversed by supplementing 22:6n-3 after weaning, when the levels of competing n-6 fatty acids in the diet and brain lipids are limited.     

Isolation and characterization of cDNAs encoding mitochondrial phosphate transporters in soybean,maize, rice,and Arabidopsis

cDNA clones encoding mitochondrial phosphate transporters were isolated from four herbaceous plants. The cDNAs for the soybean, maize and rice transporters contained entire coding regions, whereas the Arabidopsis cDNA lacked the 5 portion. The hydropathy profiles of the deduced amino acid sequences predicted the existence of six membrane-spanning domains which are highly conserved in the mitochondrial transporter family. In soybeans, the mRNA level for the transporter was high in tissues containing dividing cells. It was suggested that there are multiple copies of transporter genes in both dicots and monocots. The soybean transporter was expressed as inclusion bodies in Escherichia coli, solubilized with detergents, and then reconstituted into liposomes. The resulting proteoliposomes exhibited high phosphate transport activity. The activity was inhibited by N-ethylmaleimide, like those of mammalian phosphate transporters.