Crystal structure of chloroplastic ascorbate peroxidase from tobacco plants and structural insights into its instability

Ascorbate peroxidase (APX) is a heme-containing protein that plays a central role in scavenging H(2)O(2) in higher plants. The structure of stromal APX (sAPX) was determined at 1.6 A to an R-factor of 19.1% and an R-free-factor of 22.3%. The electrostatic potential of the gamma-channel that connects the molecular surface of sAPX to the gamma-edge of heme was more positive than that of cytosolic APX (cAPX) from pea, so sAPX might bind more easily with ascorbate than cAPX. The overall structure of sAPX was similar to those of cAPX from pea and cytochrome c peroxidase (CCP) from yeast, with a substantial difference in a loop structure located in the vicinity of the heme. The side chain of Arg169 in sAPX corresponding to His169 in cAPX and His181 in CCP extended in the opposite direction from the heme, forming two hydrogen bonds with carbonyl groups in the loop structure. The rapid inactivation of sAPX might be due to the characteristic conformation of Arg169 owing to the loop structure of sAPX.     

Prefabricated vascularized bone flap: a tissue transformation technique for bone reconstruction

In this study, an attempt was made to transform a muscle vascularized pedicle raised on host vessels into a vascularized bone flap, using recombinant human bone morphogenetic protein 2 (rhBMP-2). The purpose of this study was to produce new bone vascularized in nature to increase the survival rate of the subsequently grafted bone and to fabricate the newly formed bone into the desired shape. Silicone molds in the shape of a rat mandible were used to deliver rat bone matrix impregnated with or without rhBMP-2. A muscle pedicle the same size as the mold was raised on the saphenous vessels in the rat thigh and then sandwiched in the center of the silicone molds. The molds were sliced in half and each section was filled with rat bone matrix that was impregnated either with 25 microg of rhBMP-2 for the experimental group or with diluting material alone for the control group. The sandwiched flaps were then secured by tying them to the adjacent muscles and were harvested at 2 and 4 weeks after surgery. Three and six rats were used in the control and experimental groups at each time point, respectively. Bone formation was assessed in the ex vivo specimens by macroscopic, radiologic, and histologic evaluation. Macroscopically, the continuation of the vascular pedicle was clearly visible for both the control and experimental muscle flaps. However, no evidence of muscle-tissue transformation was observed in the control flaps, whereas all the flaps treated with rhBMP-2 produced new bone that replicated the shape of the mold exactly and had saphenous vessels supplying the newly formed bone. This study demonstrates that this experimental model has the potential to be therapeutically applied for effective bone reconstruction.     

Effects of ionic valency of interacting metal elements in ion uptake by carrot (Daucas carota cv. U.S. harumakigosun)

Interaction of elements in the course of element uptake by carrot (Daucas carota cv. U.S. harumakigosun) exerted by the addition of elements, such as Rb, Zn, and Al, was investigated. For the purpose of precise evaluation of uptake behavior, the simultaneous determination of absorption of Na, Be, Sr, Mn, Co, Zn, Ce, Pm, and Gd was conducted by the multitracer technique. For root uptakes, Al exhibited its influence on the uptake of essential elements and on the uptake of toxic or unbeneficial ones, presumably as a result of the large electric valency that caused cell membrane disintegrity. On the other hand, Zn as a divalent cation only affected the uptake of essential and beneficial elements. Rubidium, which is a monovalent cation, did not exhibit any effect on the uptake of other ions. Concerning shoot uptakes, inhibition by Zn and Al, but not by Rb, was observed for the uptake of Sr, Mn, Co, and Zn. From the present investigation, it is suggested that there exists an interaction between added ions and the elements taken into plants and that the degree of interaction increases in the increasing order of ionic valency: M+ (Rb), M2+ (Zn), and M3+ (Al).     

The N-terminal region of Sgs1, which interacts with Top3, is required for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 disruptants

The SGS1 gene of Saccharomyces (cerevisiae is a homologue of the genes affected in Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome. Disruption of the SGS1 gene is associated with high sensitivity to methyl methanesulfonate (MMS) and hydroxyurea (HU), and with hyper-recombination phenotypes, including interchromosomal recombination between heteroalleles. SGS1 encodes a protein which has a helicase domain similar to that of Escherichia coli RecQ. A comparison of amino acid sequences among helicases of the RecQ family reveals that Sgs1,WRN, and BLM share a conserved region adjacent to the C-terminal part of the helicase domain (C-terminal conserved region). In addition, Sgs1 contains two highly charged acidic regions in its N-terminal region and the HRDC (helicase and RNaseD C-terminal) domain at its C-terminal end. These regions were also found in BLM and WRN, and in Rqh1 from Schizosaccharomyces pombe. In this study, we demonstrate that the C-terminal conserved region, as well as the helicase motifs, of Sgs1 are essential for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 mutants. In contrast, the highly charged acidic regions, the HRDC domain, and the C-terminal 252 amino acids were dispensable for the complementation of these phenotypes. Surprisingly, the N-terminal 45 amino acids of Sgs1 were absolutely required for the suppression of the above phenotypes. Introduction of missense mutations into the region encoding amino acids 4-13 abolished the ability of Sgsl to complement MMS sensitivity and suppress hyper-recombination in sgs1 mutants, and also prevented its interaction with Top3, indicating that interaction with Top3 via the N-terminal region of Sgs1 is involved in the complementation of MMS sensitivity and the suppression of hyper-recombination.     

Physiological role of soluble fumarate reductase in redox balancing during anaerobiosis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, there are two isoenzymes of fumarate reductase (FRDS1 and FRDS2), encoded by the FRDS and OSM1 genes, respectively. Simultaneous disruption of these two genes results in a growth defect of the yeast under anaerobic conditions, while disruption of the OSM1 gene causes slow growth. However, the metabolic role of these isoenzymes has been unclear until now. In the present study, we found that the anaerobic growth of the strain disrupted for both the FRDS and OSM1 genes was fully restored by adding the oxidized form of methylene blue or phenazine methosulfate, which non-enzymatically oxidize cellular NADH to NAD(+). When methylene blue was added at growth-limiting concentrations, growth was completely arrested after exhaustion of oxidized methylene blue. In the double-disrupted strain, the accumulation of succinate in the supernatant was markedly decreased during anaerobic growth in the presence of methylene blue. These results suggest that fumarate reductase isoenzymes are required for the reoxidation of intracellular NADH under anaerobic conditions, but not aerobic conditions.     

Homologous pairing and ring and filament structure formation activities of the human Xrcc2*Rad51D complex

The Xrcc2 and Rad51D/Rad51L3 proteins, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) in vertebrates. The Xrcc2 and Rad51D/Rad51L3 genes, whose products interact with each other, have essential roles in ensuring normal embryonic development. In the present study, we coexpressed the human Xrcc2 and Rad51D/Rad51L3 proteins (Xrcc2 and Rad51D, respectively) in Escherichia coli, and purified the Xrcc2*Rad51D complex to homogeneity. The Xrcc2 small middle dotRad51D complex catalyzed homologous pairing between single-stranded and double-stranded DNA, similar to the function of the Xrcc3*Rad51C complex, which is another complex of the Rad51 paralogs. An electron microscopic analysis showed that Xrcc2*Rad51D formed a multimeric ring structure in the absence of DNA. In the presence of ssDNA, Xrcc2*Rad51D formed a filamentous structure, which is commonly observed among the human homologous pairing proteins, Rad51, Rad52, and Xrcc3*Rad51C.     

Cloning and analysis of the telomere and terminal inverted repeat of the linear chromosome of Streptomyces griseus

Cloning and sequencing of the telomere of Streptomyces griseus revealed five palindromic sequences in the terminal 116 nucleotides, all of which can make a hairpin loop structure. However, the end sequence cannot form the foldback secondary structure that is common in Streptomyces telomeres and is suggested to be necessary for terminal replication. Both inside ends of the terminal inverted repeat (TIR) were also cloned and sequenced. The results confirmed the size of the TIR to be 24 kb and identified two almost identical open reading frames that might have been involved in the formation of the TIR.     

A case of acute gastric mucosal lesions associated with Helicobacter heilmannii infection

A 69-year-old-woman presented with acute epigastric pain, nausea, vomiting and heartburn. Endoscopy disclosed acute gastric mucosal lesions including mucosal edema, erosions, and ulcers with blood crusts in the antrum. Touch cytology and histological assessment obtained from the affected mucosa revealed acute neutrophilic gastritis and single longer and more coiled organisms than Helicobacter pylori, suggesting Helicobacter heilmannii. Electron micropragh confirmed the characteristic morphology. Despite a positive rapid urease test, H. pylori was not isolated by culture or detected by histology and Gram smears. Based on these findings, a diagnosis of acute gastric mucosal lesions associated with H. heilmannii infection was established. This was successfully treated with a 2-week triple therapy consisting of lansoprazole, clarithromycin and metronidazole with persistent endoscopic and histological remission. This is a rare case of H. heilmannii-associated acute gastric mucosal lesions, diagnosed by morphology using touch cytology and histology. The patient might benefit from antimicrobial treatment employing the regimen effective for H. pylori.     

Correlation between serum cholesterols and trace element uptake in liver,kidney, and blood of hypercholesterolemic mice

The radioactive multitracer technique was applied to the simultaneous determination of the uptake of 17 trace elements (Be, Na, Sc, V, Cr, Mn, Fe, Co, Zn, As, Se, Rb, Sr, Y, Zr, Nb, and Ru) in the liver, kidney, and blood of hypercholesterolemic model mice. The uptakes of Be, Sc, V, Cr, Fe, As, Rb, Y, Zr, Nb, and Ru in liver increased with an increasing feeding period of a cholesterol-rich diet, whereas the uptakes of Zn and Se decreased. Feeding of the diet resulted in a marked increase in serum total cholesterol, triglycerides, and low-density lipoprotein cholesterol. The metabolism of trace elements between cholesterolemic and normal mice was compared with respect to their serum cholesterol levels. A significant positive correlation was found between the concentration of serum triglycerides and liver uptakes of Cr, Fe, and As and a negative correlation for the uptake of Zn. A significant positive correlation was found between the concentrations of serum high- and low-density lipoprotein cholesterols and kidney uptakes of Cr and Rb. A negative correlation was found between the uptake of Be in the blood and the concentration of serum triglycerides. These results suggest that cholesterolemia have some specific effects on the metabolism of some elements.     

Characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.