The Planarian HOM/HOX Homeobox Genes (Plox) Expressed along the Anteroposterior Axis

In the freshwater planarian Dugesia japonica, five cDNAs for HOM/HOX homeobox genes were cloned and sequenced. Together with sequence data on HOM/HOX homeobox genes of platyhelminthes deposited in databases, comparison of the deduced amino acid sequences revealed that planarians have at least seven HOM/HOX homeobox genes, Plox1 to Plox7 ( anarian HOM/H homeobox genes). Whole-mount in situ hybridization and RT-PCR revealed that Plox4 and Plox5 were increasingly expressed along a spatial gradient in the posterior region of intact animals. During regeneration, Plox5 was expressed only in the posterior region of regenerating body pieces, suggesting that the gene is involved in the anteroposterior patterning in planarians. Plox5 was not found to be expressed in a blastema-specific manner, which contradicts a previous report (J. R. Bayascas, E. Castillo, A. M. Muños-Mármol, and E. Saló. Development 124, 141–148, 1997). X-ray irradiation experiments showed that Plox5 was expressed at least in some cells other than neoblasts, but that the induction of Plox5 expression during regeneration might require neoblasts.     

Elevation of KL-6, a lung epithelial cell marker,in plasma and epithelial lining fluid in acute respiratory distress syndrome

KL-6 is a pulmonary epithelial mucin more prominently expressed on the surface membrane of alveolar type II cells when these cells are proliferating, stimulated, and/or injured. We hypothesized that high levels of KL-6 in epithelial lining fluid and plasma would reflect the severity of lung injury in patients with acute lung injury (ALI). Epithelial lining fluid was obtained at onset (day 0) and day 1 of acute respiratory distress syndrome (ARDS)/ALI by bronchoscopic microsampling procedure in 35 patients. On day 0, KL-6 and albumin concentrations in epithelial lining fluid were significantly higher than in normal controls (P < 0.001), and the concentrations of KL-6 in epithelial lining fluid (P < 0.002) and in plasma (P < 0.0001) were higher in nonsurvivors than in survivors of ALI/ARDS. These observations were corroborated by the immunohistochemical localization of KL-6 protein expression in the lungs of nonsurvivors with ALI and KL-6 secretion from cultured human alveolar type II cells stimulated by proinflammatory cytokines. Because injury to distal lung epithelial cells, including alveolar type II cells, is important in the pathogenesis of ALI, the elevation of KL-6 concentrations in plasma and epithelial lining fluid could be valuable indicators for poor prognosis in clinical ALI.     

Larva migrans by Baylisascaris transfuga: fatal neurological diseases in Mongolian jirds, but not in mice

Raccoon roundworms (Baylisascaris procyonis) and other Baylisascaris species cause patent or latent larva migrans (LM) in a variety of mammals and birds, including humans. It is not clear whether LM by Baylisascaris transfuga, roundworms of bears, is associated with clinical neurological disorders. To clarify this issue, ICR and BALB/c mice as well as Mongolian jirds (Meriones unguiculatus) were orally inoculated with 2,000-5,000 embryonated eggs of B. transfuga. In mice, the ascarid caused symptomatic LM of limited extent and duration, whereas the infection was fatal in jirds; i.e., they exhibited general signs such as severe depression and emaciation on days 8-11 postinfection (PI) and died, or they developed progressive and fatal neurological disorders after day 14 PI. Histological examination showed B. transfuga larvae in the brain of all mice and jirds examined, and the larvae collected from them developed to a size comparable with that of B. procyonis. There existed, however, critical differences in host reactions against larvae localized in the brain of mice and jirds; B. transfuga larvae found in mice were surrounded by granulomatous reactions and immobilized, whereas larvae found in jirds were free from any host reaction and mobile, causing extensive malacia.     

Synthesis and inhibition mechanism of Delta lac-acetogenins, a novel type of inhibitor of bovine heart mitochondrial complex I

We have synthesized Deltalac-acetogenins that are new acetogenin mimics possessing two n-alkyl tails without an alpha,beta-unsaturated gamma-lactone ring and suggested that their inhibition mechanism may be different from that of common acetogenins [Hamada et al. (2004) Biochemistry 43, 3651-3658]. To elucidate the inhibition mechanism of Deltalac-acetogenins in more detail, we carried out wide structural modifications of original Deltalac-acetogenins and characterized the inhibitory action with bovine heart mitochondrial complex I. In contrast to common acetogenins, both the presence of adjacent bis-THF rings and the stereochemistry around the hydroxylated bis-THF rings are important structural factors required for potent inhibition. The inhibitory potency of a derivative possessing an n-butylphenyl ether structure (compound 7) appeared to be superior to that of the original Deltalac-acetogenins and equivalent to that of bullatacin, one of the most potent natural acetogenins. Double-inhibitor titration of steady-state complex I activity showed that the extent of inhibition of compound 7 and bullatacin is not additive, suggesting that the binding sites of the two inhibitors are not identical. Competition tests using a fluorescent ligand indicated that the binding site of compound 7 does not overlap with that of other complex I inhibitors. The effects of compound 7 on superoxide production from complex I are also different from those of other complex I inhibitors. Our results clearly demonstrate that Deltalac-acetogenins are a novel type of inhibitor acting at the terminal electron-transfer step of bovine complex I.     

Fibroblastic cells from human periapical granulation tissue preferentially form calcified matrices in decalcified boiled rat bone

We have been studying the potential of human fibroblastic cells (HFC) from periapical granulation tissue to form a calcified matrix. Recently, we reported that inflamed periapical granulation tissue contains osteogenic cells. In the present study, we tested the hypothesis that HFC, cultured with decalcified bone (DB) of rat, might form much greater calcified matrices than with rat decalcified boiled bone (DBB), which was originally prepared as a negative control. HFC were cultured with DB or DBB in the presence or absence of 2 mM -glycerophosphate (-GP) and 50 g/ml ascorbic acid. After six weeks of culture, a number of von Kossa-positive globular structures were unexpectedly observed inside DBB, but not DB. Without HFC, such structures were never seen in DBB incubated with 2 mM -GP and 50 g/ml ascorbic acid. DB cultured with HFC under the same conditions did not show these structures. Electron-microscopic observation revealed that matrix vesicles aggregated on collagen fibrils around globular structures in DBB. Energy dispersive X-ray microanalysis confirmed that these structures were calcified matrices composed of calcium and phosphate. These results suggest that human periapical granulation tissue contains cells responsible for the formation of calcified matrices in DBB, and that DBB could serve as an excellent scaffold for the calcification of HFC, rather than DB.This work was supported by grant-in-aid (project 15689024) for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.     

Ion-selective enrichment of tyrosine-sulfated peptides from complex protein digests

We have developed a novel procedure for concentrating sulfated peptides, as a front end to mass spectrometric analysis, based on ion-selective interaction of sulfate ions with anion exchangers. Ions with a higher charge and smaller solvated ion radius, such as sulfate ions, have higher retention in an ion exchanger due to their greater degree of coulombic interactions. We tested the effectiveness of this approach for enrichment and identification of sulfated peptides using a tryptic digest of bovine serum albumin spiked with model sulfated peptide (molar ratio 20:1) and using a tryptic digest of bovine fibrinogen. Sulfated peptides are identified by mass spectrometry in which both the molecular ion and its specific fragment ion produced by facile loss of SO(3) are detected. In both experiments, sulfated peptides were strongly retained on the anion exchanger and were eluted by higher concentrations of competing ion with minimal contamination of nonsulfated peptides. Using this procedure, we determined that the 13-amino acid C-terminal peptide of the minor gamma'-chain of bovine fibrinogen contains sulfated tyrosine.     

POPK-1/Sad-1 kinase is required for the proper translocation of maternal mRNAs and putative germ plasm at the posterior pole of the ascidian embryo

Maternal mRNAs localized to specific regions in eggs play important roles in the establishment of embryonic axes and germ layers in various species. Type I postplasmic/PEM mRNAs, which are localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, such as the muscle determinant macho-1 mRNA, play key roles in embryonic development. In the present study, we analyzed the function of the postplasmic/PEM RNA Hr-POPK-1, which encodes a kinase of Halocynthia roretzi. When the function of POPK-1 was suppressed by morpholino antisense oligonucleotides, the resulting malformed larvae did not form muscle or mesenchyme, as in macho-1-deficient embryos. Epistatic analysis indicated that POPK-1 acts upstream of macho-1. When POPK-1 was knocked down, localization of every Type I postplasmic/PEM mRNA examined, including macho-1, was perturbed, showing diffuse early distribution and eventual concentration into a smaller area. This is the probable reason for the macho-1 dysfunction. The postplasmic/PEM mRNAs such as macho-1 and Hr-PEM1 are co-localized with the cortical endoplasmic reticulum (cER) and move with it after fertilization. Eventually they become highly concentrated into a subcellular structure, the centrosome-attracting body (CAB), at the posterior pole of the cleaving embryos. The suppression of POPK-1 function reduced the size of the domain of concentrated cER at the posterior pole, indicating that POPK-1 is involved in the movement of postplasmic/PEM RNAs via relocalization of cER. The CAB also shrank. These results suggest that Hr-POPK-1 plays roles in concentration and positioning of the cER, as well as in the concentration of CAB materials, such as putative germ plasm, in the posterior blastomeres.     

Autoxidation kinetic analysis of docosahexaenoic acid ethyl ester and docosahexaenoic triglyceride with oxygen sensor

The application of omega-3 polyunsaturated fatty acids (PUFAs) as food additives is restricted by their chemically quite reactive properties. However, quantitative analyses of the oxidative kinetics of PUFAs are very few compared to other studies on food chemistry. In this study, the autoxidation kinetics of ethyl docosahexaenoate (DHAEE), docosahexaenoic triglyceride (DHA oil), and emulsified DHA oil were investigated with an oxygen sensor. The autocatalytic reaction rate constants for DHAEE, DHA oil, and the emulsified DHA oil with 20% (w/v) GA, 20% SSPS, or 20% SSPS containing 5% soy protein were obtained at 35, 50, and 70 degrees C. A plot of the natural logarithm of the frequency factor, In ka0, vs. the activation energy, Ea, demonstrated that In ka0 against Ea fitted well with a single straight line both for the data from this study and for other reported results. This implies that the chemical compensation relationship holds between ka0 and Ea for PUFA and emulsified DHA oil.