Ontogenic emergence and localization of larval skin antigen molecule recognized by adult T cells of Xenopus laevis: Regulation by thyroid hormone during metamorphosis

Results from previous studies using an inbred strain of Xenopus laevis have led to the proposition that metamorphosis includes the events by which the newly differentiating adult immune system, including T lymphocytes, recognizes and eliminates larval skin cells as 'non-self'. More recently, a larval antigen targeted by adult T cells was identified as a 59 kDa protein with a specific peptide sequence. Using antisera directed against the larval antigen and the peptide, immunohistochemistry and western blotting were done to examine expression of the 59 kDa larval antigen in the skin during larval and metamorphic periods. There was no expression before Nieuwkoop and Faber stage 53. Expression was first seen at the beginning of metamorphic stage 54, when hind limbs appear, and increased thereafter, in apical and skein cells of both trunk and tail regions. In the trunk region, expression started to decrease at stage 58, until it completely disappeared at stage 62 (metamorphic climax). In the tail skin, however, expression persisted throughout the metamorphic stages. Treatment of larvae with thyroid hormone (TH) resulted in repression of expression of the 59 kDa molecule in a dose-dependent manner. Downregulation occurred earlier in the trunk than in the tail skin. These results suggest involvement in metamorphic events of an immunological mechanism: differential expression of the larval antigen in the trunk and tail skin cells due to their differing concentration of TH results in the tail, but not the trunk skin, being selectively attacked by the newly differentiating adult-type immune system.     

A G protein-coupled receptor responsive to bile acids

So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5'-O-3-thio-triphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids.     

Docosahexaenoic acid supplementation-increased oxidative damage in bone marrow DNA in aged rats and its relation to antioxidant vitamins

We compared the influence of docosahexaenoic acid (DHA) supplementation on oxidative DNA damage in bone marrow between young and aged rats. As a marker of oxidative DNA damage, 8-hydroxydeoxy-guanosine (8-OHdG) in DNA was analyzed. Young (5-week-old) and aged (100-week-old) female Wistar rats were given DHA (300mg/kg body weight/day) or vehicle (control) orally for 12 weeks. The 8-OHdG in the bone marrow in the aged DHA group was significantly higher than that in the other groups. Vitamin E concentrations, however, did not differ among the groups regardless of the DHA supplementation. Vitamin C (ascorbic acid) concentrations in the aged control group were approximately 1/2 those in the young control group. The concentrations of vitamin C tended to be higher in the young DHA group and lower in the aged DHA group when compared to their respective control groups. Changes in the concentrations of vitamin C and vitamin E in plasma were similar to those in the bone marrow. The activity of hepatic l-gulono- γ -lactone oxidase, an enzyme responsible for vitamin C synthesis, corresponded well to the concentrations of vitamin C in the bone marrow and the plasma. These results suggest that in aged rats, but not young rats, excess supplementation of DHA induces oxidative DNA damage in bone marrow and that the decrease in vitamin C synthesis in aged rats is involved in the mechanisms of DNA damage.     

In vitro abzyme evolution to optimize antibody recognition for catalysis

Enzymes have evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreasing the affinity for the ground state. To evolve abzymes toward higher catalytic activity, we have reconstructed an enzyme-evolutionary process in vitro. Thus, a phage-displayed combinatorial library from a hydrolytic abzyme, 6D9, generated by the conventional in vivo method with immunization of the transition-state analog (TSA), was screened against a newly devised TSA to optimize the differential affinity for the transition state relative to the ground state. The library format successfully afforded evolved variants with 6- to 20-fold increases in activity (kcat) as compared with 6D9. Structural analysis revealed an advantage of the in vitro evolution over the in vivo evolution: an induced catalytic residue in the evolved abzyme arises from double mutations in one codon, which rarely occur in somatic hypermutation in the immune response.     

Specific binding and stabilization of DNA and phosphorothioate DNA by amphiphilic alpha-helical peptides

Synthetic cationic peptides with amphiphilic alpha-helical structure were found to have DNA binding ability to stabilize double and triple stranded DNA. The stabilization effect was significant for cationic alpha-helical peptides indicating the importance of an electrostatic interaction of a positive charge of peptides and a negative charge of DNAs.     

Antitumor effect of photodynamic therapy with zincphyrin, zinc-coproporphyrin III, in mice

We studied the antitumor effects of photodynamic therapy (PDT) with Zincphyrin, coproporphyrin III with zinc, derived from Streptomyces sp. AC8007, in vitro and in vivo. The photokilling effect of Zincphyrin in the presence of 0.78-100 microg/ml with visible light of 27.2 mW x min/cm2 for 10 min was lower than the hematoporphyrin (Hp) used as a control with L5178Y or sarcoma-180 cells. On the other hand, Zincphyrin apparently reduced tumor growth after intraperitoneal injection at doses of 12.5-50 mg/kg with light irradiation of 75.48 mW x min/cm2 for 10 min in sarcoma-180-bearing mice. Although no mice treated with Zincphyrin died, Hp did cause the death of mice. In B-16 melanoma-bearing mice, both Zincphyrin and Hp had a similar phototherapic effect. Further improvement of the phototherapic effect was observed with the continuous administration of Zincphyrin at 12.5 mg/kg per day for 3 days. The concentration of Zincphyrin in the serum reached a maximum level of 16 microg/ml within 20 min, and the concentration remained at 4.2 microg/ml at 1 hour after the onset of treatment, indicating its rapid action in the body. No animals died after the intraperitoneal administration of Zincphyrin at 100 mg/kg plus exposure to light of 10 mW x min/cm2 for 2 hours, and the body weight of the mice did not decrease. In contrast, all animals receiving 100 mg/kg of Hp under the same conditions died. These results indicate that Zincphyrin would be a useful photosensitizer with low phototoxicity.     

Effects of buckwheat in a renal ischemia-reperfusion model

Experiments were done to find whether buckwheat extract ameliorates the renal injury induced by ischemia-reperfusion. In ischemic-reperfused control rats, the activities of antioxidative enzymes in renal tissue and blood and renal parameters deviated from the normal range, indicating dysfunction of the kidneys. In contrast, when buckwheat extract was given orally for 20 consecutive days before ischemia and reperfusion, the activities of the antioxidation enzymes superoxide dismutase, catalase, and glutathione peroxidase were higher, while thiobarbituric acid-reactive substance levels in serum and renal tissue were lower in the treated rats than in the controls. Decreased levels of urea nitrogen and creatinine in serum demonstrated a protective effect against the renal dysfunction caused by ischemia and recirculation. On the other hand, it was demonstrated that buckwheat extract had a protective effect on cultured proximal tubule cells subjected to hypoxia-reoxygenation, probably by preventing oxygen free radicals from attacking the cell membranes.     

Membrane-bound quinoprotein D-arabitol dehydrogenase of Gluconobacter suboxydans IFO 3257: a versatile enzyme for the oxidative fermentation of various ketoses

Solubilization of membrane-bound quinoprotein D-arabitol dehydrogenase (ARDH) was done successfully with the membrane fraction of Gluconobacter suboxydans IFO 3257. In enzyme solubilization and subsequent enzyme purification steps, special care was taken to purify ARDH as active as it was in the native membrane, after many disappointing trials. Selection of the best detergent, keeping ARDH as the holoenzyme by the addition of PQQ and Ca2+, and of a buffer system involving acetate buffer supplemented with Ca2+, were essential to treat the highly hydrophobic and thus labile enzyme. Purification of the enzyme was done by two steps of column chromatography on DEAE-Toyopearl and CM-Toyopearl in the presence of detergent and Ca2+. ARDH was homogenous and showed a single sedimentation peak in analytical ultracentrifugation. ARDH was dissociated into two different subunits upon SDS-PAGE with molecular masses of 82 kDa (subunit I) and 14 kDa (subunit II), forming a heterodimeric structure. ARDH was proven to be a quinoprotein by detecting a liberated PQQ from SDS-treated ARDH in HPLC chromatography. More preliminarily, an EDTA-treated membrane fraction lost the enzyme activity and ARDH activity was restored to the original level by the addition of PQQ and Ca2+. The most predominant unique character of ARDH, the substrate specificity, was highly versatile and many kinds of substrates were oxidized irreversibly by ARDH, not only pentitols but also other polyhydroxy alcohols including D-sorbitol, D-mannitol, glycerol, meso-erythritol, and 2,3-butanediol. ARDH may have its primary function in the oxidative fermentation of ketose production by acetic acid bacteria. ARDH contained no heme component, unlike the type II or type III quinoprotein alcohol dehydrogenase (ADH) and did not react with primary alcohols.     

Purification and characterization of membrane-bound quinoprotein cyclic alcohol dehydrogenase from Gluconobacter frateurii CHM 9

A quinoprotein catalyzing oxidation of cyclic alcohols was found in the membrane fraction for the first time, after extensive screening among aerobic bacteria. Gluconobacter frateurii CHM 9 was finally selected in this study. The enzyme tentatively named membrane-bound cyclic alcohol dehydrogenase (MCAD) was found to occur specifically in the membrane fraction, and pyrroloquinoline quinone (PQQ) was functional as the primary coenzyme in the enzyme activity. MCAD catalyzed only oxidation reaction of cyclic alcohols irreversibly to corresponding ketones. Unlike already known cytosolic NAD(P)H-dependent alcohol-aldehyde or alcohol-ketone oxidoreductases, MCAD was unable to catalyze the reverse reaction of cyclic ketones or aldehydes to cyclic alcohols. MCAD was solubilized and purified from the membrane fraction of the organism to homogeneity. Differential solubilization to eliminate the predominant quinoprotein alcohol dehydrogenase (ADH), and the subsequent two steps of column chromatographies, brought MCAD to homogeneity. Purified MCAD had a molecular mass of 83 kDa by SDS-PAGE. Substrate specificity showed that MCAD was an enzyme oxidizing a wide variety of cyclic alcohols. Some minor enzyme activity was found with aliphatic secondary alcohols and sugar alcohols, but not primary alcohols, differentiating MCAD from quinoprotein ADH. NAD-dependent cytosolic cyclic alcohol dehydrogenase (CCAD) in the same organism was crystallized and its catalytic and physicochemical properties were characterized. Judging from the catalytic properties of CCAD, it was apparent that CCAD was distinct from MCAD in many respects and seemed to make no contributions to cyclic alcohol oxidation.