Immunological detection of the dystrophin molecule with antibody directed against the synthetic peptide.

We synthesized a peptide designated R8 (amino acid residues 1157-1201) based on the primary structure presumed from the nucleotide sequence of the cDNA clone from the gene for Duchenne muscular dystrophy. Antibody to the synthetic R8 generated by immunization of rabbits was tested on human and mouse skeletal muscle by Western blotting analysis. The antibody reacted with a component of the 400K dystrophin of normal human and mouse skeletal muscles, but not with components of the muscles of Duchenne muscular dystrophy patients and mdx mice. Thus we established that this peptide sequence is in fact missing in the protein product 'dystrophin' encoded by the DMD gene. The antibody may prove useful for the diagnosis of the Duchenne types of muscular dystrophy.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Effect of changes in thyroid state on metabolism of thyroxine by rat placenta

We studied the effect of the state of the thyroid on T4 monodeiodination in the rat placenta, and it was compared with those in the liver and kidney. The tissues, maternal serum, and amniotic fluid were obtained from pregnant rats. The tissues were homogenized in cold 50 mM Tris-HCl buffer, pH 7.5. The homogenate (1 mg protein) was incubated at 37 degrees C for 60 min with 1 microgram T4 in the presence of 5 mM DTT. The T3 and reverse T3 generated in the reaction mixture were extracted into cold ethanol and measured by RIAs. The conversion of T4 to reverse T3 in rat placenta was not significantly changed in MMI-induced hypothyroidism or T4 induced hyperthyroidism. On the other hand, conversion of T4 to T3 in the liver and kidney were changed in parallel with the thyroid state. The concentration of reverse T3 in the amniotic fluid was increased in accordance with the increase in the maternal serum T4 concentration. These results indicate that the placental T4 inner ring deiodination is not affected by the thyroid state, and that the change in the amniotic fluid reverse T3 concentration in this study is mainly dependent upon the change in maternal thyroid function.     

Possible role of macrophage-derived soluble mediators in the pathogenesis of encephalomyocarditis virus-induced diabetes in mice.

Pancreatic islets from DBA/2 mice infected with the D variant of encephalomyocarditis (EMC-D) virus revealed lymphocytic infiltration with moderate to severe destruction of pancreatic beta cells. Our previous studies showed that the major population of infiltrating cells at the early stages of infection is macrophages. The inactivation of macrophages prior to viral infection resulted in the prevention of diabetes, whereas activation of macrophages prior to viral infection resulted in the enhancement of beta-cell destruction. This investigation was initiated to determine whether macrophage-produced soluble mediators play a role in the destruction of pancreatic beta cells in mice infected with a low dose of EMC-D virus. When we examined the expression of the soluble mediators interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) in the pancreatic islets, we found that these mediators were clearly expressed at an early stage of insulitis and that this expression was evident until the development of diabetes. We confirmed the expression of these mediators by in situ hybridization with digoxigenin-labelled RNA probes or immunohistochemistry in the pancreatic islets. Mice treated with antibody against IL-1beta or TNF-alpha or with the iNOS inhibitor aminoguanidine exhibited a significant decrease in the incidence of diabetes. Mice treated with a combination of anti-IL-1beta antibody, anti-TNF-alpha antibody, and aminoguanidine exhibited a greater decrease in the incidence of disease than did mice treated with one of the antibodies or aminoguanidine. On the basis of these observations, we conclude that macrophage-produced soluble mediators play an important role in the destruction of pancreatic beta cells, resulting in the development of diabetes in mice infected with a low dose of EMC-D virus.     

Role of Ca2+ in t-PA production by human embryonic lung diploid fibroblast, IMR-90 cells, stimulated by proteose peptone

Proteose peptone (p.peptone) remarkably induced tissue plasminogen activator (t-PA) activity in the conditioned medium of confluently cultured human embryonic lung diploid fibroblast, IMR-90 cells, in a dose-dependent manner. t-PA activity correlated well with the amount of t-PA antigen found in the conditioned medium of IMR-90 cells stimulated by p.peptone. t-PA production by IMR-90 cells stimulated by p.peptone was dependent on extracellular Ca2+ concentration and maximum t-PA production required approximately 3.6 mM extracellular Ca2+. Conversely, elimination of Ca2+ from the culture medium by EGTA, Ca2+ chelate agent, strongly inhibited t-PA production induced by p.peptone. t-PA production induced by p.peptone was inhibited in a dose-dependent manner by Verapamil, which inhibits Ca2+ uptake through the slow channels and also by W-7, an inhibitor of calmodulin. These results suggested that influx of extracellular Ca2+ into IMR-90 cells was caused by p.peptone and induced t-PA production by the cells.     

Purification and characterization of 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina

The inducible 3-ketosteroid-delta 1-dehydrogenase of Nocardia corallina which catalyzes the introduction of a double bond into the position of carbon 1 and 2 of ring A of 3-ketosteroid has been obtained in four steps with a 50% yield and 360-fold purification. The enzyme is homogeneous as judged by SDS-gel electrophoresis and is a monomeric protein with a molecular weight of 60,500. The isoelectric point of the enzyme is about 3.1. The enzyme contains 1 mol of flavin adenine dinucleotide per mol of protein, and has a typical flavoprotein absorption spectrum with maxima of 458, 362 and 268 nm. The enzyme is very stable in the absence of added cofactors, and catalyzes the dehydrogenation of delta 4-3-ketosteroids in the presence of phenazine methosulfate, which acts as an excellent electron acceptor. Potassium ferricyanide and cytochrome c did not act as electron acceptors. The delta 1-dehydrogenation was also stimulated by molecular oxygen with stoichiometric production of hydrogen peroxide and delta 1,4-3-ketosteroid. The optimum pH is 10 for dehydrogenation using phenazine methosulfate, and is between 8.5 and 10 for the oxidase reaction. The enzyme oxidizes a wide variety of 3-ketosteroids, but not 3 beta-hydroxysteroids. 3-Ketosteroids having an 11 alpha- or 11 beta-hydroxyl group were oxidized at slow rates. The purified enzyme catalyzes efficiently aromatization of the A-ring of 19-nortestosterone and 19-norandrostenedione to produce estradiol and estrone. 19-Hydroxytestosterone, 19-hydroxyandrostenedion and 19-oxotestosterone were converted to the respective phenolic steroids with cleavage of the C10 side-chain. Activities of 3-ketosteroid-delta 4-dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, 3 beta-hydroxysteroid dehydrogenase and 17 beta-hydroxysteroid dehydrogenase were not observed in the purified preparations. Properties of this novel flavoprotein enzyme are discussed.     

Spectral properties of 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina

3-Ketosteroid-delta 1-dehydrogenase from Nocardia corallina is a flavoenzyme that catalyzes 1,2-desaturation of 3-ketosteroid. The dehydrogenase generated complexes with 3-ketosteroids and phenolic steroids such as estradiol with remarkable perturbations of the visible spectrum. The enzyme did not make the adduct with sulfite ion, but could use molecular oxygen as the electron acceptor. The CD spectra of oxidized and steroid-bound enzymes exhibited positive dichroisms in the visible region which resembled those of flavoenzyme oxidases. The dehydrogenase led isosbestically to the stable red semiquinone species with large yields upon photochemical or dithionite reduction (at pH 7.4) in the presence of the steroid product, 1,4-androstadiene-3,17-dione, but in the absence of the steroid the yield of semiquinone was low and the fully reduced enzyme was obtained. Substrate titration also yielded the red flavo-semiquinone stoichiometrically and it was hard to generate the fully reduced form. The reduced enzyme was oxidized with molecular oxygen, but did not oxidize with ferricyanide. An EPR study of these half-reduced forms confirmed the presence of the radical species with the g = 2.004 signal. The dehydrogenase was rapidly reduced with an excess amount of 3-ketosteroid at about 80% yield at pH 7.4 under anaerobic conditions and the reduced species was altered to the stable red semiquinone species. The rate of this reaction was t1/2 = 28 min at pH 7.4, 130 min at pH 9.0 and 34 min at pH 6.4, respectively. These results indicate that the semiquinone species does not act directly in turnover of the dehydrogenase reaction. The results were compared with the spectral properties of general acyl-CoA dehydrogenases and acyl-CoA oxidase toward the mechanism of C1,2-dehydrogenation.     

A fibroblast-derived tumor cytotoxic factor/F-TCF (hepatocyte growth factor/HGF) has multiple functions in vitro.

We previously demonstrated that a tumor cytotoxic factor(F-TCF) purified from the culture broth of human embryonic lung diploid fibroblast, IMR-90 cells was one of the human hepatocyte growth factors (hHGFs). In the present report, we demonstrate its biological functions. F-TCF showed moderate cytotoxicity on human tumor cell lines KB, BG-1, MCF-7 and Hs913 T, and strong cytotoxicity on mouse tumor cell lines Sarcoma 180, Meth A sarcoma and P388. On the contrary, F-TCF was a potent mitogen not only for adult rat hepatocytes, but also for human endothelial cells, HUVEC and human melanocytes. Moreover, F-TCF induced the differentiation of premyelocyte leukemia, HL-60 cells into morphologically granulocyte-like cells. These biological functions suggest that F-TCF is an effector molecule responsible for inflammation and repair in injured tissues including tumor and liver.     

Effect of Caffeine-Containing Beverage Consumption on Serum Alanine Aminotransferase Levels in Patients with Chronic Hepatitis C Virus Infection: A Hospital-Based Cohort Study

Introduction

To date, there have been no prospective studies examining the effect of coffee consumption on serum alanine aminotransferase (ALT) level among individuals infected with the hepatitis C virus (HCV). We conducted a hospital-based cohort study among patients with chronic HCV infection to assess an association between baseline coffee consumption and subsequent ALT levels for 12 months.

Materials and Methods

From 1 August 2005 to 31 July 2006, total 376 HCV-RNA positive patients were recruited. A baseline questionnaire elicited information on the frequency of coffee consumption and other caffeine-containing beverages. ALT level as a study outcome was followed through the patients’ medical records during 12 months. The association between baseline beverage consumption and subsequent ALT levels was evaluated separately among patients with baseline ALT levels within normal range (≤45 IU/L) and among those with higher ALT levels (>45 IU/L).

Results

Among 229 patients with baseline ALT levels within normal range, 186 (81%) retained normal ALT levels at 12 months after recruitment. Daily drinkers of filtered coffee were three times more likely to preserve a normal ALT level than non-drinkers (OR=2.74; P=0.037). However, decaffeinated coffee drinkers had a somewhat inverse effect for sustained normal ALT levels, with marginal significance (OR=0.26; P=0.076). In addition, among 147 patients with higher baseline ALT levels, 39 patients (27%) had ALT reductions of ≥20 IU/L at 12 months after recruitment. Daily drinkers of filtered coffee had a significantly increased OR for ALT reduction (OR=3.79; P=0.034). However, in decaffeinated coffee drinkers, OR could not be calculated because no patients had ALT reduction.

Conclusion

Among patients with chronic HCV infection, daily consumption of filtered coffee may have a beneficial effect on the stabilization of ALT levels.     

Selection for floral integration and trait variation in zygomorphic flowers of Aconitum japonicum ssp. subcuneatum (Ranunculaceae)

Pollinator-mediated selection might lead to among-trait differences in the degree and pattern of floral integration and intra-flower variation. To examine the patterns of intra-flower variation in floral traits, including nectar volume, we performed a field study using the zygomorphic flowers of Aconitum japonicum ssp. subcuneatum. We investigated (1) correlations between the sizes of the left and right sepals and petals, (2) variation in floral traits among plants, within plants and within flowers, (3) effects of sexual phases on floral integration variation in floral and nectar traits, and (4) the effect of size and intra-flower variation in traits of the left and right sepals and petals on pollen removal by pollinators. Lateral sepal area, but not lower sepal area, was highly correlated between the left and right sepals. Floral traits were more integrated during the male phase than during the female phase. Nectar standing crop in male-phase flowers correlated with helmet height and lateral and lower sepal area, but in female-phase flowers it only correlated with spur length. While intra-flower variance in lateral sepal area accounted for approximately 10% of the overall variance in these traits, the variance in lower sepal area accounted for 70% of the overall variance. Lateral sepal area had a negative effect on the number of pollen grains remaining after pollinator visits. Low variance in lateral sepals within flowers and measurements of pollen removal suggest that lateral sepals play a more important role in pollen export than the other traits. Left and right sepals may be the targets of selection for symmetry in zygomorphic flowers.