Juzentaihoto extract suppresses adipocyte hypertrophy and improves hyperglycemia in KKAy mice.

Juzentaihoto is a herbal medicine with reported anti-inflammatory effects, and it is predicted to improve inflammation and insulin sensitivity within obesity. In the present study, juzentaihoto hot water extract (JTT) was administered to obese type 2 diabetic model mice (KKAy) for 56 days. In addition, the effects of JTT on the adipose tissue, glucose metabolism, and blood lipids were evaluated for examining its impact on insulin sensitivity and obesity. As a result of JTT administration, KKAy mice exhibited suppressed adipocyte hypertrophy, decreased the mRNA levels of tumor necrosis factor α, and increased the mRNA levels of adiponectin in epididymal fat tissue. In addition, fasting blood glucose levels, blood triglyceride, and total cholesterol decreased. In summary, these data indicated that JTT administration suppressed the production of inflammatory cytokines and increased adiponectin levels in the adipose tissue. Therefore, with improved insulin sensitivity, blood glucose, and lipid decreased.

Quality control of hospital preparations: Establishment of a simple and rapid method for quantifying ulinastatin in vaginal suppositories.

Ulinastatin vaginal suppositories, used to prevent threatened premature delivery, are frequently used in hospitals. However, there is no established method for quantifying ulinastatin contained in suppositories. Therefore, we investigated a simple and efficient method for quantifying ulinastatin contained in suppositories. Our analytical method involved removal of the base; optimising the enzyme inhibition reaction time and enzyme reaction time; and measuring the absorbance. The modified method was reproducible, operation time was significantly shortened, and cost was reduced to approximately 1/17 of that of the previously reported method. This simple and rapid quantitative method could contribute to the improvement of quality control of ulinastatin vaginal suppositories as an extemporaneous hospital preparation.

Behaviour and adsorptive removal of siloxanes in sewage sludge biogas.

We investigated the behaviour of siloxanes, which adversely affect biogas engines, as well as their concentration levels in sewage sludge biogas in Japan. We also performed experiments on the absorptive removal of siloxanes using various adsorbents and determined the main adsorbent characteristics required for the removal of siloxanes. The results of our study on the concentration and composition of siloxanes in biogas were similar to previous reports. Moreover, we found that the concentration of siloxanes changes in relation to the outside air temperature based on real-time measurements of siloxanes using a continuous analyser. We further speculated that the continuous analyser would accurately indicate the siloxane concentration in model biogas but overestimate the siloxane concentration in actual biogas because of positive interference by VOCs and other biogas components. In the siloxane adsorption experiment, the equilibrium uptake of both cyclic siloxanes, D4 and D5, was positively related to the BET-specific surface area of the adsorbents and the fraction of the external surface area taken up by relatively large diameter pores. We attributed the adsorption results to the fact that the siloxane molecules are generally larger than micropores; therefore, they are less susceptible to adsorption to micropores. Based on these results, we concluded that adsorbents with large BET-specific surface areas, especially those with a high external specific surface area and pores of relatively large diameters, are desired for the removal of siloxanes.

Electrolyzed-reduced water reduced hemodialysis-induced erythrocyte impairment in end-stage renal disease patients.

Chronic hemodialysis (HD) patients increase erythrocyte susceptibility to hemolysis and impair cell survival. We explored whether electrolyte-reduced water (ERW) could palliate HD-evoked erythrocyte impairment and anemia. Forty-three patients undergoing chronic HD were enrolled and received ERW administration for 6 month. We evaluated oxidative stress in blood and plasma, erythrocyte methemoglobin (metHb)/ferricyanide reductase activity, plasma metHb, and proinflammatory cytokines in the chronic HD patients without treatment (n=15) or with vitamin C (VC)- (n=15), vitamin E (VE)-coated dialyzer (n=15), or ERW treatment (n=15) during an HD course. The patients showed marked increases (15-fold) in blood reactive oxygen species, mostly H(2)O(2), after HD without any treatment. HD resulted in decreased plasma VC, total antioxidant status, and erythrocyte metHb/ferricyanide reductase activity and increased erythrocyte levels of phosphatidylcholine hydroperoxide (PCOOH) and plasma metHb. Antioxidants treatment significantly palliated single HD course-induced oxidative stress, plasma and RBC PCOOH, and plasma metHb levels, and preserved erythrocyte metHb /ferricyanide reductase activity in an order VC>ERW>VE-coated dialyzer. However, ERW had no side effects of oxalate accumulation easily induced by VC. Six-month ERW treatment increased hematocrit and attenuated proinflammatory cytokines profile in the HD patients. In conclusion, ERW treatment administration is effective in palliating HD-evoked oxidative stress, as indicated by lipid peroxidation, hemolysis, and overexpression of proinflammatory cytokines in HD patients.

Dietary and inhalation intake of lead and estimation of blood lead levels in adults and children in Kanpur, India.

This research was initiated to study lead levels in various food items in the city of Kanpur, India, to assess the dietary intake of lead and to estimate blood lead (PbB) levels, a biomarker of lead toxicity. For this purpose, sampling of food products, laboratory analysis, and computational exercises were undertaken. Specifically, six food groups (leafy vegetables, nonleafy vegetables, fruits, pulses, cereals, and milk), drinking water, and lead air concentration were considered for estimating lead intake. Results indicated highest lead content in leafy vegetables followed by pulses. Fruits showed low lead content and drinking water lead levels were always within tolerable limits. It was estimated that average daily lead intake through diet was about 114 microg/day for adults and 50 microg/day in children; tolerable limit is 250 microg/day for adults and 90 microg/day for children. The estimated lead intakes were translated into the resultant PbB concentrations for children and adults using a physiologically-based pharmacokinetic (PBPK) model. Monte Carlo simulation of PbB level variations for adults showed that probability of exceeding the tolerable limit of PbB (i.e.,10 microg/dL) was 0.062 for the pre-unleaded and 0.000328 for the post-unleaded gasoline period. The probability of exceeding tolerable limits in PbB level was reduced by a factor of 189 in the post-unleaded scenario. The study also suggested that in spite of the introduction of unleaded gasoline, children continue to be at a high risk (probability of exceeding 10 microg/dL = 0.39) because of a high intake of lead per unit body weight.

The distribution and speciation of antimony in river water, sediment and biota in Yodo River, Japan.

In 1993, the Japanese government set the guideline concentration of 2 microg l(-1) for antimony in natural water and drinking water as an item for precautionary monitoring in consideration of its chronic toxicity, however in 1999 the guideline value was tentatively canceled to examine the health risk of antimony through the accumulation of more information and data for detailed risk assessment. In this study, the distribution of antimony in the river water environment including water, sediment, aquatic plants and fishes was surveyed in the Yodo River basin. The characteristics of antimony distribution between river water and sediment were determined by the batch sorption experiments, focusing on the difference in oxidation state. This study suggests that biota in the Yodo River basin have antimony at concentrated levels. Some of the sewage treatment plants seemed to influence the concentration of antimony in river water. It was shown that the pentavalent antimony is dominant in river water and especially paramount downstream of the sewage treatment plants, though the trivalent antimony is dominant in industrial use. According to the sorption experiments of antimony on river sediment, the trivalent antimony is sorbed faster and more in partition than the pentavalent antimony.

Inhibition of thyroid hormone binding to the nuclear receptor by mobilization of free fatty acids.

Through binding to its nuclear receptor (TR), thyroid hormone (T3) activates the expression of the thyroid hormone-responsive genes that are essential for the regulation of energy consumption. Previously, we found that free fatty acids (FFAs) and their CoA esters strongly inhibited the binding of T3 to its nuclear receptor in vitro. In the present study, we have examined the physiological relevance of this inhibitory mechanism. TRs in isolated nuclei and in a solubilized free form were half-maximally inhibited with oleic acid at 120 and 2.8 microM, respectively. The lower sensitivity of the nuclear TR as compared with free TR was attributed to the nuclear envelope and the association of TR with chromatin. Among TRs in chromatin, those in the transcriptionally active chromatin exhibited the highest sensitivities to FFAs and were inhibited half-maximally by oleic acid at 10 microM. While the plasma concentration of FFAs in total was 0.4 to 1 mM, their nuclear concentration was about 5 microM. Thus, the sensitivities of TRs in active chromatin and in solubilized form were at physiological levels with respect to the nuclear FFA concentration. We further examined the effect of FFA mobilization on the T3-binding to TR in animals. Nuclear T3-binding was significantly inhibited when plasma and cellular FFAs were increased by norepinephrine in vivo. The increase in cellular FFAs and the TR-inhibition were well correlated, and much larger in the heart than in the liver and kidney. These results suggest that TR is negatively controlled by increased FFAs in a tissue-dependent manner.

Ultrastructural studies of late-stage spermatids and mature spermatozoa of the puffer fish, Takifugu niphobles (Tetraodontiformes) and the effects of osmolality on spermatozoan structure.

Ultrastructures of late-stage spermatids and spermatozoa, and of spermatozoa after exposure to various osmolalities, were studied in the puffer, Takifugu niphobles. The mature spermatozoa consisted of a head, a midpiece of many mitochondria and a flagellum with sharp sidefins, had many ring-structures just inside of the plasma membrane of cytoplasmic sleeve and triangular-structures projecting into cytoplasmic canal at the base of flagellum. In late spermatids, the rings and projections were present, but the side-fins had round ends and the cytoplasm of flagellum was amorphous. When spermatozoa were exposed to seawater, the plasma membrane became swollen in the head-midpiece region but shrank in the tail region. In 1/2 seawater, swelling in the tail occurred in some spermatozoa. In 1/3 seawater approximately isotonic to the seminal plasma, there was little change. In 1/10 seawater, the plasma membrane swelled slightly in the head region, but swelled much more in the tail region. In buffer solution, the membrane swelled in all regions, surrounding the nucleus and many sections of axoneme. Thus, function of the plasma membrane in the head-region may be different from that in the tail-region. Spermatozoa of marine fish may fertilize the eggs when the osmolality surrounding the sperm, which changes due to the mixing of seminal plasma and seawater, reaches the correct level for the spermatozoa to obtain correct structure.

Comparison of the mutations induced by p-benzoquinone, a benzene metabolite, in human and mouse cells.

Benzene is one of the chemicals widely contaminating the environment. Benzene is suggested to be a human leukemogen. When benzene is absorbed in the human body, it is metabolized firstly in the liver and subsequently in the bone marrow where it provokes initiation of leukemia. In the present study, we analyzed mutations induced by p-benzoquinone (p-BQ), a benzene metabolite, in human cells using a shuttle vector plasmid pMY189, and compared frequencies, types and spectra of the mutations with those of the mutations previously revealed in mouse cells using a similar plasmid pNY200. We found that p-BQ induces mutations in human and mouse cells at similar frequencies but with different types of mutagenesis. The proportion of tandem base mutations was significantly lower in human cells than in mouse cells. Most base substitutions were induced in G:C base pairs in both human and mouse cells. However, the proportion of G:C-->C :G transversion is significantly higher in human cells. These findings indicate that the p-BQ-induced DNA damage in human and mouse cells is processed in a different manner, and that extrapolation of mice findings on experimental benzene carcinogenesis to human cancer risk assessment should be conducted carefully.

Fine structure of micropylar region during late oogenesis in eggs of the hagfish Eptatretus burgeri (Agnatha).

Using the hagfish, Eptatretus burgeri, the fine structure of formation of the micropylar region in hagfish eggs during the late stages of oogenesis was investigated for the first time, focusing on the bottom region of the micropyle and the egg surface. During these stages, many cells penetrated through the chorion and reached a pit of the egg surface, forming a shovel-like structure in two-dimensional sections. The cells, which we called micropylar cells, were separated from the chorion by a wall of amorphous material. In the pit, another fibrous layer filled the space between the egg surface and the anterior portion of the shovel-like structure. Microvilli coming from the egg surface were embedded in this layer. In later stages, the stack of micropylar cells loosened, and a space appeared between the anterior region of the shovel-like structure and the layer on the egg surface. Microvilli decreased in length and number. The pit region appeared likely to have a role in fertilization. The structures associated with the forming micropyle were markedly different from those observed in the same region of teleost fishes. A hypothesis that hagfish might show transitional structures in gametes from protochordates to teleosts is suggested.

Molecular analysis of mutations induced by a benzene metabolite, p-benzoquinone, in mouse cells using a novel shuttle vector plasmid.

Human population has been continually exposed to benzene which is present in our environment as an essential component of petroleum. p-Benzoquinone (p-BQ) is one of the benzene metabolites and is thought to be an ultimate toxic or carcinogenic substance. For molecular analysis of carcinogen-induced mutations in mouse cells, we constructed a new shuttle vector plasmid pNY200 that has supF gene as a target of the mutations and replicates in mouse and in Escherichia coli cells. In p-BQ-treated pNY200 propagated in mouse cells, base substitutions were induced predominantly at G:C sites, and the major mutation was G:C-->A:T transition. Many tandem base substitutions were also induced at CC:GG sequences. By a postlabeling analysis and a polymerase stop assay, we confirmed that p-BQ adducts formed in DNA and mutation sites roughly correspond to the sites where the adducts were formed. Comparing data of pNY200 in mouse cells with those of the similar shuttle vector plasmid pMY189 in human cells should be important for extrapolation of data from mouse to human, because carcinogenicity of chemicals is tested in mice.

Acrosome reaction in spermatozoa of the hagfish Eptatretus burgeri (Agnatha).

Fertilization of the hagfish or myxiniformes, a member of the most primitive vertebrate group and an animal of phylogenic interest, is unknown. Here, induction of an acrosome reaction for spermatozoa in the hagfish, Eptatretus burgeri, was successfully achieved by treatment of mature spermatozoa with ionomycin and excess Ca2+. The spermatozoon produced an acrosomal process that elongated from the apex of the long sperm head. The reaction bears resemblance to that of invertebrate spermatozoa rather than that of vertebrate spermatozoa. The result provides insights into the phylogenetical changes that have occurred in this sperm reaction.

Evaluation of the Japanese health risks induced by global fallout tritium.

Radioactive tritium (3H) generated by the atmospheric nuclear detonation tests has been circulated in the global environment to cause, through various pathways, low-level world-wide contamination of the environment and potential health risks to human beings. In this study, the dynamic performance of tritium in the global environment was modeled considering its transport with foods/feeds imported from all over the world. The mathematical model was examined by comparing the numerically simulated results with the fallout monitoring data. Main results obtained in this study were as follows: (1) The mathematical model developed in this study can reproduce well the global scale tritium circulation and its concentration in food; (2) The excess fatality rate of Japanese by the dietary intake of tritium is estimated to be on the order of 10(-8) y(-1) at present, which is about two orders of magnitude smaller than that by 90Sr; and (3) About 70% of the Japanese internal radiation dose by tritium is due to drinking water. Farm products, especially imported wheat, are secondly important in the Japanese health risk evaluation; its contribution, however, becomes small if the isotopic exchange effect of tritium is considered.

Proteasomes regulate the motility of salmonid fish sperm through modulation of cAMP-dependent phosphorylation of an outer arm dynein light chain.

Proteasomes are involved in ATP-dependent regulation of sperm motility in salmonid fish. We have demonstrated here by immunoelectron microscopy that proteasomes are located at the structure of the chum salmon sperm flagellum that attaches at the base of the outer arm dynein and extends toward the plasma membrane. Furthermore, substrates and inhibitors of proteasome inhibit the cAMP-dependent phosphorylation of a 22 kDa axonemal protein in chum salmon sperm. The 22 kDa phosphoprotein was solubilized by treatment of the axoneme with a high salt solution and subsequent sucrose density gradient centrifugation of the extract revealed that it cosedimented with 19 S outer arm dynein, indicating that it is a dynein light chain. These results suggest that proteasomes modulate the activity of outer arm dynein by regulating cAMP-dependent phosphorylation of the 22 kDa dynein light chain.

A dosimetric determination of 137Cs ingestion from global fallout and the related risks to Japanese.

137Cs released from atmospheric nuclear detonation tests has been transported worldwide in the environment and finally taken up by humans through various pathways. In particular, ingestion pathways are important for evaluating the human health risks caused by the chronic global low-level radioactive contamination. In this research, the mathematical model for the evaluation of the dietary intake of 137Cs and the related risks to Japanese are proposed by coupling the previously published global 137Cs distribution model with the regional models such as various food ingestion models and the model of the domestic and international supply. Predictions from the proposed model were compared with the monitoring data of 137Cs in Japanese total diet as an attempt at validation. The major findings obtained in this research include that the proposed model is promising for evaluating the risk to Japanese health caused by the dietary intake of global radioactive fallout 137Cs, the 137Cs is taken up by Japanese mostly through farm products, the ingestion of 137Cs through imported foods is increasing, the risk to the Japanese health of inducing cancer by 137Cs internal exposure reached a maximum in 1963, gradually decreasing to the lowest present level, and the risk to infants is the highest.

Electrolyzed-reduced water scavenges active oxygen species and protects DNA from oxidative damage.

Active oxygen species or free radicals are considered to cause extensive oxidative damage to biological macromolecules, which brings about a variety of diseases as well as aging. The ideal scavenger for active oxygen should be 'active hydrogen'. 'Active hydrogen' can be produced in reduced water near the cathode during electrolysis of water. Reduced water exhibits high pH, low dissolved oxygen (DO), extremely high dissolved molecular hydrogen (DH), and extremely negative redox potential (RP) values. Strongly electrolyzed-reduced water, as well as ascorbic acid, (+)-catechin and tannic acid, completely scavenged O.-2 produced by the hypoxanthine-xanthine oxidase (HX-XOD) system in sodium phosphate buffer (pH 7.0). The superoxide dismutase (SOD)-like activity of reduced water is stable at 4 degrees C for over a month and was not lost even after neutralization, repeated freezing and melting, deflation with sonication, vigorous mixing, boiling, repeated filtration, or closed autoclaving, but was lost by opened autoclaving or by closed autoclaving in the presence of tungsten trioxide which efficiently adsorbs active atomic hydrogen. Water bubbled with hydrogen gas exhibited low DO, extremely high DH and extremely low RP values, as does reduced water, but it has no SOD-like activity. These results suggest that the SOD-like activity of reduced water is not due to the dissolved molecular hydrogen but due to the dissolved atomic hydrogen (active hydrogen). Although SOD accumulated H2O2 when added to the HX-XOD system, reduced water decreased the amount of H2O2 produced by XOD. Reduced water, as well as catalase and ascorbic acid, could directly scavenge H2O2. Reduce water suppresses single-strand breakage of DNA b active oxygen species produced by the Cu(II)-catalyzed oxidation of ascorbic acid in a dose-dependent manner, suggesting that reduced water can scavenge not only O2.- and H2O2, but also 1O2 and .OH.

Molecular cloning of a RNA binding protein, S1-1.

S1 proteins A-D constitute a nuclear protein family that are liberated rapidly in a set from chromatin by mild digestion with a DNA or RNA hydrolyzing enzyme. With an anti-S1-protein B antiserum that reacted with B2, C1 and D1, a cDNA clone, pS1-1, was obtained, which encoded a protein of 852 amino acids. The S1-1 protein, encoded within the cells by a mRNA of 3480 nt, was a novel protein and could be distinguished from the S1 proteins B, C and D by their amino acid sequences. The S-1-1 protein synthesized by in vitro translation bound to RNA homopolymers, with a preference for G and U polyribonucleotides and little for poly(A). The protein contained two tandem RNP motifs and several intriguing sequences, such as a novel repeat of five octamers with a consensus sequence DP-S(Q/G)YYY and a potentially perfect amphipathic alpha-helix of five turns with basic and acidic amino acids positioned in an ordered way. The two RNP motif sequences were similar, although homologies were low, to the RNP motif sequences of yeast NSR1 protein, animal nucleolins, Drosophila hnRNP Al and tobacco chloroplast RNP precursor protein, suggesting a functional uniqueness of the S1-1 protein in RNA metabolism and also the evolution of its RNP motif structure before plants and animals diverged. These results indicate that the S1-1 protein encoded by the cDNA is a new class of RNA binding protein.

A numerical model for the analysis and evaluation of global 137Cs fallout.

Fallout 137Cs from atmospheric nuclear detonation tests has been monitored worldwide since the late 1950's. The deviation and the correlation among these monitoring data were analyzed, and their surface deposition characteristics were estimated by the compartment model developed in this research. In the analysis, the scale of space (i.e., size of each compartment) and the degree of detail (i.e., number of compartments) were statistically determined using the global distribution data of 137Cs. The mathematical model was evaluated by comparing the numerically stimulated results with the fallout monitoring data including the 137Cs concentration in sea water. The major findings obtained in this research include that the deposition pattern of 137Cs is dependent on the latitude zone but not on the longitude, the mathematical model is promising for evaluating the dynamic performance of 137Cs in global atmospheric environment and its surface deposition, 137Cs is accumulated more in both the surface and deep ocean water of the North Pacific Ocean and the North Atlantic Ocean than that of other oceans, the 137Cs inventory is decreasing after the peak time in 1965, and the 137Cs inventory in the deep ocean water is decreasing more slowly than that in the surface ocean water.

Fine Structure of Spermatozoa of the Hagfish Eptatretus burgeri (Agnatha)1.

Live motile spermatozoa of the hagfish Eptatretus burgeri were obtained for the first time, and their fine structure was examined. The spermatozoon is characterized by an extremely long midpiece. Two of the four midpiece mitochondria are extensively elongated and extend through almost the entire length of the tail. The acrosome contains electron-dense and less dense materials in two different compartments. Amorphous subacrosomal material lies between the acrosome and the nucleus. No distinct perforatorium rod or filamentous structure was observed within the subacrosomal material. Two centrioles lie almost end to end in the nuclear fossa near the posterior end of the nucleus. The structure of the acrosomal complex in the hagfish, which is quite different from that in the lamprey, was compared to that of other chordates with respect to its function in sperm-egg interaction and phylogeny.