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Obesity is a metabolic disorder associated with many diseases. WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is a HECT-type E3 ubiquitin ligase involved in several diseases. Recently, we found that the level of WWP1 is increased in white adipose tissue in a mouse model of obesity and that obese Wwp1 knockout (KO) mice exhibit improved whole-body glucose metabolism. Here, to determine which insulin-sensitive tissues contribute to this phenotype, we investigated the levels of several insulin signaling markers in white adipose tissue, liver, and skeletal muscle of Wwp1 KO mice, which were fed a normal or high-fat diet and transiently treated with insulin. In obese Wwp1 KO mice, phosphorylated Akt levels were increased in the liver but not in white adipose tissue or skeletal muscle. Moreover, the weight and triglyceride content of the liver of obese Wwp1 KO mice were decreased. These results suggest that systemic deletion of WWP1 improves glucose metabolism via enhanced hepatic insulin signaling and suppressed hepatic fat accumulation. In summary, WWP1 participates in obesity-related metabolic dysfunction and pathologies related to hepatic steatosis via suppressed insulin signaling., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The scanning electron microscope (SEM) has been reassembled into a new type of cryo-electron microscope (cryo-TSEM) by installing a new cryo-transfer holder and anti-contamination trap, which allowed simultaneous acquisition of both transmission images (STEM images) and surface images (SEM images) in the frozen state. The ultimate temperatures of the holder and the trap reached - 190 °C and - 210 °C, respectively, by applying a liquid nitrogen slush. The STEM images at 30 kV were comparable to, or superior to, the images acquired with conventional transmission electron microscope (100 kV TEM) in contrast and sharpness. The unroofing method was used to observe membrane cytoskeletons instead of the frozen section and the FIB methods. Deep sublimation of ice surrounding unroofed cells by regulating temperature enabled to emerge intracellular fine structures in thick frozen cells. Hence, fine structures in the vicinity of the cell membrane such as the cytoskeleton, polyribosome chains and endoplasmic reticulum (ER) became visible. The ER was distributed as a wide, flat structure beneath the cell membrane, forming a large spatial network with tubular ER., (© 2021. The Author(s).)

Boiling-water extract of Korean-Saiko (Bupleuri Radix, from South Korea, Bupleurum falcatum L.) enhanced the mutagenic activity of Trp-P-1, Trp-P-2 and benzo[a]pyrene with S9mix. The boiling-water extract was fractionated with ether and then n-BuOH. Both the ether and the n-BuOH fractions also enhanced mutagenicity of Trp-P-1, respectively. The n-BuOH fraction was separated into seven fractions by silica gel chromatography and the chloroform eluate had the strongest enhancing effect on the mutagenic activity of Trp-P-1 with S9mix. The chloroform eluate fraction was further separated into five spots by thin-layer chromatography. Two of the spots had the strongest enhancing effect on the mutagenic activity of Trp-P-1. Since saikosaponin is a well known component in Bupleuri Radix, the effects of its existence were tested and saikosaponins a and c were found. The enhancement activity of saikosaponin a was very weak. The effective components are now being studied.

The toxicity of xanthene dyes were studied by various interaction between the dyes and the components in vital body. (1) An increase in the amount of Rose Bengale adsorbed on the gill of fish was followed by the increase in red corpuscle number, and it was assumed that the death of fish in xanthene dye solution was due to anoxemia. (2) Binding capacity of xanthene dyes with bovine serum albumin decreased in the following; Rose Bengale, Phloxine, Erythrosine, Eosine and Uranine. This order was quite coincident with the toxicity compared by TLm values. (3) From the results of rec-assay test by use of Bacillus subtilis, it was confirmed that Phloxine and Rose Bengale had DNA-damaging capacity related to the mutagenecity.

Reason of acute toxicity to fish caused by coal-tar dyes and their industrial effluents were studied from the three points of view. 1) Partition coefficient (n-octanol/water) was determined on several coal-tar dyes, and it was found that the larger the ratio was, the stronger was toxicity to fish. 2) As a result of determination about existent form of xanthene dyes under photo-irradiation, it was clarified that dyes in solution were in the form of excited triplet state and halogen in dye molecule was liberated in free form. 3) When coal-tar dyes were treated with sodium hypochlorite, harmful chloroform was detected from dye solution. Chloroform and its homologous compounds showed the low TLm value and had the serious effect on fish.

Biological tests using Orizias latipes (LC50 and oxygen uptake test), Moina macrocopa (LC50), and Dugesia japonica (head regeneration test and LC50) were carried out in order to clarify the mutual relationship of these test methods. The oxygen uptake rate of O. latipes was not effective to assess chemical toxicity. Adding the results of the growth inhibition test of Tetrahymena pyriformis (Yoshioka, Y., Ose, Y., and Sato, T. (1985). Sci. Total Environ. 43, 149-157), the correlation coefficients between each two test methods were calculated. The test results except EC50 and LC50 of D. japonica showed a good relation to each other. We determined the solubility and the n-octanol/water partition coefficient (P) of some chemicals used in the test. Log P interpreted the toxicity in mol/liter unit but not in mg/liter. Solubility was not a useful descripter neither in mol/liter nor in mg/liter unit.

The acute toxicity of photodecomposed products of Erythrosine, Eosine, Phloxine and Rose Bengale were studied, since it was found that toxicity of these dyes to fish increased after the dyes had been photoirradiated. Photodecomposed products of the dyes were isolated and identified with UV, IR, NMR spectra and the acute toxicity of those compounds were determined by TLm test. As results of these studies, it became clear that the toxicity of photodecomposed organic products (dehalogenated compounds of dyes) were lower than the mother compounds. The increases in toxicity of the xanthene dyes by photo-irradiation were attributed to the liberated halogens by irradiation.

Biochemical degradation test of food coal-tar dyes using sludge was studied in order to evaluate their safety. It was found that some dyes were little decomposed under aerobic condition but four azo dyes were readily decomposed under anaerobic condition. These were Food Yellow No. 4, No. 5, and Food Red No. 2, No. 102. Decomposed products of these four azo dyes by sludge under anaerobic condition were identified as sulfonilic, naphthionic acids and so on.

Twenty kinds of coal-tar dyes were subjected to median tolerance limit (TLm) test by use of Himedaka (Oryzias latipes) for the comparision of their acute toxicities. It became clear that 4 kinds of halogens substituted xanthene compounds dyes showed strong acute toxicities. From the fact that uranine had the lower acute toxicity than halogens substituted compounds and the toxicities of these 4 dyes increased through irradiation, it was assumed that halogen atoms in dyes might be responsible for these strong acute toxicities to fish.

The test method of "activated sludge, respiration inhibition test" proposed by the OECD was critically carried out and compared with other test methods. Investigation of test conditions showed that the moderate deviation from the test conditions defined by the OECD Test Guidelines did not have much effect on the result, and some modifications were proposed to improve the method. This method had a poor detection limit compared with the LC50 test with Oryzias latipes and EC50 of the growth inhibition test with Tetrahymena pyriformis. The susceptivity of the method was particularly poor for the chemicals which were highly toxic in the other two tests.

Forty preparations of the extracts from 28 kinds of Asian herbs were tested for ability to inhibit the activities of murine retroviral reverse transcriptase and human deoxyribonucleic acid (DNA) polymerases. Among the 40 extracts, 35 inhibited reverse transcriptase activity and 29 inhibited DNA polymerase alpha activity. The inhibitory potencies of these extracts were expressed as the 50% inhibition concentrations (IC50), at which the enzyme activities were inhibited by 50%. Very strong inhibitions were observed with the extracts from Millettia pachycarpa (Leguminosae) and Mallotus apelta (Euphorbiaceae) as shown by their low IC50 values for reverse transcriptase (0.4-0.5 micrograms/ml) and DNA polymerase alpha (0.9-1.4 micrograms/ml). Enzyme kinetic analysis revealed that the mode of inhibition of reverse transcriptase by these two extracts was competitive with respect to the template.primer [poly(rA).oligo(dT)] and noncompetitive with respect to deoxythymidine triphosphate (dTTP) substrate. Besides reverse transcriptase and DNA polymerase alpha, DNA polymerase I and ribonucleic acid (RNA) polymerase from E. coli were inhibited by these two extracts. These results indicate that the herb extracts contain as yet unidentified substance(s) which inhibit the activities of reverse transcriptase and cellular DNA polymerases.