Isolation and characterization of diploid clones from adult and newborn rat liver cell lines.

A high frequency of diploid and near-diploid clones were developed from cell lines derived from adult and newborn rat liver using micropipettes. There were some differences in morphology, biochemical properties and growth rate between clones. Cloned cells had low levels of tyrosine transaminase activity, glucose-6-phosphatase activity and albumin content. A diploid clone and pseudodiploid clone derived from adult rat liver cell line were positive for alpha-fetoprotein.     

Effect of 3′-methyl-4-dimethylaminoazobenzene in the induction of malignant transformation and of 8-azaguanine-resistant mutations and chromosomal aberrations in a diploid clone derived from normal rat liver cells in culture

Summary The effect of 3′methyl-4-dimethylaminoazobenzene (3′-Me-DAB) in the induction of malignant transformation and of 8-azaguanine-resistant mutations and chromosomal aberrations was studied in a diploid strain derived from normal rat liver cells. The cells were malignantly transformed by treatment with 3′-Me-DAB 1.7 μg/ml for 130 to 221 d or 1.7 μg/ml for 53 d followed by 24.9 μg/ml for 27 to 77 d. The untreated control cells did not transform spontaneously until the 232nd d in culture. Some properties of the 3′-Me-DAB-treated cells were compared to those of untreated control cells but no reliable marker for predicting the tumorigenic potential of the cells was found. The single addition of 3′-Me-DAB caused little induction of 8-azaguanine-resistant mutations and chromosomal aberrations to the cells. However, mutations and chromosomal aberrations were significantly induced byN-acetoxy-4-methylaminoazobenzene, an active metabolite of 4-dimethylaminoazobenzene or 3′-Me-DAB in the presence of liver microsomes. This study was supported by a grant for cancer research from the Japanese Ministry of Education.     

Degradation of Polycarbonate by a Polyester-Degrading Strain, Amycolatopsis sp. Strain HT-6

Amycolatopsis sp. strain HT-6, a poly(tetramethylene succinate) (PTMS)-degrading actinomycete, was observed to degrade poly(tetramethylene carbonate) (PTMC). In a liquid culture with 150 mg of PTMC film, 59% degradation was achieved, but with a low yield of cell growth. On the other hand, PTMS copolymerized with a small amount of PTMC, forming a copolyester carbonate (PEC) that was completely and rapidly degraded with a high yield of cell growth.     

Photosynthetic accumulation of poly-(hydroxybutyrate) by cyanobacteria--the metabolism and potential for CO2 recycling.

Regulatory mechanism in PHB [poly-(hydroxybutyrate)] accumulation by cyanobacteria, especially by a thermophilic isolate, Synechococcus MA19 was reviewed in comparison with a genetically engineered strain. The strain, MA19 accumulates PHB under nitrogen starved and photoautotrophic conditions (MA19-N). Little PHB synthase activity was detected in crude extracts from the cells grown in nitrogen sufficient conditions (MA19 + N). The activity was detected exclusively in membrane fractions from MA19 + N. The change of the enzyme activity was insensitive to chloramphenicol, which suggests post-translational activation. In vitro, acetyl phosphate activated PHB synthase in membrane fractions from MA19 + N, and the extent of activation depended on the concentration of acetyl phosphate. Phosphotransacetylase which catalyzes the conversion of acetyl-CoA to acetyl phosphate was detected in crude extracts from MA19-N but not in those from MA19 + N. These results suggested that intracellular acetyl phosphate concentration could be controlled, depending on C-N balance and intracellular acetyl-CoA concentration. On the contrary, in genetically-engineered cyanobacterium (transformant with PHB synthesizing genes from Ralstonia eutropha), it did not seem to be PHB synthase but acetyl-CoA flux that limits PHB synthesis. The closer association of PHB granules with thylakoid membranes in MA19 is suggested than that in the genetically-engineered cyanobacterium, which may reflect the difference of distribution of PHB synthase. Transposon-mutagenesis was used to acquire mutants of its altered PHB regulatory mechanism. PHA production by cyanobacteria was considered from the aspects of photobioreactors.     

Primary culture of liver cancer tissues with or without transcatheter arterial embolization and establishment of a cell strain.

More epithelial-like cells are found in primary cultures of transcathetel arterial embolization (TAE)-untreated human liver cancer tissues than in those of TAE-treated tissues. A new cell strain, HUH-33, established from the former, grows slowly and is untransplantable into nude mice and secretes alpha-fetoprotein, albumin and some other tumor markers. Chromosome numbers are widely distributed. HUH-33 expresses hepatocyte type keratin and contains desmosome- or intermediate filament bundle-like structures.     

Properties of a bacterium which degrades solid poly(tetramethylene succinate)-co-adipate, a biodegradable plastic

Various microorganisms were screened for their ability to degrade poly(tetramethylene succinate)-co-(tetramethylene adipate) (PBSA). Strain BS-3, which was newly isolated from a soil sample, was selected as the best strain. From taxonomical studies, the strain was tentatively ascribed to belong to the genus Acidovorax, most probably to the species A. delafieldii. Strain BS-3 could degrade both solid and emulsified PBSA, and also emulsified poly(tetramethylene succinate). During the degradation, a lipase activity was observed in the culture broth. This lipase activity was induced more strongly by PBSA than by tributyrin or triolein which are typical substrates of lipase. These observations strongly suggest that this lipase was involved in the PBSA biodegradation in strain BS-3.     

Minimal Internal Radiation Exposure in Residents Living South of the Fukushima Daiichi Nuclear Power Plant Disaster

Following the Fukushima nuclear power plant disaster, assessment of internal radiation exposure was indispensable to predict radiation-related health threats to residents of neighboring areas. Although many evaluations of internal radiation in residents living north and west of the crippled Fukushima nuclear power plant are available, there is little information on residents living in areas south of the plant, which were similarly affected by radio-contamination from the disaster. To assess the internal radio-contamination in residents living in affected areas to the south of the plant or who were evacuated into Iwaki city, a whole body counter (WBC) screening program of internal radio-contamination was performed on visitors to the Jyoban hospital in Iwaki city, which experienced less contamination than southern areas adjacent to the nuclear plant. The study included 9,206 volunteer subjects, of whom 6,446 were schoolchildren aged 4–15 years. Measurements began one year after the incident and were carried out over the course of two years. Early in the screening period only two schoolchildren showed Cs-137 levels that were over the detection limit (250 Bq/body), although their Cs-134 levels were below the detection limit (220 Bq/body). Among the 2,760 adults tested, 35 (1.3%) had detectable internal radio-contamination, but only for Cs-137 (range: 250 Bq/body to 859 Bq/body), and not Cs-134. Of these 35 subjects, nearly all (34/35) showed elevated Cs-137 levels only during the first year of the screening. With the exception of potassium 40, no other radionuclides were detected during the screening period. The maximum annual effective dose calculated from the detected Cs-137 levels was 0.029 and 0.028 mSv/year for the schoolchildren and adults, respectively, which is far below the 1 mSv/year limit set by the government of Japan. Although the data for radiation exposure during the most critical first year after the incident are unavailable due to a lack of systemic measurements, the present results suggest that internal radio-contamination levels more than one year after the incident were minimal for residents living south of the crippled Fukushima nuclear plant, and that the annual additional effective doses derived from internal Cs contamination were negligible. Thus, internal radio-contamination of residents living in southern radio-contaminated areas appears to be generally well controlled.     

Imaging mass spectrometry reveals characteristic changes in triglyceride and phospholipid species in regenerating mouse liver

After partial hepatectomy (PH), regenerating liver accumulates unknown lipid species. Here, we analyzed lipids in murine liver and adipose tissues following PH by thin-layer chromatography (TLC), imaging mass spectrometry (IMS), and real-time RT-PCR. In liver, IMS revealed that a single TLC band comprised major 19 TG species. Similarly, IMS showed a single phospholipid TLC band to be major 13 species. In adipose tissues, PH induced changes to expression of genes regulating lipid metabolism. Finally, IMS of phosphatidylcholine species demonstrated distribution gradients in lobules that resembled hepatic zonation. IMS is thus a novel and power tool for analyzing lipid species with high resolution.     

Binding of NAD+ and l-Threonine Induces Stepwise Structural and Flexibility Changes in Cupriavidus necator l-Threonine Dehydrogenase

Crystal structures of short chain dehydrogenase-like l-threonine dehydrogenase from Cupriavidus necator (CnThrDH) in the apo and holo forms were determined at 2.25 and 2.5 Å, respectively. Structural comparison between the apo and holo forms revealed that four regions of CnThrDH adopted flexible conformations when neither NAD⁺ nor l-Thr were bound: residues 38–59, residues 77–87, residues 180–186, and the catalytic domain. Molecular dynamics simulations performed at the 50-ns time scale revealed that three of these regions remained flexible when NAD⁺ was bound to CnThrDH: residues 80–87, residues 180–186, and the catalytic domain. Molecular dynamics simulations also indicated that the structure of CnThrDH changed from a closed form to an open form upon NAD⁺ binding. The newly formed cleft in the open form may function as a conduit for substrate entry and product exit. These computational results led us to hypothesize that the CnThrDH reaction progresses by switching between the closed and open forms. Enzyme kinetics parameters of the L80G, G184A, and T186N variants also supported this prediction: the k_cat/K_{m, l-Thr} value of the variants was >330-fold lower than that of the wild type; this decrease suggested that the variants mostly adopt the open form when l-Thr is bound to the active site. These results are summarized in a schematic model of the stepwise changes in flexibility and structure that occur in CnThrDH upon binding of NAD⁺ and l-Thr. This demonstrates that the dynamical structural changes of short chain dehydrogenase-like l-threonine dehydrogenase are important for the reactivity and specificity of the enzyme.