An inter-laboratory collaborative study by the Non-Genotoxic Carcinogen Study Group in Japan, on a cell transformation assay for tumour promoters using Bhas 42 cells

The Bhas promotion assay is a cell culture transformation assay designed as a sensitive and economical method for detecting the tumour-promoting activities of chemicals. In order to validate the transferability and applicability of this assay, an inter-laboratory collaborative study was conducted with the participation of 14 laboratories. After confirmation that these laboratories could obtain positive results with two tumour promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and lithocholic acid (LCA), 12 coded chemicals were assayed. Each chemical was tested in four laboratories. For eight chemicals, all four laboratories obtained consistent results, and for two of the other four chemicals, only one of the four laboratories showed inconsistent results. Thus, the rate of consistency was high. During the study, several issues were raised, each of which were analysed step-by-step, leading to revision of the protocol of the original assay. Among these issues were the importance of careful maintenance of mother cultures and the adoption of test concentrations for toxic chemicals. In addition, it is suggested that three different types of chemicals show positive promoting activity in the assay. Those designated as T-type induced extreme growth enhancement, and included TPA, mezerein, PDD and insulin. LCA and okadaic acid belonged to the L-type category, in which transformed foci were induced at concentrations showing growth-inhibition. In contrast, M-type chemicals, progesterone, catechol and sodium saccharin, induced foci at concentrations with little or slight growth inhibition. The fact that different types of chemicals similarly induce transformed foci in the Bhas promotion assay may provide clues for elucidating mechanisms of tumour promotion.     

Dosing-time-dependent differences in lipopolysaccharide-induced liver injury in rats

Dosing-time-dependent differences in lipopolysaccharide (LPS)-induced liver injury were examined in rats housed under a 12 h light:dark (LD) cycle. LPS (5 mg/kg) was intravenously injected into different groups of rats at 2, 14, or 20 h after light on (HALO). Elevations in serum liver enzymes after 14 HALO were significantly greater than those after 2 HALO. These parameters were lower in rats given LPS at 20 HALO, compared to 14 HALO. The number of polymorphonuclear cells (PMN) in the liver and the amount of hepatic myeloperoxidase activity, which reflects the number of PMN in liver tissues, was significantly greater in the 14 than in the 2 HALO group. In addition, hepatic interleukin-6 (IL-6) production in the 14 HALO group was enhanced compared to that in the 2 HALO trial. These results suggest that LPS-induced liver injury is greater during the early active than during the early resting period. Dosing-time-dependent variation in the accumulation of PMN in the liver and, potentially, subsequent IL-6 production in liver tissues might be involved in this phenomenon.     

Inhibitory mechanisms of highly purified vitamin B2 on the productions of proinflammatory cytokine and NO in endotoxin-induced shock in mice

Inhibitory effects of highly purified vitamin B2 (riboflavin-5'-sodium phosphate, >97%) on the interleukin (IL)-6, macrophage inflammatory protein (MIP)-2 and nitric oxide (NO) in LPS-induced shock mice were evaluated. Vitamin B2 at 20 mg/kg (protective effect on mice mortality induced by LPS), intravenously administered 6 h after LPS injection, significantly decreased the plasma elevated levels of IL-6 and MIP-2 at 9 and 12 h. In addition, vitamin B2 lowered the tissue concentration and the mRNA expression of IL-6 in lung and those of MIP-2 in liver at 9 h. Vitamin B2 also reduced concentration of MIP-2 concentration in lung, and inhibited mRNA expression in kidney, respectively. Vitamin B2 decreased the plasma elevated NO levels in accordance with a reduction in expression of inducible NO synthase (iNOS) both at 21 and 24 h. Accordingly, the reduction in elevated plasma cytokine levels and NO based on the inhibitory effect on local cytokine mRNA expression and iNOS would be responsible for the anti-septic effect of vitamin B2.     

Dual roles for DNA polymerase eta in homologous DNA recombination and translesion DNA synthesis

Chicken B lymphocyte precursors and DT40 cells diversify their immunoglobulin-variable (IgV) genes through homologous recombination (HR)-mediated Ig gene conversion. To identify DNA polymerases that are involved in Ig gene conversion, we created DT40 clones deficient in DNA polymerase eta (poleta), which, in humans, is defective in the variant form of xeroderma pigmentosum (XP-V). Poleta is an error-prone translesion DNA synthesis polymerase that can bypass UV damage-induced lesions and is involved in IgV hypermutation. Like XP-V cells, poleta-disrupted (poleta) clones exhibited hypersensitivity to UV. Remarkably, poleta cells showed a significant decrease in the frequency of both Ig gene conversion and double-strand break-induced HR when compared to wild-type cells, and these defects were reversed by complementation with human poleta. Our findings identify a DNA polymerase that carries out DNA synthesis for physiological HR and provides evidence that a single DNA polymerase can play multiple cellular roles.     

Enzymatic properties of rhea lysozyme

Rhea lysozyme was analyzed for its enzymatic properties both lytic and oligomer activities to reveal the structural and functional relationships of goose type lysozyme. Rhea lysozyme had the highest lytic activity at pH 6, followed by ostrich and goose at pH 5.5-6, whereas the optimum of cassowary was at pH 5. pH profile was correlated to the net charge of each molecule surface. On the other hand, the pH optimum for oligomer substrate was found to be pH 4, indicating the mechanism of rhea catalysis as a general acid. The time-course of the reaction was studied using beta-1,4-linked oligosaccharide of N-acetylglucosamine (GlcNAc) with a polymerization degree of n ((GlcNAc)n) (n=4, 5, and 6) as the substrate. This enzyme hydrolyzed (GlcNAc)6 in an endo-splitting manner, which produced (GlcNAc)3+(GlcNAc)3 predominating over that to (GlcNAc)2+ (GlcNAc)4. This indicates that the lysozyme hydrolyzed preferentially the third glycosidic linkage from the nonreducing end. Theoretical analysis has shown the highest rate constant value at 1.5 s-1 with (GlcNAc)6. This confirmed six substrate binding subsites as goose lysozyme (Honda, Y., and Fukamizo, T., Biochim. Biophys. Acta, 1388, 53-65 (1998)). The different binding free energy values for subsites B, C, F, and G from goose lysozyme might responsible for the amino acid substitutions, Asn122Ser and Phe123Met, located at the subsite B.     

Functions of family-22 carbohydrate-binding modules in Clostridium josui Xyn10A

Clostridium josui xylanase Xyn10A is a modular enzyme comprising two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module (CM), a family-9 CBM, and two S-layer homologous modules, consecutively from the N-terminus. To study the functions of the family-22 CBMs, truncated derivatives of Xyn10A were constructed: a recombinant CM polypeptide (rCM), a family-22 CBM polypeptide (rCBM), and a polypeptide composed of the family-22 CBMs and CM (rCBM-CM). Recombinant proteins were characterized by enzyme and binding assays. rCBM-CM showed the highest activity toward xylan and weak activity toward some polysaccharides such as barley beta-glucan and carboxymethyl-cellulose. Although rCBM showed an affinity for insoluble and soluble xylan as well as barley beta-glucan and Avicel in qualitative binding assays, removal of the CBMs negligibly affected the catalytic activity and thermostability of the CM.     

Unbiased estimation of relative reproductive success of different groups: evaluation and correction of bias caused by parentage assignment errors

Parentage assignment is widely applied to studies on mating systems, population dynamics and natural selection. However, little is known about the consequence of assignment errors, especially when some parents are not sampled. We investigated the effects of two types of error in parentage assignment, failing to assign a true parent (type A) and assigning an untrue parent (type B), on an estimate of the relative reproductive success (RRS) of two groups of parents. Employing a mathematical approach, we found that (i) when all parents are sampled, minimizing either type A or type B error insures the minimum bias on RRS, and (ii) when a large number of parents is not sampled, type B error substantially biases the estimated RRS towards one. Interestingly, however, (iii) when all parents were sampled and both error rates were moderately high, type A error biased the estimated RRS even more than type B error. We propose new methods to obtain an unbiased estimate of RRS and the number of offspring whose parents are not sampled (zW(z)), by correcting the error effects. Applying them to genotypic data from steelhead trout (Oncorhynchus mykiss), we illustrated how to estimate and control the assignment errors. In the data, we observed up to a 30% assignment error and a strong trade-off between the two types of error, depending on the stringency of the assignment decision criterion. We show that our methods can efficiently estimate an unbiased RRS and zW(z) regardless of assignment method, and how to maximize the statistical power to detect a difference in reproductive success between groups.     

Mechanism of proliferation arrest of embryonic cells of Xenopus by diterpene compounds

Three diterpene compounds isolated from the anti-cancer herbal medicine kansui, namely, kansuinin B, 20-OD-ingenol Z, and 20-OD-ingenol E, specifically inhibited the proliferation of isolated embryonic cells from Xenopus embryos. We conducted a cytologic study to determine the mechanism underlying the arrest of the cellular proliferation by these compounds. While kansuinin B and 20-OD-ingenol Z treatment decreased the cell numbers in the S phase and the M phase substages of the cell cycle, 20-OD-ingenol E inhibited mitosis.