Twenty-six-Week carcinogenicity study of chloroform in CB6F1 rasH2-transgenic mice

The carcinogenic potential of chloroform was evaluated in a short-term carcinogenicity testing system using CB6F1 rasH2-Tg (rasH2-Tg) mice. Chloroform was administered to rasH2-Tg males at doses of 28, 90, or 140 mg/kg and rasH2-Tg females at 24, 90, or 240 mg/kg by oral gavage for 26 weeks. Wild-type (non-Tg) male and female mice received doses of 140 mg/kg and 240 mg/kg, respectively. N-methyl-N-nitrosourea was administered to rasH2-Tg mice by single intraperitoneal injection (75 mg/kg) as a positive control. In both the rasH2-Tg and non-Tg mice, there was no significant increase in the incidence of neoplastic lesions by chloroform treatment. The incidence of hepatocellular foci in the rasH2- and non-Tg females receiving 240 mg/kg was increased. Forestomach tumors and malignant tumors occurred in most of the rasH2-mice in the positive control group. Swelling or vacuolation of hepatocytes, a toxic change induced by chloroform, occurred in both the rasH2-Tg and non-Tg mice. It is concluded that chloroform, a putative human noncarcinogen, did not show evidence of carcinogenic potential in the present study using rasH2-Tg mice. This study suggests that the rasH2-Tg mouse model may not be appropriate for detecting nongenotoxic carcinogens. However, the sensitivity of rasH2-Tg mice to nongenotoxic carcinogens should be assessed with consideration of the results from the other ILSI-HESI project studies.     

Organ design for generation and reception of CO: lessons from the liver

Carbon monoxide (CO) is synthesized in vivo by heme oxygenase. Although for many years CO had been regarded as potentially toxic waste, recent studies have indicated that it is a signaling molecule with important physiological functions. Nitric oxide (NO), another diatomic diffusible gas, is regarded as an established signaling molecule. Structural similarities between CO and NO have led many investigators to draw analogies between the two gaseous mediators. Whereas the NO signaling system has been well defined as to its receptor molecule, soluble guanylate cyclase, the CO system has been conceived to require further tuning with respect to identifying its receptor molecules and its downstream effectors. Furthermore, there has been little quantitative information to argue for a physiological role of CO in vivo. This review, therefore, focuses on recent developments on both physiologic and pathophysiologic roles of CO in the model of isolated perfused liver of rats where endogenous production of CO is actually estimated. This model has revealed that CO acts as an endogenous vasorelaxant in the liver and that effects of CO are at least in part cyclic GMP-dependent. It has also provided answers to many questions of hepatobiliary functions that had not been resolved because of the complexity introduced by the interplay between NO and CO.     

Molecular characterisation of glutamate dehydrogenase gene defects in Japanese patients with congenital hyperinsulinism/hyperammonaemia

Congenital hyperinsulinism and hyperammonaemia (CHH) is caused by dysregulation of glutamate dehydrogenase (GDH). We characterised the GDH gene in two Japanese patients with CHH. Patient 1 showed late-onset and mild hypoglycaemic episodes and mild hyperammonaemia, compared with patient 2. In GDH activity of lymphoblasts, patient 1 showed twofold higher basal GDH activity than control subjects and mild insensitivity for GTP inhibition. Patient 2 showed severe insensitivity for GTP inhibition, and similar allosteric stimulation by ADP in the controls. Genetic studies identified heterozygous and de novo L413V and G446D mutations in patients 1 and 2, respectively. COS cell expression study confirmed that both mutations were disease-causing gene. The insensitivity for GTP inhibition in L413V and G446D was emphasised in COS cell expression system as a result of the dosage effect of mutant GDH gene. L413V showed less impairment of GDH than G446D based on biochemical and genetic results, which was consistent with the clinical phenotype. Based on the structure of bovine GDH, G446D was located in GTP binding site of pivot helix and its surroundings, while L413V was located in alpha-helix of antenna-like structure. These different locations of mutations gave different effects on GDH enzyme. The antenna-like structure plays an important role in GDH activity.     

The loss of subtypic determinants in alleles, d/y or w/r, on hepatitis B surface antigen

Hepatitis B surface antigen possesses the group-specific determinant called a and one or another member from each of two pairs of allelic determinants, d and y as well as w and r, thereby creating the four major subtypes, adw, adr, ayw and ayr. In the sequence of major surface antigen polypeptides made of 226 amino acid residues, lysine or arginine at amino acid position 122 specifies d or y determinant, and lysine or arginine at position 160 specifies w or r determinant, respectively. By means of site-directed mutagenesis and expression of mutant genes in cultured cells, the mechanism for the loss of subtypic determinants on surface antigens was investigated at the molecular level. A rare sample of surface antigen of subtype ad, devoid of w or r determinant, had asparagine at position 160. When it was converted to lysine, the surface antigen of subtype adw was obtained. Two samples of surface antigen were subtyped as ar. They lacked d determinant, despite having lysine at position 122 which usually specified it. They differed from all reported sequences of surface antigen in amino acid 144 or 145. They displayed d determinant when amino acid 144 was converted from glutamic acid to aspartic acid, or when amino acid 145 was changed from alanine to glycine. These results indicate that the key amino acid residue at position 122 or 160 is indispensable for the expression of subtypic determinants and that some distant residues are also crucially involved in conforming them.     

Further characterization of memory T cells existing in a case of CD8 deficiency

CD8 deficiency is a rare primary immunodeficiency caused by a defect of ZAP-70, which plays a pivotal role in T cell activation. We previously reported the existence of memory phenotype-CD4+ T cells in a case of CD8 deficiency, which demonstrates that activation signals through ZAP-70 are not essential to the phenotypic conversion of T cells from "naive" to "memory." In this study, we further characterized CD45RO+ T cells in a CD8 deficient patient. We showed that the patient's CD45RO+ T cell population had a wide variety of T cell receptor Vbeta-chain gene usage, and contained few clonally expanded T cells, while many clonally expanded T cells were present in the memory T cell population of age-matched healthy children. These results suggest that various kinds of antigens were involved in the differentiation of the patient's T cells, and that the differentiation into memory T cells was not accompanied by profound T cell proliferation. Moreover, our findings confirmed that the patient's CD45RO+CD4+ T cells had acquired effector-cytokine producing ability, indicating that there exists an alternative activation pathway which is independent of ZAP-70 for the acquisition of effector-cytokine producing ability.     

Epithelioid leiomyosarcoma of the external deep soft tissue

Epithelioid leiomyosarcoma in the external deep soft tissue is extremely rare. Most epithelioid leiomyosarcomas occur in the uterus. We present a case of epithelioid leiomyosarcoma occurring in the muscle of the thigh of a 78-year-old man. Histologically, the tumor predominantly consisted of round or polygonal cells arranged in sheets with a focal spindle cell component. Immunohistochemical analysis revealed that the tumor cells expressed vimentin, alpha-smooth muscle actin, and alpha-sarcomeric actin. The tumor was negative for desmin, S100 protein, glial fibrillary acidic protein, pan-keratin, epithelial membrane antigen, CAM 5.2, HMB-45, leukocyte common antigen, factor VIII-associated antigen, and CD34. Electron microscopically, some tumor cells contained abundant actin-type filaments in their cytoplasm.     

Enkephalin-like immunoreactive neurons and fibers in the ventromedial hypothalamic nucleus

Enkephalin-like immunoreactive (ENK-IR) neurons and fibers in the rat ventromedial hypothalamic nucleus (VMH) were examined by light and electron microscopy using the peroxidase-antiperoxidase immunocytochemistry. There were groups of ENK-IR neurons present in the ventrolateral part of the VMH, and such neurons were scattered elsewhere. These neurons had perikarya 10-25 microns in diameter with moderately developed cell organelles and enfolded nuclei that were often distributed eccentrically placed in the cell. The perikarya and dendrites contained diffuse, large-cored vesicles (LCV) (60-230 nm with a predominance in the 60-80 nm). ENK-IR neurons received synaptic inputs on the soma and dendrites from unlabeled axonal boutons containing many small, clear vesicles and occasional LCV. The ultrastructural features of the ENK-IR cells in the VMH seemed to correspond to the "common cells" described by Millhouse. Dense ENK-IR fibers were distributed in this nucleus throughout the rostrocaudal and ventrodorsal areas. Axonal boutons containing numerous small, clear vesicles, and dispersed LCV generally made synaptic contacts with the cell bodies and dendrites of unlabeled neurons. The findings suggest that opioid peptides directly influence VMH neurons through synaptic contacts.