Incorporation of biological information in cancer risk assessment: Example — Vinyl chloride

Vinyl chloride (VC) is used as an example to demonstrate how biological information can be incorporated into quantitative risk assessment. The information included is the pharmacokinetics of VC in animals and humans and the data-generated hypothesis that VC primarily affects the initiation stage of the multistage carcinogenesis. The emphasis in this paper is on the improvement of risk assessment methodology rather than the risk assessment of VC per se.Sufficient data are available to construct physiologically-based pharmacokinetic models for both animals and humans. These models are used to calculate the metabolized dose corresponding to exposure scenarios in animals and in humans.On the basis of the data on liver angiosarcomas and carcinomas in rats, the cancer risk per unit of metabolized dose is comparable, irrespective of routes (oral or inhalation) of exposure. The tumor response from an intermittent/partial lifetime exposure is shown to be consistent with that from a lifetime exposure when VC is assumed to affect the first (initiation) stage of the multistage carcinogenic process. Furthermore, the risk estimates calculated on the basis of animal data are shown to be consistent with the human experience.     

An eight-nucleotide sequence in the potato virus X 3' untranslated region is required for both host protein binding and viral multiplication.

Gel retardation and UV-cross-linking techniques were used to demonstrate that two tobacco proteins, with approximate molecular masses of 28 and 32 kDa, bind to a site within the 3' region of potato virus X (PVX) genomic RNA. The protein binding is specific, in that a 50-fold excess of unlabeled probe prevents formation of the complexes but no reduction is observed with a 2,000-fold molar excess of yeast tRNA. Complex formation is inhibited by poly(U) but is relatively unaffected by poly(A), poly(G), or poly(C-I). PVX RNA-host protein complex formation occurs in vitro at salt concentrations up to 400 mM. Deletion mapping indicates that the proteins bind within the 3' untranslated region (UTR) of PVX genomic RNA and that an 8-nucleotide U-rich sequence (5'-UAUUUUCU) is required for the binding. Deletion of the 8-nucleotide U-rich region from the 3' UTR of a sensitive PVX reporter virus that carries the luciferase gene in place of the PVX coat protein gene results in a more than 70,000-fold reduction in luciferase expression in tobacco protoplasts. RNA probes carrying the sequence GCGC in place of the central four contiguous uridines of the 8-nucleotide U-rich motif fail to bind host protein at detectable levels, and the same mutation, when introduced into the PVX reporter virus, eliminates viral multiplication. Mutations of 1 or 2 nucleotides within the same four uridines reduced both binding of host proteins and replication of reporter virus. These results indicate that the 8-nucleotide U-rich motif within the PVX 3' UTR is important for some aspect of viral multiplication and suggest that host protein binding plays a role in the process.     

Origin and phylogeny of Oryza species with the CD genome based on multiple-gene sequence data

The CD genome species in the genus Oryza are endemic to Latin America, including O. alta, O. grandiglumis and O. latifolia. Origins and phylogenetic relationship of these species have long been in dispute and are still ambiguous due to their homogeneous genome type, similar morphological characteristics and overlapping distribution. In the present study, we sequenced two chloroplast fragments (matK and trnL-trnF) and portions of three nuclear genes (Adh1, Adh2 and GPA1) from sixteen accessions representing seven species with the C, CD, and E genomes, as well as one G genome species as the outgroup. Phylogenetic analyses using parsimony and distance methods strongly supported that the CD genome originated from a single hybridization event, and that the C genome species (O. officinalis or O. rhizomatis instead of O. eichingeri) served as the maternal parent while the E genome species (O. australiensis) was the paternal donor during the formation of CD genome. In addition, the consistent phylogenetic relationships among the CCDD species indicated that significant divergence existed between O. latifolia and the other two (O. alta and O. grandiglumis), which corroborated the suggestion of treating the latter two as a single species or as taxa within species.We thank Tao Sang of Michigan State University (East Lansing, USA) and Bao-rong Lu of Fudan University (Shanghai, China) for their encouragement and assistance. We are also grateful to the International Rice Research Institute (Manila, Philippines) for providing plant material for this study. This research was supported by the Chinese Academy of Sciences (kscxz-sw-101A), the National Natural Science Foundation of China (30025005) and the Program for Key International S & T Cooperation Project of P. R. China (2001CB711103).     

Tissue distribution and kininogen gene expression after acute-phase inflammation

A kinin-directed monoclonal antibody to kininogens has been developed by the fusion of murine myeloma cells with mouse splenocytes immunized with bradykinin-conjugated hemocyanin. The hybrid cells were screened by an enzyme-linked immunosorbent assay (ELISA) and a radioimmunoassay (RIA) for the secretion of antibodies to bradykinin. Ascitic fluids were produced and purified by a bradykinin-agarose affinity column. The monoclonal antibody (IgG1) bound to bradykinin, Lys-bradykinin, Met-Lys-bradykinin, and kininogens in ELISA. Further, this target-directed monoclonal antibody recognized purified low and high molecular weight bovine, human, or rat kininogens and T-kininogen in Western blotting. After turpentine-induced acute inflammation, rat kininogen levels increased dramatically in liver and serum as well as in the perfused pituitary, heart, lung, kidney, thymus, and other tissues, as identified by the kinin-directed kininogen antibody in Western blot analyses. The results were confirmed by measuring kinin equivalents of kininogens with a kinin RIA. During an induced inflammatory response, rat kininogens were localized immunohistochemically with the kinin-directed monoclonal antibody in parenchymal cells of liver, in acinar cells and some granular convoluted tubules of submandibular gland, and in the collecting tubules of kidney. Northern and cytoplasmic dot blot analyses using a kinin oligonucleotide probe showed that kininogen mRNA levels in liver but not in other tissues increase after turpentine-induced inflammation. The results indicated that rat kininogens are distributed in various tissues in addition to liver and only liver kininogen is induced by acute inflammation. The target-directed kininogen monoclonal antibody is a useful reagent for studying the structure, localization, and function of kininogens or any protein molecule containing the kinin moiety.     

Abundance of mRNAs encoding HMG1/HMG2 class high-mobility-group DNA-binding proteins are differentially regulated in cotyledons of Pharbitis nil

The abundance of an mRNA encoding an HMG1/2 protein from Pharbitis nil (HMG1) has been previously shown to be regulated by light and an endogenous rhythm in cotyledons. A second Pharbitis nil HMG cDNA (HMG2) was characterized. The sequence of HMG2 was 82% and 86% identical to HMG1 at the nucleotide and amino acid level, respectively. As with HMG1, HMG2 mRNA was detected in all vegetative tissues and was most abundant in roots. However, unlike HMG1, HMG2 mRNA abundance did not increase upon transfer of cotyledons to darkness and did not exhibit regulation by an endogenous circadian rhythm when maintained in continuous darkness over a 68 h period. Similarly, while the abundance of HMG1 mRNA during a dark period that induced photoperiodically controlled flowering was dramatically affected by brief light exposure (night break), this treatment had no effect on HMG2 mRNA abundance. Collectively, these data are consistent with a role of HMG1 in contributing to the circadian-regulated and/or dark-regulated gene expression with constitutive expression of HMG2 playing a housekeeping role in the general regulation of gene expression in Pharbitis nil cotyledons.     

Conformationally altered aortic myosin light chains

Aorta smooth myosin contains two types of light chain, LC20 and LC17, which fold together with the N-terminal region of each heavy chain to form the globular head region of myosin. We demonstrate an altered conformation of LC20 after its separation from heavy chain by high concentrations of urea, on the basis of the following evidende: 1) A polyclonal antibody against LC20 was not able to recognize this conformationally altered form; 2) Myosin reconstituted from heavy chains and urea-dissociated light chains exhibited extremely low ATPase activity. Circular dichroism unfolding profiles showed that light chains dissociated from heavy chains by SDS appeared to be more stable than those generated by urea dissociation.     

带γδ和αβ受体的T细胞（TCR）在人胎儿组织内分布的研究

本文用细胞免疫化学方法,在冰冻切片上,检测了胎儿不同组织和器官内带γδ和αβ受体的Ｔ细胞（ＴＣＲ）的分布,结果发现ＴＣＲ细胞的分布与,般Ｔ细胞不同,有相对固定的分布区,如胸腺内ＴＣＲ细胞主要分布在皮筋质交界处和髓质部;脾脏内的γδＴ主要位于边缘区,而αβＴ主要位于动脉周围淋巴鞘,在红髓和血窦两种细胞共存;淋巴结内只有少数ＴＣＲ细胞位于滤泡间或副皮质,滤泡内则未见。消化管内的ＴＣＲ细胞主要分布在小肠的固有膜,而胃、大肠和阑尾的固有膜内很少见;肝内ＴＣＲ细胞主要集中在血管和血窦周围;皮肤切片内的少数ＴＣＲ细胞见于真皮内,表皮基底层细胞内未见。这些细胞在胎儿期的免疫皮应及其生理功能还不清楚。     

γ—亚麻酸生产菌原生质体的制备及诱变条件的研究

将γ─亚麻酸生产菌Ｃｕｎｎｉｎｇｈａｍｅｌｌａｓｐｐ．８７６１进行原生质体化,并用物理方法进行诱变,使其在形态上产生了变异,γ─亚麻酸含量有所增加。本文报道了该菌株原生质体的制备,再生条件,以及诱变后形态变化,发酵性能。     

Kallikrein activation of a high molecular weight atrial peptide

Mammalian atrial extracts contain bioactive peptides that exert profound effects upon renal function and isolated smooth muscle preparations. Gel filtration chromatography of rat atrial extract separates the activity into two peaks having apparent molecular weights of 20,000 to 30,000 and less than 10,000. Mild proteolytic treatment (trypsin 1 U/ml) of the high molecular weight fraction enhances the smooth muscle relaxant activity of this fraction and concomitantly reduces the apparent molecular weight of this fraction to less than 10,000. In this report we show that urinary and submaxillary kallikrein enhances the activity of rat atrial extracts in a similar fashion. Pretreatment of the high molecular weight fraction with either kallikrein (1 microgram/ml) enhances the smooth muscle relaxant activity of this fraction. Similar treatment of the low molecular weight fraction had no effect. The enhancement of the bioactivity of the high molecular weight substance(s) by the kallikreins was abolished by aprotinin but was unaffected by soybean trypsin inhibitor. These results suggest that exogenous addition of tissue kallikrein activates a high molecular weight peptide by limited proteolysis. Analysis of the kallikrein-treated high molecular weight peptide fraction by gel filtration indicates that the biological activity comigrates with the low molecular weight peptides present in the original atrial extract.