遗传性瞳孔异位小鼠及其突变基因的染色体定位

目的研究乙烷基亚硝基脲(ENU)诱变获得的遗传性瞳孔异位(B6-24#)小鼠的病变特征、遗传模式及突变基因在染色体上的位置。方法用瞳孔异常杂合子与正常C57BL/6(B6)小鼠配种,记录后代突变小鼠与正常小鼠数目,采用卡方检验确定遗传模式。繁殖[(B6×D2)×B6]F2及少量的[(B6×D2)F1×D2]F2突变小鼠,用平均分布于小鼠常染色体上的39个微卫星标记对突变基因进行染色体扫描,采用计算最大优势值(LODS)法判定突变基因与微卫星是否连锁并根据重组率计算突变基因的位置。结果初步诊断B6-24#为遗传性瞳孔异位,呈单基因显性遗传,外显率为85·71%。基因组扫描发现,微卫星D2Mit17与突变基因最大LODS为3·27,确定连锁。继续选用接近突变基因的D2Mit398、D2Mit259与其进行连锁分析,结果突变基因与D2Mit398、D2Mit259的重组率分别为5·45%±2·16%、0·90%±0·90%。突变基因被定位于距着丝粒约64·5cM处。在此区域附近未发现明显的候选基因。结论研究成功定位了小鼠瞳孔异位基因,有望为人类类似疾病提供一种新的小鼠模型。     

QTL analysis and genomewide mutagenesis in mice: complementary genetic approaches to the dissection of complex traits

Quantitative genetics and quantitative trait locus (QTL) mapping have undergone a revolution in the last decade. Progress in the next decade promises to be at least as rapid, and strategies for fine-mapping QTLs and identifying underlying genes will be radically revised. In this Commentary we address several key issues: first, we revisit a perennial challenge—how to identify individual genes and allelic variants underlying QTLs. We compare current practice and procedures in QTL analysis with novel methods and resources that are just now being introduced. We argue that there is no one standard of proof for showing QTL = gene; rather, evidence from several sources must be carefully assembled until there is only one reasonable conclusion. Second, we compare QTL analysis with whole-genome mutagenesis in mice and point out some of the strengths and weakness of both of these phenotype-driven methods. Finally, we explore the advantages and disadvantages of naturally occurring vs mutagen-induced polymorphisms. We argue that these two complementary genetic methods have much to offer in efforts to highlight genes and pathways most likely to influence the susceptibility and progression of common diseases in human populations.     

Perinatal Toxicology: Its Recognition and Fundamentals

Perinatal toxicology is the study of aberrant or toxic responses to environmental agents when exposure occurs from conception through the neonatal period. The current increased interest in perinatal toxicology reflects the concern of both society and the individual in the resultant deficits induced by exposure to agents in the workplace, home. environment and by therapeutic intervention during early development. Not only is differentiation during organogenesis (the first eight weeks in human gestation) a highly susceptible period to the induction of malformations, but the fetal/neonatal developmental phases are just as sensitive for certain developmental deficits. Long-term postnatal evaluations are in most instances critical for the documentation of fetal/neonatal functional defects, which include carcinoogenesis, behavioral impairment, endocrine and immune dysfunction, Ethylnitrosource, estrogens (diethylstilbestrol), and cadmium are discussed in relationship to specificity of their fetal effects, long-term follow-up, and possible mechanisms of toxic action.     

Exposure to Ethylnitrosourea Before Implantation Induces Congenital Malformations in Mouse Fetuses

Abstract The effects of ethylnitrosourea (ENU) on the development of preimplantation mouse embryos were investigated. ICR mice were treated intraperitoneally with single doses of 25, 50 or 100 mg ENU/kg body weight on day 0, 1, 2 or 3 of gestation, or with single doses of 25, 50 or 75 mg ENU/kg on day 8 of gestation. The uterine contents were examined on day 18 of gestation, and viable fetuses were inspected for external and skeletal malformations. No significant differences were observed in the number of implants between the ENU-treated groups on day 1, 2 or 3 of gestation and controls, while the number of implants in all of the groups treated with ENU on day 0 of gestation was significantly decreased compared to that in the control group. The frequencies of early postimplantation deaths were significantly increased in all of the groups treated with ENU on each gestational day before implantation, compared to the control frequencies. ENU treatment before implantation caused dose-dependent increases in the incidence of externally or skeletally malformed fetuses. Cleft palate, exencephaly and umbilical hernia were the most common types of external malformations in the groups treated with ENU before implantation and in the control group. The skeletal malformations seen in the ENU-treated groups were malformed vertebrae, malformed ribs, and bending of appendicular skeleton. Fused ribs was the most common skeletal malformation seen in the control fetuses. The type distributions of external and skeletal malformations induced by the treatment with ENU before implantation is quite different from those of fetal malformations induced by the treatment with ENU at the organogenesis stage. The results in the present study demonstrate that embryos before implantation in the uterus are susceptible with regard to the induction of congenital malformations by chemicals, and I propose that a large portion of the external malformations in fetuses treated at the preimplantation stages is the result of increased yields of spontaneously-occurring malformations.     

Comparison of methods for detecting mitomycin C- and ethyl nitrosourea-induced germ cell damage in mice: Sperm enzyme activities,sperm motility,testis weight

Testes weights, sperm motility and enzyme activities in single sperm were compared with respect to their ability to detect either developmental or mutational damage to germ cells. Male mice were injected i.p. with 2.5 mg/kg mitomycin C (MC) or 50 or 100 mg/kg ethylnitrosourea (ENU) or saline and were then killed at times such that sperm derived from treated vas sperm (SZ), spermatids (ST), preleptotene-late-spermatogonial cells (PLSG), spermatogonial cells (SG), or spermatogonial stem cells (SGS) could be evaluated. Testis weights decreased significantly as early as 1 wk after treatment, with the greatest decrease reached 3–4 wk after treatment, followed by recovery to normal levels 10–15 wk after treatment. We conclude that testis weight, which is easily obtained, is a sensitive indicator of germ cell damage by these agents. Sperm from each animal were evaluated for sperm motility, acrosin activity, succinic dehydrogenase (SDH) activity with or without the competitive inhibitor malonate or after exposure to 60°C for 10 min. The latter two assays were to detect sperm enzymes resistant to the inhibitor or heat. The presence of the acrosin protein was also detected immunologically. Sperm motility decreased most from treatment of PLSG and SG. After MC or ENU treatment, the greatest loss of acrosin activity and of the acrosin protein was also noted in sperm derived from treated PLSG and SG. MC and ENU failed to induce SDH activity in single sperm resistant to 60°C heat inhibition or to inhibition by malonate. Of the sperm assays, acrosin activity proved to be the most sensitive indicator of germ cell damage and was the simplest to measure.     

Supraependymal Cell Clusters Induced by ENU Treatment in Developing Rat Retina

ABSTRACT Supraependymal cell clusters in the retina induced by treatment with ethylnitrosourea in fetal and. neonatal rats are described. Following the early cytotoxic effects of this drug on the matrix layer, the supraependymal cell clusters began to form a protrusion from the matrix layer into the intraretinal space through the defective portion of the outer limiting membrane. The supraependymal cell clusters consisted of matrix cells and neurons, or matrix cells, neurons, photoreceptor-like cells, and glial cells depending on the time of treatment. The clusters became smaller in size, but they remained at least for 7 days after treatment. Our findings indicate that defect of the outer limiting membrane induced by the cytotoxic effect of ethylnitrosourea may be an essential factor for the formation of supraependymal cell clusters, and that the clusters can be regarded as a type of tissue malformation or dysontogenetic anomaly.     

Nerve growth factor and neural oncology

The precise role of the nerve growth factor protein (NGF) during the growth and development of the human nervous system is not determined. Although it appears to influence a number of neural functions, its mechanism of action is poorly understood. A number of researchers have proposed that NGF may be involved in several pathological conditions including cancer. It has been shown that NGF is secreted by certain sarcoma (23), neuroblastoma (113), and glioma (7,102,136) cell lines and can bind to neuroblastoma and metastatic melanoma cell lines (42). Neuroblastoma (136,181) and pheochromocytoma (165) cells in vitro can be induced by NGF to differentiate toward a morphologically "more benign" state and appropriate NGF treatment of rats can reduce the number of chemically induced gliomas and neurinomas (174,178). NGF can also reduce the growth of intracerebrally inoculated anaplastic glioma cells (172). Anti-NGF treatment of rats (178) and mice (179) can alter the tumor distribution observed following ethylnitrosourea or benzo(a)pyrene treatment (10). In humans, it has been reported that serum levels of NGF are usually elevated in persons "at risk" for neurofibromatosis (156). The precise nature of the NGF role is not known in these instances. Further understanding of the action of NGF could be of clinical importance.     

The Viracept-EMS case: Impact and outlook

This perspective first considers the potential impact of the Viracept-EMS case in the framework of the current understanding of the low-dose effects of DNA-reactive chemicals and the approaches used to estimate health risks from genotoxins occurring as impurities in pharmaceutical products or as contaminants in the environment or workplace. It also presents an outlook on the nature of additional research building upon the Viracept-EMS case to test assumptions underlying thresholded dose–response relationships and to establish biologically based risk assessment models in lieu of default models for DNA-reactive compounds.     

In vivo mutation in gene A of splenic lymphocytes from phiX174 transgenic mice

Single-burst analysis was applied to a forward assay for gene A mutation in splenic lymphocytes of phiX174 transgenic mice for the purpose of optimizing analytical parameters for identifying in vivo mutations. The effect of varying the cutoff value for an in vivo burst on induced mutant frequency, fold increase, and the significance of the difference between control and N-ethyl-N-nitrosourea (ENU)-treated mice was calculated by two different methods. The plating density was reduced to an average of less than 10 background mutant plaques per aliquot in order to separate in vitro bursts. The spectrum of mutations contributing < 60 plaques per aliquot from control animals was not significantly different from the control spectra from E. coli or transgenic phiX174 cells in culture. The mutant spectra from ENU-treated animals was highly different between mutant bursts of > 80 plaques per aliquot compared to mutations contributing < 60 plaques per aliquot (P < 0.000001), the former fitting the spectrum expected for ENU-induced mutations. The latter spectrum was also different from control animals and E. coli (P < 0.000001), suggesting the difference was caused by ex vivo mutation. With the mutations found in this study, the total number of reported target sites for gene A is now 33. The results support the interpretation that, in contrast to results for the lacI transgene, 100% of mutants isolated in gene A from control animals and cells were fixed in E. coli. We attribute the difference between the two genes to hot-spot sites for mutation in gene A and to a testable hypothesis that the mosaic plaque assay for the lacI transgene underestimates the frequency of ex vivo mutants.     

Distinctive expression of midkine in the repair period of rat brain during neurogenesis: immunohistochemical and immunoelectron microscopic observations

Distinctive expression of midkine (MK) was observed during the repair period of fetal brain neuroepithelium. MK is a heparin-binding growth factor that occurs as a product of a retinoic acid-inducible gene, and has a molecular mass of 13 kDa. MK expression was examined immunohistochemically and by immunoelectron microscopy during a period of repair in developing rat brain at the neurogenesis stage. Injury was induced in rat fetuses by transplacental administration of ethylnitrosourea (ENU) on embryonic Day (E) 16, and histological changes were examined up to 48 hr thereafter (i.e., up to E 18). In normal rat fetuses, MK immunostaining was observed in the cytoplasm and radial and horizontal processes of all cells in the neuroepithelium (NE), subventricular zone (SV), and intermediate zone (IMZ). In ENU-administered brains, cells in the NE, SV, and IMZ were damaged severely, especially 16-24 hr after ENU administration. The remaining neuroepithelial cells, with the exception of those in M-phase and the tips of processes at the ventricular surface, were negative for MK immunohistochemistry 16-24 hr after the administration of ENU. Forty-eight hours after the administration, the cytoplasm and processes of cells in the NE, SV, and IMZ were MK immunopositive. Our previous data reported that the cell cycle of most NE cells is synchronized to the S-phase 16 hr after ENU administration and to the M-phase at 24 hr, and many NE cells were recovered 48 hr after ENU administration. The previous results taken together with the present results indicate that: (1) MK expression does not increase during the repair period of the NE, being different from adults; (2) MK expression is likely to be suppressed at S-phase according to the condition of the NE; and (3) MK expression is not essential for every cell cycle phase of NE cells; but (4) is necessary to maintain the M-phase of NE cells.