A new allele of Gli3 and a new mutation,circletail (Crc), resulting from a single transgenic experiment

While generating transgenic lines, transgene-linked mutations can occur, which are caused by an insertional mutation at a given locus. More rarely, mutations unlinked to the transgene insertion site are observed. In the process of generating a mouse overexpressing the enzyme tyrosinase, we have obtained one transgenic line that appears to carry a semidominant insertional mutation at the Gli3 (extra toes) locus, characterized by polydactyly and skeletal malformations. Additionally, the transgenic line contained a second mutation, Crc (circletail), which appears to be unlinked to the transgene insertion site. Heterozygous Crc mice are incompletely penetrant for a circled-tail phenotype, while all homozygous Crc/Crc mice die at birth of a severe neural tube defect (craniorachischisis). Anatomical evidence from a Crc/Crc; Gli3/+ fetus indicates that these two genes may interact.     

磁共振磁敏感加权成像在颅脑疾病中的应用研究

目的探讨磁敏感加权成像在脑部疾病中的临床应用价值。方法对65例临床疑是脑血管病变患者行常规T1WI、T2WI、DWI、SWI序列及增强T1WI、MRA,探讨SWI序列在显示小出血灶、小静脉及含铁血黄素、钙化等顺磁性物质的优越性。结果①海绵状血管瘤,SWI能鉴别出血与血管,发现更多的小出血灶;②动静脉畸形,SWI能够发现更多的细小静脉向大静脉引流;③急性脑梗死,SWI可发现小的出血灶;④脑肿瘤,SWI显示出小的引流静脉;⑤帕金森病,SWI能显示脑内多发异常低信号铁沉积。结论 SWI对低流量血管畸形、小静脉结构、多发细小出血以及铁钙沉积十分敏感,为常规MRI的重要补充,应用于中枢神经系统疾病的诊断和鉴别诊断。     

Pregnancy outcomes after maternal exposure to simvastatin and lovastatin

BACKGROUND: Our objective was to determine the frequency of adverse outcomes after maternal exposure to simvastatin and/or lovastatin during pregnancy in postmarketing experience. METHODS: We reviewed the Merck & Co., Inc. (West Point, PA) pharmacovigilance database for reports of exposure to simvastatin or lovastatin during pregnancy. The reports were classified as prospective (reported prior to pregnancy outcome) or retrospective (reported after pregnancy outcome) and were evaluated for timing of exposure, outcome, congenital anomalies, and other events. Outcome rates were calculated for prospective pregnancies. RESULTS: We identified 477 reports (386 prospective and 91 retrospective) with 225 prospective outcomes reported: 154 live born infants, 49 elective abortions, 18 spontaneous abortions, and 4 fetal deaths. Six congenital anomalies were reported: chromosomal translocation, trisomy 18, hypospadias, duodenal atresia, cleft lip, and skin tag. The rate of congenital anomalies (congenital anomalies/live births plus fetal deaths) was 3.8%, which is similar to the background population rate (3.2%; relative ratio, 1.21; 95% 1-sided upper confidence interval [CI], 2.02). There were 13 retrospective reports describing a range of congenital anomalies. No specific pattern of anomalies was identified in either the prospective or retrospective reports. Rates for other outcomes were similar to background rates. CONCLUSIONS: Although the number of reports was relatively small, there was no evidence of a notable increase in congenital anomalies in women exposed to simvastatin or lovastatin versus the general population. Greater reporting of congenital abnormalities in the retrospective cohort is not unexpected and may reflect a reporting bias. Drugs should be used during pregnancy only if the benefits outweigh the risks. Simvastatin and lovastatin remain contraindicated during pregnancy.     

Nicotine-induced embryonic malformations mediated by apoptosis from increasing intracellular calcium and oxidative stress

BACKGROUND: Tobacco smoking by women during pregnancy increases the risk of congenital birth defects in the infants. Among the smoke products, nicotine is believed to be the major teratogenic factor that perturbs embryonic development. However, the role of nicotine in embryonic malformations has not been addressed, and the mechanisms by which nicotine affects embryonic development remain to be delineated. METHODS: To investigate the effects of nicotine on early embryogenesis, murine embryos at embryonic day (E) 8.5 were dissected out of the uteri, cultured in a roller bottle system, and treated with nicotine (0.6-6 microM) or vehicle. Embryonic morphogenesis and growth were examined in terms of structural morphology and crown/rump length, respectively. Programmed cell death (apoptosis) was assessed using LysoTracker Red staining of whole mount embryos and TUNEL assay of tissue sections. Changes in intracellular calcium concentration ([Ca2+]i) and reactive oxygen species (ROS) production were assessed using fluorescent dyes (Flu-4, AM; H2DCFDA, respectively) under a confocal microscope. To further investigate the role of intracellular calcium and ROS in nicotine-induced embryopathy, embryos were treated with BAPTA-AM (2 microM) to inhibit [Ca2+]i elevation and ascorbic acid (vitamin C; 100 microg/ml) to scavenge ROS in presence of nicotine (6 microM). RESULTS: The embryos treated with nicotine in 3-6 microM were smaller than those treated with vehicle. Most of the embryos had open neural tube in the anterior (brain) regions. The embryos treated with 6 microM nicotine also exhibited severe defects in the posterior trunk, resembling caudal dysplasia. Excessive apoptosis was observed in the deformed structures. Significant increases in [Ca2+]i and ROS were seen in the tissues that had higher levels of apoptosis. Furthermore, inhibition of [Ca2+]i and scavenging of ROS significantly reduced embryonic malformation and apoptotic rates in the embryos. CONCLUSIONS: Nicotine affects embryonic development in a concentration-dependent manner. The nicotine-induced embryonic malformations are, in part, a result of excessive cell death. Nicotine increases [Ca2+]i and ROS level, which play a role in nicotine-induced embryonic apoptosis and malformations. These studies identify the molecular pathway of nicotine action in embryonic apoptosis and malformations, and provide a promising approach for ameliorating the teratogenic effects of nicotine.     

Teratological studies of prenatal exposure of mice to a 20 kHz sawtooth magnetic field

In order to evaluate the importance of gestational age in possible effects due to exposure to a 20 kHz sawtooth magnetic field, pregnant ICR mice at gestational 2.5-15.5 days post-coitus, which is the most sensitive stage for the induction of major congenital malformations, were exposed in a carrousel irradiator. The mice were exposed to a 20 kHz intermediate frequency (IF) sawtooth magnetic field had a 6.5 microT peak intensity for 8 h/day. The animals were sacrificed on the 18th day of gestation; and the fetuses were examined for mortality, growth retardation, changes in head size, and other morphological abnormalities. From the above conditions, it is concluded that the exposure to a 20 kHz sawtooth magnetic field with 6.5 microT peak intensity does not inflict any adverse effect on fetuses of pregnant mice.     

Polycomb homologs are involved in teratogenicity of valproic acid in mice

BACKGROUND: Valproic acid (VPA) is widely used to treat epilepsy and bipolar disorder and is also a potent teratogen, but its teratogenic mechanisms are unknown. We have attempted to describe a fundamental role of the Polycomb group (Pc-G) in VPA-induced transformations of the axial skeleton. METHODS: Pregnant NMRI mice were given a single subcutaneous injection of vehicle or VPA (800 mg/kg) on gestation day (GD) 8. The expression of genes encoding Polycomb and trithorax groups was measured by quantitative real-time RT-PCR using total RNA isolated from the embryos exposed to vehicle or VPA for 1, 3, and 6 hr. In addition, the use of two less teratogenic antiepileptic chemicals valpromide (VPD) and valnoctamide (VCD) provide reliable evidence to support the relationship between VPA teratogenicity and the Polycomb group. RESULTS: At a teratogenic level, VPA inhibits the expression of the Polycomb group genes, including Eed, Ezh2, Zfp144, Bmi1, Cbx2, Rnf2, and YY1 in the mouse embryos. In contrast, neither VPD nor VCD have significant effects on the expression of those genes affected by VPA. The trithorax group (trx-G) gene MLL, which is known to be required to maintain homeobox gene expression such as the Polycomb gene, is not affected by a teratogenic dose of VPA. CONCLUSIONS: We propose that, during embryonic development, VPA may affect the gene silencing pathway mediated by the Polycomb group complex. The epigenetic mechanism of VPA teratogenicity on anteroposterior patterning is suspected.     

Dog as a model in studies on human hereditary diseases and their gene therapy

During the last 15 years spectacular progress has been achieved in knowledge on the dog genome organization and the molecular background of hereditary diseases in this species. A majority of canine genetic diseases have their counterparts in humans and thus dogs are considered as a very important large animal model in human biomedicine. Among canine monogenic diseases with known causative gene mutations there are two large groups classified as retinal dystrophies and lysosomal storage diseases. Specific types of these diseases are usually diagnosed in a single or several breeds. A well known disorder, restricted to a single breed, is congenital stationary night blindness described in Briards. This disease is a counterpart of Leber amaurosis in children. On the other hand, one of the most common monogenic human diseases (Duchenne muscular dystrophy), has its canine counterparts in several breeds (e.g., the Golden retriever, Beagle and German short-haired pointer). For some of the canine diseases gene therapy strategy was successfully applied, e.g., for congenital stationary night blindness, rod-cone dystrophy and muccopolysaccharydoses type I, IIIB and VII. Since phenotypic variability between the breeds is exceptionally high, the dog is an interesting model to study the molecular background of congenital malformations (e.g., dwarfism and osteoporosis imperfecta). Also disorders of sexual development (DSD), especially testicular or ovotesticular DSD (78,XX; SRY-negative), which is widely distributed across dozens of breeds, are of particular interest. Studies on the genetic background of canine cancers, a major health problem in this species, are also quite advanced. On the other hand, genetic studies on canine counterparts of major human complex diseases (e.g., obesity, the metabolic syndrome and diabetes mellitus) are still in their infancy.     

Induced pluripotent stem (iPS) cells from human fetal stem cells (hFSCs)

Introduction

(1) Human embryonic stem (ES) cells are pluripotent but are difficult to be used for therapy because of immunological, oncological and ethical barriers. (2) Pluripotent cells exist in vivo, i.e., germ cells and epiblast cells but cannot be isolated without sacrificing the developing embryo. (3) Reprogramming to pluripotency is possible from adult cells using ectopic expression of OKSM and other integrative and non-integrative techniques. (4) Hurdles to overcome include i.e stability of the phenotype in relation to epigenetic memory.

Sources of data

We reviewed the literature related to reprogramming, pluripotency and fetal stem cells.

Areas of agreement

(1) Fetal stem cells present some advantageous characteristics compared with their neonatal and postnatal counterparts, with regards to cell size, growth kinetics, and differentiation potential, as well as in vivo tissue repair capacity. (2) Amniotic fluid stem cells are more easily reprogrammed to pluripotency than adult fibroblast. (3) The parental population is heterogeneous and present an intermediate phenotype between ES and adult somatic stem cells, expressing markers of both.

Areas of controversy

(1) It is unclear whether induced pluripotent stem (iPS) derived from amniotic fluid stem cells are fully or partially reprogrammed. (2) Optimal protocols to ensure highest efficiency and phenotype stability remains to be determined. (3) The “level” of reprogramming, fully vs partial, of iPS derived from amniotic fluid stem cells remain to be determined.

Growing points

Banking of fully reprogrammed cells may be important both for (1) autologous and allogenic applications in medicine, and (2) disease modeling.