Modelling Germline Mosaicism and Different New Mutation Rates Simultaneously for Appropriate Risk Calculations in Families with Duchenne Muscular Dystrophy

For several genetic diseases two biological phenomena have been recognised as important: germline mosaicism; and different new mutation rates in males and females depending on mutation type. Both principles have been investigated separately and their influence on risk estimation in families has been exemplified in the literature. The aim of this paper is to present a general model that includes mosaicism and different new mutation rates. Mosaicism is introduced by defining additional alleles at the disease locus in combination with adapted segregation rules. Taking Duchenne muscular dystrophy as an example, we derive the conditions which have to be fulfilled for a population in mutation selection equilibrium. Our approach describes the model at the population level and not in individual subjects. This has the advantage of being able to use well known algorithms for the calculation of likelihoods in pedigrees, and to include additional diagnostic information such as marker genotypes and carrier deletion test results. We demonstrate the impact of the new model on a typical pedigree. In families where the patient is not available, the distinction between point mutations and deletions is important, since often molecular diagnostic tests for females can only screen for deletions. Negative deletion test results can now be included in the risk calculations.     

Comparing histological data from different brains: Sources of error and strategies for minimizing them

The recent development of brain atlases with computer graphics templates, and of huge databases of neurohistochemical data on the internet, has forced a systematic re-examination of errors associated with comparing histological features between adjacent sections of the same brain, between brains treated in the same way, and between brains from groups treated in different ways. The long-term goal is to compare as accurately as possible a broad array of data from experimental brains within the framework of reference atlases. Main sources of error, each of which ideally should be measured and minimized, include intrinsic biological variation, linear and nonlinear distortion of histological sections, plane of section differences between each brain, section alignment problems, and sampling errors. These variables are discussed, along with approaches to error estimation and minimization in terms of a specific example—the distribution of neuroendocrine neurons in the rat paraventricular nucleus. Based on the strategy developed here, the main conclusion is that the best long-term solution is a high-resolution 3D computer graphics model of the brain that can be sliced in any plane and used as the framework for quantitative neuroanatomy, databases, knowledge management systems, and structure–function modeling. However, any approach to the automatic annotation of neuroanatomical data—relating its spatial distribution to a reference atlas—should deal systematically with these sources of error, which reduce localization reliability.     

应用HMM和加权距离判别法的真核基因识别程序研究

根据现代科学对基因的认识，应用了隐马尔科夫模型(HMM)的算法，以大量核酸序列为信息来源，通过计算机计算来寻找未知基因的大体位置；再通过基因的固有结构特征及密码子使用的偏向性，使用加权距离判别法来准确地定位基因，以图形及文本的形式输出，从而极大地方便了实验室的研究工作。考虑到基因的许多特征还不为人们所了解，而且不同物种之间基因结构又有一定的差异，所以还开发了程序自学习功能，不断地存储已知的基因，再据此改变一些已有固有数据，以便更好地适应和了解不同生物基因结构的特异性，更加准确地寻找未知基因的位置。     

脂肪来源间充质干细胞对大鼠肝移植的免疫调节作用

目的探讨大鼠脂肪来源间充质干细胞(MSC)对大鼠肝移植术后急性排斥反应的作用。方法分离、培养SD大鼠MSC,体外混合淋巴细胞培养(MLC)体系中,研究MSC对Wistar大鼠T细胞增殖的抑制作用。以SD与Wistar大鼠为供受体建立肝移植模型。随机分为MSC处理组与空白对照组,术后第7天检测肝功能、血清白细胞介素(IL)-2和白细胞介素(IL)-10水平、肝组织病理形态及肝细胞凋亡。结果体外MLC中,Wistar大鼠T细胞增殖明显受抑,抑制率为48.44%。实验组血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、总胆红素(TBIL)、IL-2、IL-10分别为(134.2±45.0)、(162.5±30.5)U/L、(30.6±5.4)μmol/L、(187.35±18.26)、(193.95±37.62)μg/L;对照组上述指标分别为(355.6±54.3)、(296.4±71.2)U/L、(145.7±28.6)μmoL/L、(295.73±57.15)、(75.12±11.23)μg/L,两组差异有统计学意义(P<0.05);病检提示实验组排斥反应较对照组明显减轻;脱氧脲核苷酸缺口末端标记(TUNEL)检测提示实验组肝细胞凋亡程度明显低于对照组(P<0.05)。结论供体来源MSC能明显抑制MLC体系中受体源T细胞的增殖,并能显著减轻大鼠肝移植术后急性排斥反应。     

Hepatic Disposition Characteristics of Electrically Charged Macromolecules in Rat in Vivo and in the Perfused Liver

The effect of electric charge on the hepatic disposition of macromolecules was studied in the rat. Charged derivatives of dextran (T-70) and bovine serum albumin (BSA), mitomycin C–dextran conjugates (MMC-D), and lactosaminated BSA (Lac-BSA) were employed as model macromolecules. After intravenous injection, cationic macromolecules were rapidly eliminated from plasma because of their extensive hepatic uptake, while anionic and neutral macromolecules were slowly eliminated. Cationic macromolecules were recovered from parenchymal and nonparenchymal hepatic cells at a cellular uptake (per unit cell number) ratio of 1.4–3.2, while that of Lac-BSA was 14. During liver perfusion using a single-pass constant infusion mode, cationic macromolecules were continuously extracted by the liver, with extraction ratios at steady-state (E _ss) ranging between 0.03 and 0.54, whereas anionic and neutral macromolecules were almost completely recovered in the outflow at steady state. The E _ss for cationized BSA (Cat-BSA) and cationic MMC-Dcat were concentration dependent and decreased at low temperatures and in the presence of colchicine and cytochalasin B. The possible participation of the internalization process in the uptake of cationic macromolecules by hepatocytes was suggested.     

Effect of the free radical scavenger MCI-186 on spinal cord reperfusion after transient ischemia in the rabbit

Objective: Paraplegia remains a serious complication of aortic operations. The production of free radicals during reperfusion after transient ischemia is believed to induce secondary spinal neuronal injury, resulting in paraplegia. The aim of the present study was to clarify the protective effect and method of administration of antioxidants on the neurological and histological outcome in the animal model for reperfusion injury after transient spinal cord ischemia. Methods: New Zealand white rabbits underwent surgical exposure of the abdominal aorta that was clamped for 15 minutes to achieve spinal cord ischemia. Group A animals received two 10 mg/kg doses of 3-methyl-l-phenyl-2-pyrazolin-5-one (MCI-186) at the time of release of the aortic clamp and 30 minutes later. In group B, MCI-186, 5 mg/kg, was given three times, at the time of aorta clamp release, 30 minutes and 12 hours later. In group C (control group), one dose of vehicle was administered. Neurological status was assessed using modified Tarlov’s score until 168 hours after operation. Spinal cord sections were examined microscopically to determine the extent of ischemic neuronal damage. Results: Groups A and B animals had better neurological function than group C (p(0.001). In contrast, group C animals exhibited paraplegia or paraparesis with marked neuronal necrosis. The number of surviving neurons within examined sections of the spinal cord was significantly greater in group B than in group C (p(0.001). Conclusion: In a 15-minute ischemia-reperfusion model using rabbits, systemic repetitious administration of MCI-186, a free radical scavenger, was found to have a protective effect on the spinal cord neurons both neurologically and histologically. We postulate that the drug minimizes the delayed neuronal cell death for reperfusion injury after transient ischemia by reducing the free radical molecules. Moreover, it was thought that we could protect delayed neuronal cell death more effectively by administering MCI-18612 hours later.     

制作缺血性中风气虚血瘀证大鼠模型的实验研究

目的 探索建立缺血性中风气虚血瘀证大鼠模型的制作方法和评价体系。     

两种隐匿阴茎大鼠模型的建立及比较

目的:建立稳定的大鼠隐匿阴茎模型,为探索阴茎包埋对海绵体结构和功能的影响提供实验动物模型。方法:90只2周龄雄性SD大鼠随机均分为A、B、C3组,A组采用阴茎根部内荷包缝合法,B组采用包皮折叠缝合法包埋阴茎,C组为假手术组。在180d内观察两种方法的包埋效果。结果:A组术后4只死于急性尿潴留,5只因尿道口周围软组织感染、皮肤破溃导致包埋失败,3只因包埋过松阴茎伸出;B组术后1只死于麻醉,2只死于急性尿潴留;因阴茎发育和勃起,A组有7只、B组有10只阴茎伸出;C组1只死于麻醉。A组和B组中其余大鼠均有较好的包埋效果,A组包埋成功率为36.7%,B组为56.7%,而且可以在实验中任何时候解除包埋。结论:包皮折叠缝合法和阴茎根部内荷包缝合法均能建立稳定的、且与人类隐匿阴茎自然病程较为一致的2周龄大鼠隐匿阴茎动物模型。     

Development of a Simple Model of Extrinsic Denervation of the Small Bowel in Mouse

Small bowel transplantation (SBT) is associated with poorly understood enteric dysfunction. The study of SBT in mice is hindered by the technical difficulty of orthotopic SBT in the mouse. Our aim was to develop an easy preparation of extrinsic denervation of the entire jejunoileum in mice as a model of orthotopic SBT. All neurolymphatic tissues accompanying the superior mesenteric artery (SMA) and vein (SMV) were ligated just distal to the middle colic vessels. The SMA and SMV were then stripped of investing adventitia, and the mesentery to jejunum and colon were transected radially. Jejunum and colon were not transected and reanastomosed. To confirm extrinsic denervation 1, 3, and 6 months later, segments of small bowel were stained for protein gene product 9.5 (PGP9.5) and tyrosine hydroxylase (TH). Tyrosine hydroxylase immunoreactive intensity was then quantified using a semiquantitative analysis. Immunohistochemical fluorescence showed persistence of PGP9.5 immunoreactivity confirming enteric nerves in jejunoileum; however, there was no TH immunoreactivity in jejunoileum in denervated mice despite the expected preservation of TH immunoreactivity in the still-innervated duodenum at 1 month. At 3 months, sparse immunoreactivity for TH was present, and by 6 months, reinnervation of TH-containing nerves appeared similar to controls. Quantification of intensity at each time-point further confirmed this trend. This technique in the mouse accomplishes a complete extrinsic denervation of jejunoileum early postoperatively (1 and 3 months); reinnervation occurs by 6 months. This is an easily learned murine model of orthotopic SBT. Presented at the American Gastroenterological Association during Digestive Disease Week in Los Angeles, CA, as a poster presentation on May 23 2006. Abstract published in GastroenterologyE 2006; 130:A604.