Germline mutations of the MEN1 gene in familial multiple endocrine neoplasia type 1 and related states

Familial multiple endocrine neoplasia type 1 (FMEN1) is an autosomal dominant trait characterized by tumors of the parathyroids, gastro- intestinal endocrine tissue, anterior pituitary and other tissues. We recently cloned the MEN1 gene and confirmed its identity by finding mutations in FMEN1. We have now extended our mutation analysis to 34 more unrelated FMEN1 probands and to two related states, sporadic MEN1 and familial hyperparathyroidism. There was a high prevalence of heterozygous germline MEN1 mutations in sporadic MEN1 (8/11 cases) and in FMEN1 (47/50 probands). One case of sporadic MEN1 was proven to be a new MEN1 mutation. Eight different mutations were observed more than once in FMEN1. Forty different mutations (32 FMEN1 and eight sporadic MEN1) were distributed across the MEN1 gene. Most predicted loss of function of the encoded menin protein, supporting the prediction that MEN1 is a tumor suppressor gene. No MEN1 germline mutation was found in five probands with familial hyperparathyroidism, suggesting that familial hyperparathyroidism often is caused by mutation in another gene or gene(s).      

The 5′-terminal 32 basepairs conserved between genome segments A and B contain a major promoter element of infectious bursal disease virus

Summary.  The regions of the infectious bursal disease virus (IBDV) genome with regulatory function are not known. In the present study, progressively deleted lengths of the 5′ noncoding region of segment A were constructed in pGL3 vectors having SV40 enhancer or promoter, and a luciferase (LUC) reporter gene. Transient transfections of the constructs made in a promoter-less pGL3-Enhancer vector when transfected in Vero cells and the lysates assayed for LUC expression, allowed the localization of maximal activity to the 32-nucleotide stretch (precursor polyprotein ORF positions −131 to −100), which is highly conserved at the 5′ end of both genome segments. This fragment, when evaluated in parallel in an enhancer-less pGL3-Promoter vector demonstrated no activity. To determine if this region is recognized by IBDV replicative proteins, we engineered modifications in an enhancer-less pGL3-Promoter vector where the terminal 32-bp fragment, the full-length noncoding region, or the noncodig region with the 32-bp fragment deleted was positioned in either the plus-sense or the minus-sense orientation immediately downstream of the SV40 promoter and upstream of the LUC gene. Transfections of these constructs in IBDV-infected and uninfected Vero cells resulted in the endogenous generation of recombinant viral-LUC RNAs containing the 5′ terminal viral RNA sequences in either the plus-sense or the minus-sense orientation. LUC assays of the infected cell lysates showed up-regulated expression of LUC only with constructs containing the 32-bp fragment in the minus-sense orientation. Deletion of this 32-bp fragment abolished such LUC expression. We therefore conclude that the 5′-terminal 32 base pairs of genomic segment A contain a major promoter element in IBDV. In addition, our results show that IBDV replicative proteins recognize and transcribe single-stranded RNA in vivo. Accepted June 25, 1997 Received May 14, 1997     

Simple and Rapid Method for Production of Whole-Virus Antigen for Serodiagnosis of Caprine Arthritis-Encephalitis Virus by Enzyme-Linked Immunosorbent Assay

Polyethylene glycol (PEG) was used to produce whole-virus antigen derived from tissue culture cells infected with a Canadian strain of caprine arthritis-encephalitis virus. PEG antigen batches were obtained after precipitation and concentration of infected tissue culture material with PEG 8000 and final treatment with sodium dodecyl sulfate. The optimum time of harvest of tissue culture extracted material to produce the maximum amount of viral proteins was determined in roller bottles, after cocultivation of infected and noninfected fetal lamb corneal cells. Samples from day 9 to day 25 postculture were collected and processed. By Western blotting, the optimum time of harvest was found to be day 25 following the coculture. Two large batches of PEG antigen were prepared at the optimum time of harvest. Both batches gave similar results when tested by Western blotting and enzyme-linked immunosorbent assay (ELISA), using reference control sera from infected and noninfected goats. For further testing in ELISA, cutoff values and ratios were determined for PEG batch 1, using 200 known serum samples from goats free of the disease. The PEG antigen batch was compared with an in-house ELISA antigen in a kinetic mode, using 498 serum samples from field goats. The in-house ELISA antigen was produced following two rounds of ultracentrifugation and treatment with sodium dodecyl sulfate (R. A. Heckert, W. B. McNab, S. M. Richardson, and M. R. Briscoe, Can. J. Vet. Res. 56:237–241, 1992). The PEG antigen batch was found suitable for ELISA, with a relative specificity of 100% and a relative sensitivity of 99.4% compared to the in-house ELISA antigen. This method of antigen production for ELISA was found to be rapid, inexpensive, and reliable for the diagnosis of caprine-arthritis encephalitis, without requiring the use of sophisticated laboratory equipment.     

Coronary artery anomalies diagnosed by magnetic resonance angiography

Coronary artery anomalies are uncommon entities that may be associated with sudden death. Because of its 2‐D projection imaging nature, conventional X‐ray coronary angiography may not accurately delineate the origins and course of aberrant coronary arteries with respect to the great vessels. Non‐invasive, cross‐sectional imaging techniques such as coronary CT angiography and magnetic resonance angiography are increasingly used in clinical practice to diagnose coronary artery anomalies. Although this study reviews coronary artery anatomy and selected anomalies as seen with true fast imaging with steady‐state precession magnetic resonance angiography, the information provided is equally applicable to electrocardiogram‐gated coronary CT angiography.     

人脐血CD34+ 细胞静脉移植对急性心肌梗死大鼠血流动力学指标和超声心动图指标的影响

目的:观察人脐带血CD34 细胞移植治疗急性心肌梗死大鼠的可行性及其对心功能的影响。方法:实验于2006-03/10在首都医科大学附属北京友谊医院完成。①选取11～13周龄雄性SD大鼠40只,随机数字表法分为假手术组10只、模型对照组15只、细胞移植组15只。脐血由北京市脐血干细胞库提供,采自无妊娠并发症、身体健康、新生儿足月分娩(37～40周)孕妇,均签署知情同意书,自愿捐献用于科学研究和临床治疗。②一次性血袋抽取孕妇脐带血80～120mL,按5∶1比例加入60g/L羟乙基淀粉,4℃420r/min离心8min;去除红细胞,留取上层含有核细胞的血浆层,4℃1180r/min离心10min,去除血浆,留取有核细胞,测CD34 含量为0.35%～0.42%。③模型对照组、细胞移植组大鼠建立急性心肌梗死模型,麻醉后切开第4、5肋间肌,暴露心脏,在动脉圆锥和左心耳间、距左心耳下1mm处(相当于左前降支近段)用Prolene线结扎。假手术组只挂线不结扎。根据左室前壁颜色变白、活动减弱和心电图ST段明显抬高作为急性心肌梗死模型成功标志。④术后3h内,细胞移植组经尾静脉注射0.5mL人脐血干细胞悬液(含干细胞2×1010L-1),模型对照组经尾静脉注射0.5mL生理盐水。饲养30d。⑤分别于术前、术后1d和术后30d进行超声心动图检测。并于术后30d进行血流动力学检测。结果:假手术组挂线过程中死亡1只,模型对照组造模及细胞移植过程死亡4只,细胞移植组造模及细胞移植过程死亡5只。①人脐血CD34 细胞移植30d后对大鼠血流动力学的影响:与假手术组比较,模型对照组左室收缩压、左室压力最大变化率均明显减小(t=2.16～5.14,P均<0.05),左室舒张末压、心率均明显增加(t=2.01～8.86,P<0.01或0.05);与模型对照组比较,细胞移植组左室收缩压、左室压力最大变化率均明显增加(t=2.72～2.35,P均<0.05),左室舒张末压明显减小(t=4.24,P<0.01),心率无明显变化(t=1.67,P>0.05)。②人脐血CD34 细胞移植前后超声心动图检查结果:与假手术组比较,术前模型对照组和细胞移植组各指标无明显变化;术后1,30d模型对照组和细胞移植组左室舒张末期内径、左室收缩末期内径、左室舒张末容积、左室收缩末容积均明显增加(t=2.14～9.98,P<0.05或0.01),左室前壁厚度、左室后壁厚度、左室射血分数、左室短轴缩短率均明显减小(t=2.52～14.23,P<0.05或0.01)。与模型对照组比较,术前和术后1d细胞移植组各指标无明显变化,但术后30d左室舒张末期内径、左室收缩末期内径、左室舒张末容积、左室收缩末容积均明显减小(t=2.07～7.04,P<0.05或0.01),左室前壁厚度、左室后壁厚度、左室射血分数、左室短轴缩短率均明显增加(t=3.22～9.85,P均<0.01)。结论:在未使用免疫抑制剂的情况下,人脐带血CD34 细胞静脉移植可明显改善急性心肌梗死大鼠各项心功能指标,未见明显不良反应。