Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

反复种植失败的相关对策新进展

辅助生殖技术的迅速发展使众多不孕症患者借助体外受精-胚胎移植(IVF-ET)及其衍生技术获得了后代。然而很大一部分妇女经历多次优质胚胎移植亦不能获得妊娠,反复种植失败(RIF)已经成为阻碍妊娠率进一步提高的瓶颈问题,且日益受到生殖医学界的广泛关注。就目前的条件而言,对RIF患者给予药物或者机械操作以提高子宫内膜容受性,行宫腹腔镜检查排除宫腔及盆腔病变以改善胚胎种植环境,通过辅助孵化、选择性囊胚移植、植入前胚胎遗传学筛查、共培养等技术提高胚胎着床能力都有可能改善和提高其种植率及妊娠率。RIF成为了我们亟待解决的问题,现综述近年有关反复种植失败的相关对策新进展。     

Inactivation of purified human alpha 2-antiplasmin and purified human C1 inhibitor by synthetic fibrinolytic agents

3-Hydroxypropyl flufenamide (Flu-HPA) is one of a series of flufenamic acid derivatives that enhances blood clot lysis in vitro. Studies of possible mechanisms of action of Flu-HPA were undertaken. The profibrinolytic activity of Flu-HPA in clot lysis assays was found to be dependent on plasminogen. The influence of Flu-HPA on the ability of purified alpha 2-antiplasmin to inhibit purified plasmin was studied. Plasmin activity was determined using 125I-fibrin plates or the spectrophotometric tripeptide substrate, Val-Leu-Lys-paranitroanilide. At Flu-HPA concentrations greater than 1 mM, the inhibitory activity of alpha 2-antiplasmin was abolished in a time-dependent and concentration- dependent manner. The influence of Flu-HPA on the ability of purified Cl inhibitor to inhibit purified plasma kallikrein and beta-Factor XIIa was also studied. Cl inhibitor activity was abolished by Flu-HPA at concentrations greater than 2 mM. Notably, Flu-HPA up to 60 mM did not affect the amidolytic activities of plasmin, kallikrein, or beta-Factor XIIa. Flu-HPA did not release enzyme activity from preformed complexes of either alpha 2-antiplasmin and plasmin of Cl inhibitor and kallikrein. A water-soluble derivative of flufenamic acid, N-flufenamyl- glutamic acid, also inactivated alpha 2-antiplasm and Cl inhibitor. This inactivation was shown to be reversible. These results indicate that synthetic fibrinolytic compounds such as flufenamic acid derivatives may promote fibrinolysis by directly inactivating alpha 2- antiplasmin and Cl inhibitor.     

Mini-plasminogen: a mechanism for leukocyte modulation of plasminogen activation by urokinase

Urokinase activation of blood fibrinolysis involves polymorphonuclear leukocytes. To determine if a leukocyte proteinase can modulate plasminogen activation, plasminogen was digested with leukocyte elastase. A major product was a small, approximately 34,000 dalton fragment (mini-plasminogen), without lysine-binding function, but with fibrin-binding activity. After urokinase activation, the resulting mini- plasmin had amidolytic activity for a tripeptide plasmin substrate and fibrinolytic activity. By 125I-fibrin assay, activities of mini-plasmin and plasmin (12 nmole/liter) were 38 and 20 ng fibrin lysed/min, respectively. Lysis times of fibrin clots containing urokinase, and mini-plasminogen or plasminogen (800 nmole/liter), were 282 and 290 sec, respectively. Mini-plasmin and plasmin were inhibited similarly by epsilon-aminocaproic acid and normal plasma, but differed in responses to gel filtration fractions of plasma containing alpha 2-antiplasmin and alpha 2-macroglobulin, the primary and secondary plasmin inhibitors. With purified inhibitors, mini-plasmin required higher concentrations of, or longer preincubation with, alpha 2-antiplasmin, and lower concentrations of, or shorter preincubation with, alpha 2- macroglobulin, to produce inhibition equivalent to that observed with plasmin. Leukocyte elastase digests plasminogen to generate a mini- plasminogen which, when activated by urokinase, has a novel pattern of response to the major plasmin inhibitors in plasma.     

Membrane fluidity changes accompanying phagocytosis in normal and in chronic granulomatous disease polymorphonuclear leukocytes

We have studied membrane fluidity changes in polymorphonuclear leukocytes (PMN) during phagocytosis. Membrane fluidity was assessed by electron spin resonance (ESR) using a nitroxide-substituted stearic acid analog (5DS) as a spin probe. PMN from normal subjects and from 3 CGD patients (2 males, 1 female) were incubated in Kreb's Ringers phosphate with or without opsonized zymosan. ESR spectra were obtained and the order parameter (S), which is inversely related to membrane fluidity, was calculated. Without zymosan addition, S for normal (0.638) and for CGD (0.635) were not significantly different (p less than 0.35). The S values indicate that under resting conditions the molecular environment of the CGD membrane is similar to that of normal PMN membranes. However, with addition of opsonized zymosan, the normal, but not the CGD, PMN showed a significant increase (CGD, S = 0.638; normal, S = 0.647; p less than 0.001). This change in S for the normals is consistent with a more restricted movement of 5DS. Treatment of normal PMN with a mixture of scavengers specific for H2O2 (catalase, 1600 U/ml), O2-.(superoxide dismutase, 100 micrograms/ml), and for HO., (sodium benzoate, 1mM) during zymosan stimulation gave S values similar to those of resting cells. Catalase alone also lowered S value, suggesting that H2O2 was instrumental in causing the initial S value increase. This idea was supported by studies in which CGD cells were incubated with zymosan in the presence of glucose oxidase, an enzyme that catalyzes glucose oxidation resulting in the direct reduction of molecular oxygen to H2O2. Our results indicate that reduced O2 by- products, particularly H2O2, can cause altered biophysical properties of PMN membrane during phagocytosis.     

Percutaneous cholecystography in children

Percutaneous cholecystography was performed on 13 children who had biliary system abnormalities: two had biliary hypoplasia, five had sclerosing cholangitis, three had cirrhosis, two had distal choledochal obstruction, and one had an obstructed portoenterostomy. In 12 patients transcholecystic cholangiography showed, without significant complications, the intra-and extrahepatic bile ducts. In one patient with primary sclerosing cholangitis, the intrahepatic bile ducts were not opacified satisfactorily; dilatation of the gallbladder required surgical drainage. The transcholecystic technique is indicated when the intrahepatic bile ducts are either mildly dilated or not dilated.     

Three-dimensional rotational spinal angiography in the evaluation and treatment of vascular malformations

BACKGROUND AND PURPOSE: Conventional spinal angiography, although useful in providing angioarchitectural details of spinal vascular disease, has limitations. The advent of 3D angiography has provided a better comprehension of angioarchitectural detail when evaluating the intracranial circulation. The purpose of this study was to evaluate the usefulness of 3D angiography in the diagnosis and treatment of vascular malformations of the spine. METHODS: This retrospective analysis included 17 3D spinal angiograms acquired in 14 consecutive patients examined at our institution for a spinal vascular lesion, which included nine spinal cord arteriovenous malformations (AVMs), one perimedullary arteriovenous fistula (AVF), three spinal dural AVFs, and one nerve root AVM. 3D angiography was obtained with apnea under general anesthesia by using a 14-second acquisition and 200 degrees rotation of the gantry during injection of 300 mg I/mL nonionic contrast material at a rate of 0.5-3.5 mL/s. Multiple reconstructed images were obtained with or without opacification of the surrounding structures. These images were then evaluated by the interventionalists at the time of the procedure and compared with findings obtained by conventional subtraction angiography. RESULTS: 3D angiography was useful in differentiating intramedullary lesions from perimedullary surface lesions; detecting arterial, nidal, or venous aneurysms; and evaluating the 3D structure of the lesion as well as the relationship between the malformation and its draining veins or surrounding bony structures. In specific situations, it obviated the need for contrast-enhanced conventional or 3D CT, as well as for lateral or oblique angiographic views, which are sometimes difficult to obtain with good quality. No 3D angiography-related complications were experienced. Some limitations in the definition of small vessel anatomy in the reconstructed images were noted. CONCLUSION: In this small series of patients, 3D angiography was safe and useful for evaluation of the 3D vascular anatomy of spinal vascular malformations.