Assessment of aldehyde dehydrogenase in viable cells

Cytosolic aldehyde dehydrogenase (ALDH), an enzyme responsible for oxidizing intracellular aldehydes, has an important role in ethanol, vitamin A, and cyclophosphamide metabolism. High expression of this enzyme in primitive stem cells from multiple tissues, including bone marrow and intestine, appears to be an important mechanism by which these cells are resistant to cyclophosphamide. However, although hematopoietic stem cells (HSC) express high levels of cytosolic ALDH, isolating viable HSC by their ALDH expression has not been possible because ALDH is an intracellular protein. We found that a fluorescent aldehyde, dansyl aminoacetaldehyde (DAAA), could be used in flow cytometry experiments to isolate viable mouse and human cells based on their ALDH content. The level of dansyl fluorescence exhibited by cells after incubation with DAAA paralleled cytosolic ALDH levels determined by Western blotting and the sensitivity of the cells to cyclophosphamide. Moreover, DAAA appeared to be a more sensitive means of assessing cytosolic ALDH levels than Western blotting. Bone marrow progenitors treated with DAAA proliferated normally. Furthermore, marrow cells expressing high levels of dansyl fluorescence after incubation with DAAA were enriched for hematopoietic progenitors. The ability to isolate viable cells that express high levels of cytosolic ALDH could be an important component of methodology for identifying and purifying HSC and for studying cyclophosphamide-resistant tumor cell populations.     

Accuracy of supplementary serologic testing for human T-lymphotropic virus types I and II in US blood donors. Retrovirus Epidemiology Donor Study

Blood donations in the United States have been screened for antibody to human T-lymphotropic virus type I (HTLV-I) by HTLV-I enzyme immunoassay (EIA) since November 1988. Specimens repeatedly found to be reactive by EIA undergo confirmation by supplementary serologic tests. We assessed the accuracy of blood center testing of 994 HTLV-I EIA repeat-reactive specimens in five US blood centers between November 1988 and December 1991. Of 410 confirmed HTLV-I/II donations, 407 (99.3%) were infected with HTLV-I/II, as determined by polymerase chain reaction (PCR) (403 cases) and by repeat serologic testing (4 cases). The three false- positive results occurred in the first year of testing. Of 425 HTLV- indeterminate specimens, 6 (1.4%) were found to be infected by PCR (5 with HTLV-II and 1 with HTLV-I). None of 159 confirmatory test-negative donations was PCR positive. Of HTLV-I/II-seropositive specimens, 80.2% to 95.4% could be typed as HTLV-I or HTLV-II by type-specific serologic assays. These results support recommendations that HTLV-I/II- seropositive donors should be advised that they are infected with HTLV- I, HTLV-II, or HTLV-I/II (depending on results of type-specific assays). HTLV-indeterminate donors should be advised that their results only rarely indicate HTLV infection. HTLV confirmatory test-negative donors should be reassured that they are not infected with HTLV-I or HTLV-II.     

股骨近端锁定加压钢板治疗股骨转子间骨折

目的 探讨股骨近端锁定加压钢板（LCP）内固定治疗股骨转子间骨折的临床疗效.方法 采用股骨近端LCP治疗23例股骨转子间骨折的患者.随访观察骨折愈合时间,按Harris评分标准评价疗效.结果 23例均获随访,时间6~12（9±1.4）个月.骨折愈合时间14~20（17±1.7）周.髋关节Harris评分：优13例,良9例,一般1例.结论 股骨近端LCP内固定治疗股骨转子间骨折创伤小、出血少、对骨膜影响小,符合解剖形态,临床疗效满意.     

Detection of the hemoglobin E mutation using the color complementation assay: application to complex genotyping

The color complementation assay (CCA) is a method of allele-specific DNA amplification by which competitive priming and extension of fluorescently labeled oligonucleotide primers determine the color of DNA amplification product. This diagnostic method precludes the need for radioisotopes, electrophoresis, and multiple high-stringency reaction conditions. The multiplicity of mutant globin genes present in Southeast Asians complicates clinical diagnosis and underscores the importance of DNA-based diagnostic methods. We have applied CCA to distinguish beta A and beta E alleles. Competing 15mer primers were a fluorescein-labeled complement to beta A and a rhodamine-labeled complement to beta E, identical except for their central nucleotides. A common unlabeled primer was used to amplify DNA product, the color of which was determined by the perfectly complementary primer. Color photography and spectrofluorometry, as well as a method of black-white photography that we developed to distinguish fluorescein- and rhodamine- labeled DNA, were used to record results. We applied CCA to define the complex genotype of a Thai woman with thalassemia intermedia, 96% HbE, and 4% HbF whose possible genotypes included several permutations of alpha-thalassemia, beta-thalassemia, and beta E genes. zeta-Globin gene mapping of DNA doubly digested with Bg/II and Asp 718 showed the -alpha 3.7/--SEA genotype, and CCA confirmed homozygous beta E/beta E. The CCA is useful for diagnosing the compound hemoglobin genotypes of Southeast Asians and could be applied also to prenatal diagnosis in this population.     

Diagnostic role of an immunoassay-detected polymorphism of factor IX for potential carriers of hemophilia B

In hemophilia B, assays based on a monoclonal antifactor IX specific for the Thr-148 variant of an exonic polymorphism have diagnosed carriers in selected families by either establishing linkage or by indicating the presence or absence of a given normal factor IX. The sensitivity of the immunoassays for detecting heterozygous women was explored by comparing results from immunoassays with solid-phase polyclonal v the monoclonal antifactor IXs. Factor IX with the normal Ala-148 variant gave a flat dilution curve, qualitatively distinct from factor IX with the Thr-148 variant in the monoclonal assay. The two were indistinguishable in the polyclonal assay. Mixtures of equal amounts of the two types gave an intermediate result, about half as reactive in the monoclonal as compared with the polyclonal assay system. Whereas mixtures with 10% Ala-148 and 90% Thr-148 factor IXs could not readily be distinguished from Thr-148 factor IX plasma, as little as 1% of the Thr-148 protein was detected in Ala-148 factor IX plasma. The frequency of the Ala-148 variant varied in individuals with different ethnic backgrounds; it was found in 29% of white, 12% of black, and none of Asian blood donors' factor IX genes in Seattle. Only 4% of samples from South African black men were nonreactive (ie, Ala- 148). The Thr/Ala-148 dimorphism is in strong linkage disequilibrium with Taql restriction fragment length polymorphisms (RFLPs). Three recombinations were noted in normal white genes and one in a normal black factor IX gene (less than 2% of those examined). In 34 white families with at least one woman being a possible carrier, genetically, the immunoassay results were informative in 18. RFLP analyses were informative in eight of the 15 families tested. In five families each, assignment of carrier status was made to a woman by only DNA or only immunoassay results, whereas the other approach was noninformative. The immunoassays provide a rapid, inexpensive screening test and complement DNA analysis in white women who are potential carriers of hemophilia B.     

胰腺癌组织VEGF和MVD表达与CT灌注成像的关系

血管内皮生长因子作为肿瘤血管生成的一种主要调控因子,与微血管密度密切相关．胰腺癌组织的血管内皮生长因子和微血管密度的测定对于判断患者的预后,指导临床治疗和判断疗效等具有重要作用．CT灌注成像可以通过无创伤检查反映胰腺癌的肿瘤血管构成, 在治疗前后提供有价值依据,对胰腺癌的诊治有重要意义．     

免疫磁珠分选骨髓衍生肝干细胞亚群c-Kit+lin-

目的:利用免疫磁性细胞分选系统分离纯化骨髓衍生肝干细胞亚群c-Kit lin-。方法:实验于2006-07/08在南方医科大学实验动物中心完成。6～8周龄的SPF级纯系BALB/C雄性小鼠10只,体质量18～20g。收集小鼠股骨骨髓细胞,利用免疫磁性细胞分选系统,通过两步法分选纯化c-Kit lin-:将获取的lin-细胞悬液8℃条件下1500r/min离心10min,弃上清,按80μL/107加入Buffer重悬细胞。按20μL/107加生物素抗体磁珠,混匀,4℃冰箱孵育15min,按1mL/107加入Buffer洗细胞1次,8℃条件下1500r/min离心10min,弃上清,按500μL/108加入Buffer重悬细胞。Buffer500μL润MS柱,悬液过柱后,Buffer500μL/次洗柱3次,柱子脱离磁场,加1mLBuffer,用配套柱塞推出柱中的c-Kit lin-细胞,收集到c-kit lin-细胞,细胞计数。取2.0×106个细胞分成10等份,流式细胞仪分析c-Kit lin-细胞纯度,计算回收率,评估纯化效率,苔盼兰染色检测纯化前后的细胞活力。计算活细胞的百分率。细胞纯度和细胞回收率的计算:细胞纯度=分离产物中的阳性细胞数/分离细胞的总细胞数×100%,细胞回收率=分离产物中的阳性细胞数/起始标本阳性细胞总数×100%。结果:10只小鼠均进入结果分析。利用免疫磁性细胞分选系统分选出的骨髓衍生肝干细胞亚群c-Kit lin-细胞纯度和回收率分别为(77.98±2.34)%,75.40%,纯化前后细胞活力不受影响。结论:免疫磁性细胞分选系统能有效分选骨髓衍生肝干细胞亚群c-Kit lin-,纯度和回收率高,且不影响细胞活力。     

The brain‐derived neutrophic factor val66met polymorphism and sudden unexpected infant death

Aim: Findings of hypoxia prior to death and involvement of a dysregulation of the serotonergic network in sudden infant death syndrome (SIDS) may indicate that brain‐derived neutrophic factor (BDNF) also is of importance with regard to sudden unexpected infant death. Based on this, the purpose of this study was to investigate the BDNF val66met polymorphism in SIDS cases, cases of infectious death and controls. Methods: The polymorphism was investigated in 163 SIDS cases, 34 cases of infectious death and 121 controls, using real‐time PCR and fluorescence melting curve analysis. Results: There were no differences in val66met genotype distribution between neither the SIDS cases nor the cases of infectious death and controls (p = 0.95 and p = 0.52, respectively). Conclusion: The study indicates that the val66met polymorphism is not important for sudden unexpected infant death. However, several other SNPs in the BDNF gene, as well as in other genes involved in this pathway, including G‐protein, have to be investigated to fully exclude any involvement of BDNF in SIDS.     

金龙胶囊配合辨证用药治疗中晚期恶性肿瘤40例

1临床资料2003-12/2004-10采用北京建生药业生产的金龙胶囊配合辨证用药治疗中晚期癌症40例.Karnofsky评分≥40分,预计生存期>2 mo.