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.     

Redox-dependent complex formation by an ATP-dependent activator of the corrinoid/iron-sulfur protein

Movement, cell division, protein biosynthesis, electron transfer against an electrochemical gradient, and many more processes depend on energy conversions coupled to the hydrolysis of ATP. The reduction of metal sites with low reduction potentials (E^0′ < -500 mV) is possible by connecting an energetical uphill electron transfer with the hydrolysis of ATP. The corrinoid-iron/sulfur protein (CoFeSP) operates within the reductive acetyl-CoA pathway by transferring a methyl group from methyltetrahydrofolate bound to a methyltransferase to the [Ni-Ni-Fe₄S₄] cluster of acetyl-CoA synthase. Methylation of CoFeSP only occurs in the low-potential Co(I) state, which can be sporadically oxidized to the inactive Co(II) state, making its reductive reactivation necessary. Here we show that an open-reading frame proximal to the structural genes of CoFeSP encodes an ATP-dependent reductive activator of CoFeSP. Our biochemical and structural analysis uncovers a unique type of reductive activator distinct from the electron-transferring ATPases found to reduce the MoFe-nitrogenase and 2-hydroxyacyl-CoA dehydratases. The CoFeSP activator contains an ASKHA domain (acetate and sugar kinases, Hsp70, and actin) harboring the ATP-binding site, which is also present in the activator of 2-hydroxyacyl-CoA dehydratases and a ferredoxin-like [2Fe-2S] cluster domain acting as electron donor. Complex formation between CoFeSP and its activator depends on the oxidation state of CoFeSP, which provides evidence for a unique strategy to achieve unidirectional electron transfer between two redox proteins.Energy transduction is fundamental for life. Aerobic and anaerobic organisms depend on coupling ATP hydrolysis to movement, activation of metabolites, or peptide bond formation, among others. Several metal-containing enzymes, such as nitrogenase, radical-dependent β,α-dehydratases, the related benzoyl-CoA reductases, and different cobalamin-dependent methyltransferases are able to convert unreactive molecules by acting in a low-potential regime. The highly energetic electrons required for these reactions (1–3) are injected by ATPases that enable the transfer of electrons against the redox potential gradient, driven by the hydrolysis of ATP. Three different types of reductive metallo-ATPase have been described so far.The enzyme nitrogenase is by reducing dinitrogen with six electrons to ammonia at the heart of the global nitrogen cycle (1, 4, 5). Nitrogenase consists of the dinitrogenase, also called MoFe protein for the predominant Mo-containing variant, and the dinitrogenase reductase, called Fe protein (1, 4–6). The Fe protein is a homodimer covalently linked through a [4Fe-4S] cluster bound within the dimer interface. Both monomers are able to bind and hydrolyze ATP in a cleft containing a P loop. For electron transfer (ET) between Fe and MoFe proteins to occur, the reduced Fe protein binds MgATP and forms a complex with the MoFe protein positioning the electron-donating [4Fe-4S] cluster and electron-accepting P cluster within the typical limits for physiological ET (< 15 Å) (1, 4, 7, 8). Hydrolysis of two ATP molecules initiates a one-electron transfer between both partners (9, 10). Conformational changes of the Fe protein induced by ATP hydrolysis are believed to act as switches for the association/dissociation of the Fe:MoFe protein complex and the delivery of electrons (8–11). The Fe protein is bifunctional and also acts as a molybdate/homocitrate insertase during the maturation of nitrogenase (5, 12).Benzoyl CoA reductases and 2-hydroxyacyl CoA dehydratases rely on homologous metallo-ATPases to catalyze the reduction of benzoyl-CoA or the β/α-dehydration of 2-hydroxyacyl-CoA compounds via formation of ketyl radicals (2). The structure of the homodimeric activator of 2-hydroxyglutaryl-CoA dehydratase revealed a [4Fe-4S] cluster covalently linking the two monomers, on a first glance resembling the Fe protein (13). The structure of the activator also showed it to be a member of the ASKHA (acetate and sugar kinases/heat shock protein 70/actin) superfamily. ASKHA proteins catalyze phosphoryl transfers or hydrolysis of ATP in a variety of biological contexts and are distinct from the P loop containing switch-type NTPases to which the Fe protein of nitrogenase belongs (13, 14). The binding of two MgATP molecules to the reduced activator is supposed to induce a conformational change and drives formation of the complex with the dehydratase. ATP hydrolysis likely increases the reducing power of the reduced [4Fe-4S] cluster of the activator, enabling the one-electron transfer to the low-potential [4Fe-4S] cluster of the dehydratase (2, 15). Unlike the 2-hydroxyacyl-CoA dehydratase system, the reduction of benzoyl-CoA is a two-step ET requiring a stoichiometric consumption of ATP (3).Recently, a third class of electron-transferring metallo-ATPases has been discovered (16–18). This enzyme class belongs to the COG3894 protein family and has been termed reductive activases for corrinoid enzymes (RACE) (17). The genome of several anaerobic microorganisms, which encode corrinoid-dependent methyltransferases and enzymes of the reductive acetyl-CoA pathway, also encode for proteins homologous to the two investigated RACE proteins with their characteristic binding motifs for one Fe/S cluster (17, 18). Bacterial RACE proteins typically show [2Fe-2S] cluster-binding-motifs, as in the veratrol O-demethylase system of Acetobacterium dehalogenans (16), whereas in archaea, as in the activator of the methylamine methyltransferase of the methanogenic archaeon Methanosarcina barkeri [4Fe-4S] cluster-binding motifs are more abundant (17, 18).The anaerobic hydrogenogenic bacterium Carboxydothermus hydrogenoformans is able to convert CO₂ into cellular carbon compounds via the reductive acetyl-CoA pathway (also known as the Wood–Ljungdahl pathway) (19–21). The corrinoid/iron-sulfur protein (CoFeSP) connects the methyl and carbonyl branch of this pathway by accepting a methyl group from methyltetrahydrofolate bound to a methyltransferase and donating it to the Ni,Fe-containing acetyl-CoA synthase (22, 23). Three redox states are known for the corrinoid cofactor of CoFeSP: The nucleophilic Co(I) acts as a methyl-acceptor, Co(II) is an oxidized inactive state, and CH₃ - Co(III) acts as the methyl donor of acetyl-CoA synthase (22, 23). The occasional oxidation of Co(I) to Co(II) inactivates CoFeSP, which has to be reactivated by a one-electron reduction (23, 24). The low midpoint potential needed to reduce Co²⁺ to Co¹⁺ (< -504 mV at pH 7.4) (25) can be achieved in vitro using either chemical reducing agents such as Na-dithionite (DT), Ti³⁺ citrate, via photoreduction with deazariboflavin as a catalyst or enzymatically with electrons generated by the oxidation of CO to CO₂ by carbon monoxide dehydrogenase (22, 26). An ATP-dependent reactivation of CoFeSP has not been reported so far.An open reading frame (orf7), situated between the structural genes coding for the CoFeSP subunits CfsA and CfsB of Moorella thermoacetica (27), codes for a member of the COG3894 protein family and contains the putative [2Fe-2S] cluster-binding motif CX₅CX₂CX_nC (17, 18). The genome of C. hydrogenoformans contains a similar arrangement of genes coding for enzymes of the reductive acetyl-CoA pathway as M. thermoacetica, including a homolog of Orf7 (CHY_1224 assigned as COG3894). To test whether an ATP-dependent reductive activator is operative in the reductive acetyl-CoA pathway, we established the heterologous production of the Orf7 homolog and investigated its activity, structure, and selective complex formation with CoFeSP. Furthermore, we reveal its relationship to known ATPases including the activator of 2-hydroxyacyl-CoA dehydratases and compare its strategy to achieve unidirectional electron transport with the other types of ATP-dependent activators/reductases.     

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．