Glyoxysomal malate-dehydrogenase and malate synthase from Soybean cotyledons (Glycine max. L.): Enzyme association, antibody-production and cDNA cloning

In order to investigate a possible association between soybean malate synthase (MS; L-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD(+) oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionic-strength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (M(r)) of 670000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M, 510 000 and of gMDH as a dimer of M, 73 000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a lambda ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X(6))HL motif also found in plant and mammalian thiolases.

Gene Expression Profiling of Desmoid Tumors by cDNA Microarrays and Correlation with Progression-Free Survival.

PURPOSE: Because desmoid tumors exhibit an unpredictable clinical course, translational research is crucial to identify the predictive factors of progression in addition to the clinical parameters. The main issue is to detect patients who are at a higher risk of progression. The aim of this work was to identify molecular markers that can predict progression-free survival (PFS). EXPERIMENTAL DESIGN: Gene-expression screening was conducted on 115 available independent untreated primary desmoid tumors using cDNA microarray. We established a prognostic gene-expression signature composed of 36 genes. To test robustness, we randomly generated 1,000 36-gene signatures and compared their outcome association to our define 36-genes molecular signature and we calculated positive predictive value (PPV) and negative predictive value (NPV). RESULTS: Multivariate analysis showed that our molecular signature had a significant impact on PFS while no clinical factor had any prognostic value. Among the 1,000 random signatures generated, 56.7% were significant and none was more significant than our 36-gene molecular signature. PPV and NPV were high (75.58% and 81.82%, respectively). Finally, the top two genes downregulated in no-recurrence were FECH and STOML2 and the top gene upregulated in no-recurrence was TRIP6. CONCLUSIONS: By analyzing expression profiles, we have identified a gene-expression signature that is able to predict PFS. This tool may be useful for prospective clinical studies. Clin Cancer Res; 21(18); 4194-200. ©2015 AACR.

Sju röda stunder

Digitoitu 15. 7. 2008.

The application of cDNA and tissue microarray methods in the study of human carcinomas

Currently, numerous high-throughput technologies are available for the study of human carcinomas. In literature, many variations of these techniques have been described. The common denominator for these methodologies is the high amount of data obtained in a single experiment, in a short time period, and at a fairly low cost. However, these methods have also been described with several problems and limitations. The purpose of this study was to test the applicability of two selected high-throughput methods, cDNA and tissue microarrays (TMA), in cancer research. Two common human malignancies, breast and colorectal cancer, were used as examples. This thesis aims to present some practical considerations that need to be addressed when applying these techniques. cDNA microarrays were applied to screen aberrant gene expression in breast and colon cancers. Immunohistochemistry was used to validate the results and to evaluate the association of selected novel tumour markers with the outcome of the patients. The type of histological material used in immunohistochemistry was evaluated especially considering the applicability of whole tissue sections and different types of TMAs. Special attention was put on the methodological details in the cDNA microarray and TMA experiments. In conclusion, many potential tumour markers were identified in the cDNA microarray analyses. Immunohistochemistry could be applied to validate the observed gene expression changes of selected markers and to associate their expression change with patient outcome. In the current experiments, both TMAs and whole tissue sections could be used for this purpose. This study showed for the first time that securin and p120 catenin protein expression predict breast cancer outcome and the immunopositivity of carbonic anhydrase IX associates with the outcome of rectal cancer. The predictive value of these proteins was statistically evident also in multivariate analyses with up to a 13.1- fold risk for cancer specific death in a specific subgroup of patients.

Brunnshusteaterns program : Röda rosor

Käsinkirjoitettu merkintä vuosiluvusta

RDA (Resource Description and Access) – kuvailuohjeiden kansainvälinen ja kansallinen käyttöönotto

Marjatta Autio-Tuulen esitys Kuvailun tiedotuspäivillä Helsingissä 20.3.2013.

Uusien RDA-kenttien käsittely tietuepoiminnassa

Ulla Ikäheimon esitys Kansalliskirjaston kirjastoverkkopalveluiden Asiantuntijaseminaarissa 23.4.2013 Helsingissä.

RDA:n suhteet (funktiotermit) ja Melinda

Ulla Ikäheimon esitys Asiantuntijaseminaarissa 26.11.2013 Helsingissä.

RDA and Linked Data

Gordon Dunshiren esitys Kuvailupäivillä 25.3.2014 Helsingissä.

Kokonaiskuva RDA:n tuomasta muutoksesta

Tuula Haapamäen esitys Kuvailupäivillä 25.3.2014 Helsingissä.