Studies on the reaction specificity of ALOX15 orthologs from lower vertebrates

Einleitung: Arachidonsäurelipoxygenasen (ALOX-Isoformen) sind Dioxygenasen, die mehrfach ungesättigte Fettsäuren zu den entsprechenden Hydroperoxyfettsäurederivaten oxygenieren. Im humanen Genom gibt es sechs funktionelle ALOX-Gene (ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5), die für funktionell unterschiedliche Isoenzyme kodieren. Die Reaktionsspezifität von ALOX15-Orthologen vieler höherer Vertebraten (Säugetiere) ist gut charakterisiert, wohingegen es nur wenige Daten über das Vorhandensein von ALOX-Isoformen bei Knochenfischen gibt. Auch wurde bislang nicht untersucht, ob der oxidative Lipidstoffwechsel in kausaler Beziehung zur beschleunigten Alterung von N. furzeri, einem beliebten Modellorganismus in der Altersforschung, steht. Hieraus ergaben sich für die vorliegende Arbeit folgende konkrete Fragestellungen: i) Kommen ALOX-Gene in Knochenfischen weit verbreitet vor und können hier echte ALOX15-Gene nachgewiesen werden? ii) Können Knochenfisch ALOX-Isoformen rekombinant exprimiert werden und unterscheiden sich ihre katalytischen Eigenschaften von denen höherer Wirbeltiere? iii) Folgen die Knochenfisch ALOX-Isoformen den Hypothesen, die zur Erklärung der Reaktionsspezifität anderer ALOX-Isoformen entwickelt wurden? iv) Lassen sich aus dem ALOX-Stoffwechsel von N. furzeri Schlussfolgerungen zum Mechanismus der beschleunigten Alterung dieser Tiere ableiten? Methodik: Zur Beantwortung dieser Fragen wurden ausgewählte ALOX-Isoformen verschiedener Knochenfische als rekombinante N-terminale His-tag Fusionsproteine in pro- und eukaryotischen Expressionssystemen exprimiert und hinsichtlich ihrer protein-chemischen und enzymatischen Eigenschaften charakterisiert. Ergebnisse: ALOX-Gene kommen in den Genomen von Knochenfischen weit verbreitet vor, wobei jedoch keines der kodierten Enzyme zweifelsfrei als ALOX15-Ortholog identifiziert werden konnte. Die Knochenfisch ALOX-Isoformen konnten rekombinant exprimiert werden, wobei sich ihre katalytischen Eigenschaften teilweise von denen höherer Wirbeltiere unterschieden. Die Knochenfisch ALOX-Isoformen folgen weder vollumfänglich dem Triadenkonzept der ALOX15-Orthologen noch der A-vs.-G-Hypothese der Stereospezifität. Im Rahmen dieser Arbeit konnte der Zusammenhang zwischen dem ALOX-Stoffwechsel von N. furzeri und der beschleunigten Alterung dieser Knochenfischspezies nicht abschließend geklärt werden. Schlussfolgerung: ALOX-Isoformen kommen bei Knochenfischen weit verbreitet vor, jedoch stimmen die durch die Datenbanken vorgenommenen Annotierungen nicht immer mit den in vitro Untersuchungen zur Reaktionsspezifität überein. Die Beobachtung, dass die von uns untersuchten Knochenfisch ALOX-Isoformen nicht dem Triadenkonzept folgen, welches auf alle bisher identifizierten Säugetier ALOX15-Orthologen anwendbar ist, spricht dafür, dass es sich bei diesen Proteinen eher nicht um Funktionsäquivalente der Säugetier ALOX15-Orthologen handelt.Introduction: Arachidonate lipoxygenases (ALOX isoforms) are dioxygenases which catalyse the oxygenation of polyunsaturated fatty acids to the corresponding hydroperoxy derivatives. The human genome involves six functional ALOX genes (ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5), which encode for functional distinct isozymes. The reaction specificity of ALOX15 orthologs of higher vertebrates such as mammals has been well characterized. In contrast, little is known about the presence of ALOX isoforms in bony fish. Moreover, it has not yet been explored whether there is a causal relationship between the oxidative lipid metabolism and the accelerated aging process of N. furzeri, which is a popular model organism in age research. This lack of knowledge resulted in the following specific questions for the present dissertation: i) Are ALOX genes widely distributed in bony fish and can true ALOX15 genes be detected in these vertebrates? ii) Is it possible to express bony fish ALOX isoforms as functional recombinant proteins and do their catalytic properties differ from those of higher vertebrates? iii) Do the bony fish ALOX isoforms follow the existing hypotheses which explain the reaction specificity of other ALOX isoforms? iv) Is the ALOX metabolism of N. furzeri related to accelerated aging of this bony fish species? Methodology: To answer these questions, selected bony fish ALOX isoforms were expressed as recombinant N-terminal his-tag fusion proteins in pro- and eucaryotic expression systems and they were characterized in respect to their protein-chemical and catalytic properties. Results: ALOX genes occur frequently in different bony fish species. We expressed putative ALOX15 orthologs of three different bony fish species as catalytically active recombinant N-terminal his-tag fusion proteins but neither of the characterized enzymes exhibited a high degree of functional similarity with mammalian ALOX15 orthologs despite their database annotation as ALOX15. The putative bony fish ALOX15 orthologs did neither follow the Triad Concept of reaction specificity of ALOX15 orthologs nor the A-vs.-G-Hypothesis of stereospecificity. Comparison of the functional characteristics of the three putative bony fish ALOX15 orthologs did not provide any evidence for the assumption that the enzyme of N. furzeri is involved in the accelerated aging process of this bony fish species. Conclusions: ALOX genes occur frequently in bony fish genomes, but the functional characteristics of the putative bony fish ALOX15 orthologs are clearly different from those of mammalian ALOX15 isoforms. Our observation, that the putative bony fish ALOX15 orthologs do not follow the Triad Concept which is applicable for all mammalian ALOX15 orthologs tested so far strongly suggests that these enzymes may not be considered functional equivalents of mammalian ALOX15 orthologs in bony fish

Bony Fish Arachidonic Acid 15-Lipoxygenases Exhibit Different Catalytic Properties than Their Mammalian Orthologs, Suggesting Functional Enzyme Evolution during Vertebrate Development

The human genome involves six functional arachidonic acid lipoxygenase (ALOX) genes and the corresponding enzymes (ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5) have been implicated in cell differentiation and in the pathogenesis of inflammatory, hyperproliferative, metabolic, and neurological disorders. In other vertebrates, ALOX-isoforms have also been identified, but they occur less frequently. Since bony fish represent the most abundant subclass of vertebrates, we recently expressed and characterized putative ALOX15 orthologs of three different bony fish species (Nothobranchius furzeri, Pundamilia nyererei, Scleropages formosus). To explore whether these enzymes represent functional equivalents of mammalian ALOX15 orthologs, we here compared a number of structural and functional characteristics of these ALOX-isoforms with those of mammalian enzymes. We found that in contrast to mammalian ALOX15 orthologs, which exhibit a broad substrate specificity, a membrane oxygenase activity, and a special type of dual reaction specificity, the putative bony fish ALOX15 orthologs strongly prefer C-20 fatty acids, lack any membrane oxygenase activity and exhibit a different type of dual reaction specificity with arachidonic acid. Moreover, mutagenesis studies indicated that the Triad Concept, which explains the reaction specificity of all mammalian ALOX15 orthologs, is not applicable for the putative bony fish enzymes. The observed functional differences between putative bony fish ALOX15 orthologs and corresponding mammalian enzymes suggest a targeted optimization of the catalytic properties of ALOX15 orthologs during vertebrate development

In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR

<p>Abstract</p> <p>Background</p> <p>Housekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes.</p> <p>Results</p> <p>The expression of the potential reference genes was examined in matched malignant and non-malignant tissue specimens from 25 patients with clear cell renal cell carcinoma. Quality assessment of isolated RNA performed with a 2100 Agilent Bioanalyzer showed a mean RNA integrity number of 8.7 for all samples. The between-run variations related to the crossing points of PCR reactions of a control material ranged from 0.17% to 0.38%. The expression of all genes did not depend on age, sex, and tumour stage. Except the genes TATA box binding protein (<it>TBP</it>) and peptidylprolyl isomerase A (<it>PPIA</it>), all genes showed significant differences in expression between malignant and non-malignant pairs. The expression stability of the candidate reference genes was additionally controlled using the software programs geNorm and NormFinder. <it>TBP </it>and <it>PPIA </it>were validated as suitable reference genes by normalizing the target gene <it>ADAM9 </it>using these two most stably expressed genes in comparison with up- and down-regulated housekeeping genes of the panel.</p> <p>Conclusion</p> <p>Our study demonstrated the suitability of the two housekeeping genes <it>PPIA </it>and <it>TBP </it>as endogenous reference genes when comparing malignant tissue samples with adjacent normal tissue samples from clear cell renal cell carcinoma. Both genes are recommended as reference genes for relative gene quantification in gene profiling studies either as single gene or preferably in combination.</p

The Reaction Specificity of Mammalian ALOX15 Orthologs is Changed During Late Primate Evolution and These Alterations Might Offer Evolutionary Advantages for Hominidae

Arachidonic acid lipoxygenases (ALOXs) have been implicated in the immune response of mammals. The reaction specificity of these enzymes is decisive for their biological functions and ALOX classification is based on this enzyme property. Comparing the amino acid sequences and the functional properties of selected mammalian ALOX15 orthologs we previously hypothesized that the reaction specificity of these enzymes can be predicted based on their amino acid sequences (Triad Concept) and that mammals, which are ranked in evolution below gibbons, express arachidonic acid 12-lipoxygenating ALOX15 orthologs. In contrast, Hominidae involving the great apes and humans possess 15-lipoxygenating enzymes (Evolutionary Hypothesis). These two hypotheses were based on sequence data of some 60 mammalian ALOX15 orthologs and about half of them were functionally characterized. Here, we compared the ALOX15 sequences of 152 mammals representing all major mammalian subclades expressed 44 novel ALOX15 orthologs and performed extensive mutagenesis studies of their triad determinants. We found that ALOX15 genes are absent in extant Prototheria but that corresponding enzymes frequently occur in Metatheria and Eutheria. More than 90% of them catalyze arachidonic acid 12-lipoxygenation and the Triad Concept is applicable to all of them. Mammals ranked in evolution above gibbons express arachidonic acid 15-lipoxygenating ALOX15 orthologs but enzymes with similar specificity are only present in less than 5% of mammals ranked below gibbons. This data suggests that ALOX15 orthologs have been introduced during Prototheria-Metatheria transition and put the Triad Concept and the Evolutionary Hypothesis on a much broader and more reliable experimental basis

Вторая фаза исследования по проведению недельного цикла химиотерапии с использованием паклитаксела и карбоплатина у больных с метастатическим раком мочевого пузыря

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The effects inhibiting the proliferation of cancer cells by far-infrared radiation (FIR) are controlled by the basal expression level of heat shock protein (HSP) 70A

We developed a tissue culture incubator that can continuously irradiate cells with far-infrared radiation (FIR) of wavelengths between 4 and 20 μm with a peak of 7–12 μm, and found that FIR caused different inhibiting effects to five human cancer cell lines, namely A431 (vulva), HSC3 (tongue), Sa3 (gingiva), A549 (lung), and MCF7 (breast). Then, in order to make clear the control system for the effect of FIR, the gene expression concerned to the inhibition effect by FIR were analyzed. In consequence, basal expression level of HSP70A mRNA was higher in A431 and MCF7 cells than in the FIR-sensitive HSC3, Sa3, and A549 cells. Also, the over expression of HSP70 inhibited FIR-induced growth arrest in HSC3 cells, and an HSP70 siRNA inhibited the proliferation of A431 cells by irradiation with FIR. These results indicate that the effect of a body temperature range of FIR suppressing the proliferation of some cancer cells is controlled by the basal expression level of heat shock protein (HSP) 70A. This finding suggested that FIR should be very effective medical treatment for some cancer cells which have a low level of HSP70. Still more, if the level of HSP70 in any cancer of a patient was measured, the effect of medical treatment by FIR can be foreseen for the cancer

Interleukin-2/interferon-α2a/13-retinoic acid-based chemoimmunotherapy in advanced renal cell carcinoma: results of a prospectively randomised trial of the German Cooperative Renal Carcinoma Chemoimmunotherapy Group (DGCIN)

We performed a prospectively randomised clinical trial to compare the efficacy of four subcutaneous interleukin-2-(sc-IL-2) and sc interferon-α2a (sc-IFN-α2a)-based outpatient regimens in 379 patients with progressive metastatic renal cell carcinoma. Patients with lung metastases, an erythrocyte sedimentation rate ⩽70 mm h−1 and neutrophil counts ⩽6000 μl−1 (group I) were randomised to arm A: sc-IL-2, sc-IFN-α2a, peroral 13-cis-retinoic acid (po-13cRA) (n=78), or arm B: arm A plus inhaled-IL-2 (n=65). All others (group II) were randomised to arm C: arm A plus intravenous 5-fluorouracil (iv-5-FU) (n=116), or arm D: arm A plus po-Capecitabine (n=120). Median overall survival (OS) was 22 months (arm A; 3-year OS: 29.7%) and 18 months (arm B; 3-year OS: 29.2%) in group I, and 18 months (arm C; 3-year OS: 25.7%) and 16 months (arm D; 3-year OS: 32.6%) in group II. There were no statistically significant differences in OS, progression-free survival, and objective response between arms A and B, and between arms C and D, respectively. Given the known therapeutic efficacy of sc-IL-2/sc-INF-α2a/po-13cRA-based outpatient chemoimmunotherapies, our results did not establish survival advantages in favour of po-Capecitabine vs iv-5-FU, and in favour of short-term inhaled-IL-2 in patients with advanced renal cell carcinoma receiving systemic cytokines

Prediction of Locally Advanced Urothelial Carcinoma of the Bladder Using Clinical Parameters before Radical Cystectomy - A Prospective Multicenter Study

Introduction: We aimed at developing and validating a pre-cystectomy nomogram for the prediction of locally advanced urothelial carcinoma of the bladder (UCB) using clinicopathological parameters. Materials and Methods: Multicenter data from 337 patients who underwent radical cystectomy (RC) for UCB were prospectively collected and eligible for final analysis. Univariate and multivariate logistic regression models were applied to identify significant predictors of locally advanced tumor stage (pT3/4 and/or pN+) at RC. Internal validation was performed by bootstrapping. The decision curve analysis (DCA) was done to evaluate the clinical value. Results: The distribution of tumor stages pT3/4, pN+ and pT3/4 and/or pN+ at RC was 44.2, 27.6 and 50.4%, respectively. Age (odds ratio (OR) 0.980; p < 0.001), advanced clinical tumor stage (cT3 vs. cTa, cTis, cT1; OR 3.367; p < 0.001), presence of hydronephrosis (OR 1.844; p = 0.043) and advanced tumor stage T3 and/or N+ at CT imaging (OR 4.378; p < 0.001) were independent predictors for pT3/4 and/or pN+ tumor stage. The predictive accuracy of our nomogram for pT3/4 and/or pN+ at RC was 77.5%. DCA for predicting pT3/4 and/or pN+ at RC showed a clinical net benefit across all probability thresholds. Conclusion: We developed a nomogram for the prediction of locally advanced tumor stage pT3/4 and/or pN+ before RC using established clinicopathological parameters