In vivo targets of S-thiolation in Chlamydomonas reinhardtii

Glutathionylation is the major form of S-thiolation in cells. This reversible redox post-translational modification consists of the formation of a mixed disulfide between a free thiol on a protein and a molecule of glutathione. This recently described modification, which is considered to occur under oxidative stress, can protect cysteine residues from irreversible oxidation, and alter positively or negatively the activity of diverse proteins. This modification and its targets have been mainly studied in non-photosynthetic organisms so far. We report here the first proteomic approach performed in vivo on photosynthetically competent cells, using the eukaryotic unicellular green alga Chlamydomonas reinhardtii with radiolabeled [(35)S]cysteine to label the glutathione pool and diamide as oxidant. This method allowed the identification of 25 targets, mainly chloroplastic, involved in various metabolic processes. Several targets are related to photosynthesis, such as the Calvin cycle enzymes phosphoglycerate kinase and ribose-5-phosphate isomerase. A number of targets, such as chaperones and peroxiredoxins, are related to stress responses. The glutathionylation of HSP70B, chloroplastic 2-Cys peroxiredoxin and isocitrate lyase was confirmed in vitro on purified proteins and the targeted residues were identified.     

New antibiotic with typical plant anthraquinone structure obtained studying terrestrial and marine Streptomycetes

In our screening for new antibiotics from bacteria, the streptomycete isolate M097 from Jiaozhou Bay in China was found to produce aloesaponarin II (1a) and 1,6-dihydroxy-8-hydroxymethyl-anthraquinone (2). Similarly, a terrestrial streptomycete GW24/1694 produced 1a and its methyl ether, the new compound 1-hydroxy-6-methoxy-8-methyl-anthraquinone (1b). All structures were derived by spectrochemical analysis and by comparison with reference data. The results showed that the marine streptomycete isolate M097 and the terrestrial streptomycete GW24/1694 could be a promising material for studying the biosynthetic pathway of polyketides.     

Computer-guided surgery for gastric carcinoma

Lymphadenectomy offers the only hope for cure when lymph nodes are involved. In gastric cancer, three approaches have been pursued to preoperatively predict node status in individual patients, modern radiological imaging techniques, sentinel node and technique that uses a computerized database of information to convert a large amount of information and experience to a treatment decision for an individual patient. The aim of this study was to evaluate accuracy in preoperative prediction of lymph node status in selected patients with the help of computer analysis for stage-appropriate surgery. With the help of computer programs Win Estimate and Microsoft Access, we constructed an artificial neural network that calculated a statistical prediction of nodal status in an observed patient with preoperatively gathered data. In 110 patients who have undergone R0 resection with D2 lymphadenectomy, the differences between the individual results generated by artificial neural network calculation and the actual data were compared. The accuracy of computerized predictions of N0 stage for study group is 91%, sensitivity 94% and specificity 87%. The results of accuracy of computerized preoperative prediction of N2 stage are 88%, with sensitivity 94% and specificity 88%. Preoperative analyses of patient data and tumour characteristics offers a rational approach to individualizing tumour therapy where the extent of lymph node dissection is tailored to the type, site, and stage of the tumour, thereby minimizing the disadvantages associated with the extensive operative procedure.     

Immunohystochemical expression of cancer/testis antigens (MAGE-A3/4, NY-ESO-1) in non-small cell lung cancer: the relationship with clinical-pathological features

The aim of this study was to explore the expression of cancer/testis tumor associated antigens (C/T TAAs) MAGE-A 3/4 and NY-ESO-1 in lung squamous cell carcinoma and adenocarcinoma, and to evaluate their association with the standard clinical-pathological features of surgically treated lung cancer patients. The study included 80 patients with non-small cell lung cancer (40 adenocarcinomas, 40 squamous cell carcinomas) who had undergone surgery in the period between 2002 and 2005. The MAGE-A3/4 and NY-ESO-1 antigen expression was analyzed immunohistochemically (IHC). The results showed MAGE-A3/4 and NY-ESO-1 positive staining in 65.1% and 23.3% of squamous cell carcinomas and 18.9% and 10.8% of adenocarcinomas, respectively. A statistically higher MAGE-A3/4 expression was observed in planocellular bronchial carcinoma (p < 0.001), while no difference was found in the expression of NY-ESO-1 in adenocarcinoma and planocellular carcinoma (p = 0.144). A significant association was found between the MAGE-A3/4 expression and presence of tumor necrosis in squamous cell cancer specimens (p = 0.001), but not in adenocarcinoma (p = 0.033). A statistically significant association was noted between the NY-ESO-1 expression and positive hilar and mediastinal lymph nodes in adenocarcinoma (p = 0.025) whereas it was not the case in squamous cell carcinoma. Non-small cell lung cancer frequently expresses cancer/testis tumor associated antigens. Our results demonstrate that the MAGE-A3/4 and NY-ESO-1 expression was significant associated with prognostic factors of poor outcome of disease (presence of tumor necrosis and lymph node metastasis). As C/T antigens are important for inducing a specific immune reaction in lung cancer patients, there is an intention to form a subgroup of patients in the future, whose treatment would be enhanced by specific immunotherapy based on the observed scientific results.     

Solution structure of a let-7 miRNA:lin-41 mRNA complex from C. elegans

let-7 microRNA (miRNA) regulates heterochronic genes in developmental timing of the nematode Caenorhabditis elegans. Binding of miRNA to messenger RNA (mRNA) and structural features of the complex are crucial for gene silencing. We herein present the NMR solution structure of a model mimicking the interaction of let-7 miRNA with its complementary site (LCS 2) in the 3′ untranslated region (3′-UTR) of the lin-41 mRNA. A structural study was performed by NMR spectroscopy using NOE restraints, torsion angle restraints and residual dipolar couplings. The 33-nt RNA construct folds into a stem–loop structure that features two stem regions which are separated by an asymmetric internal loop. One of the stems comprises a GU wobble base pair, which does not alter its overall A-form RNA conformation. The asymmetric internal loop adopts a single, well-defined structure in which three uracils form a base triple, while two adenines form a base pair. The 3D structure of the construct gives insight into the structural aspects of interactions between let-7 miRNA and lin-41 mRNA.     

Inhibition of NaV1.6 sodium channel currents by a novel series of 1,4-disubstituted-triazole derivatives obtained via copper-catalyzed click chemistry

We have synthesized and evaluated a series of 1,4-disubstituted-triazole derivatives for inhibition of the rat Na_V1.6 sodium channel isoform, an isoform thought to play an important role in controlling neuronal firing. Starting from a series of 2,4(1H)-diarylimidazoles previously published, we decided to extend the SAR study by replacing the imidazole with a different heterocyclic scaffold and by varying the aryl substituents on the central aromatic ring. The 1,4-disubstituted 1,2,3-triazoles were prepared employing the copper-catalyzed azide–alkyne cycloaddition (CuAAC). Many of the new molecules were able to block the rNa_v1.6 currents at 10 μM by over 20%, displaying IC₅₀ values ranging in the low micromolar, thus indicating that triazole can efficiently replace the central heterocyclic core. Moreover, the introduction of a long chain at C4 of the central triazole seems beneficial for increased rNa_v1.6 current block, whereas the length of N1 substituent seems less crucial for inhibition, as long as a phenyl ring is not direcly connected to the triazole. These results provide additional information on the structural features necessary for block of the voltage-gated sodium channels. These new data will be exploited in the preparation of new compounds and could result in potentially useful AEDs.     

GMP Production of Liposomes—A New Industrial Approach

A new scalable liposome production system is presented, which is based on the ethanol injection technique. The system permits liposome manufacture regardless of production scale, as scale is determined only by free disposable vessel volumes. Once the parameters are defined, an easy scale up can be performed by just changing the process vessels. These vessels are fully sterilizeable and all raw materials are transferred into the sanitized and sterilized system via 0.2 μm filters to guarantee an aseptic production.

Liposome size can be controlled by the local lipid concentration at the injection point depending on process parameters like injection pressure, lipid concentration and injection rate. These defined process parameters are furthermore responsible for highly reproducible results with respect to vesicle diameters and encapsulation rates Compared to other technologies like the film method which is normally followed by size reduction through high pressure homogenization, ultrasonication or extrusion, no mechanical forces are needed to generate homogeneous and narrow distributed liposomes.

Another important advantage of this method is the suitability for the entrapment of many different drug substances such as large hydrophilic proteins by passive encapsulation, small amphiphilic drugs by a one step remote loading technique or membrane association of antigens for vaccination approaches     

Influence of Postprandial Intragastric Pressures on Drug Release from Gastroretentive Dosage Forms

Despite extensive research in the field of gastroretentive dosage forms, this “holy grail” of oral drug delivery yet remained an unmet goal. Especially under fasting conditions, the reproducible retention of dosage forms in the stomach seems to be an impossible task. This is why such systems are often advised to be taken together with food. But also the postprandial motility can contribute significantly to the failure of gastroretentive dosage forms. To investigate the influence of postprandial pressure conditions on drug release from such systems, we used a novel in vitro dissolution tool, the dissolution stress test device. With the aid of this device, we simulated three different intragastric pressure profiles that may occur after postprandial intake. These transit scenarios were based on recently obtained, postprandial SmartPill® data. The tested systems, Glumetza® 1000 and Madopar® HBS 125, are marketed dosage forms that are based on different approaches to achieve proper gastric retention. All three transit scenarios revealed a highly pressure-sensitive drug release behavior, for both drugs. For Madopar® HBS 125, nearly complete drug release was observed even after early occurring pressures. Glumetza® 1000 seemed to be more resistant to these, most likely due to incomplete wetting of the system. On the contrary to these findings, data from standard dissolution tests using the paddle apparatus displayed controlled drug release for both systems for about 6 h. Based on these results, it can be doubted that established gastroretentive systems stay intact over a longer period of time, even under postprandial conditions.