SpA: web-accessible spectratype analysis: data management, statistical analysis and visualization

SUMMARY: SpA is a web-accessible system for the management, visualization and statistical analysis of T-cell receptor spectratype data. Users upload data from their spectratype analyzers to SpA, which saves the raw data and user-defined supplementary covariates to a secure database. The statistical engine performs several data analyses and statistical summaries. The visualization engine displays spectratype histograms in a Java applet and in an image file suitable for download. All of these results are also saved to the database and remain accessible to the user. Additional statistical tools specific to the analysis of multiple spectratypes are also available through the SpA interface. AVAILABILITY: The service is freely accessible via the web at http://www.duke.edu/~kepler/spa.html. Additional technical support and specialized statistical analysis and consultation are available by arrangement with the authors and, depending on the service requested, may be subject to fee.     

Biodiesel production from triolein and short chain alcohols through biocatalysis

Oleic acid alkyl esters (biodiesel) were synthesised by biocatalysis in solvent-free conditions. Different commercial immobilised lipases, namely Candida antarctica B, Rizhomucor miehei, and Pseudomonas cepacia, were tested towards the reaction between triolein and butanol to produce butyl oleate. Pseudomonas cepacia lipase resulted to be the most active enzyme reaching 100% of conversion after 6h. Different operative conditions such as reaction temperature, water activity, and reagent stoichiometric ratio were investigated and optimised. These conditions were then used to investigate the effect of linear and branched short chain alcohols. Methanol and 2-butanol were the worst alcohols: the former, probably, due to its low miscibility with the oil and the latter because secondary alcohols usually are less reactive than primary alcohols. Conversely, linear and branched primary alcohols with short alkyl chains (C(2)--C(4)) showed high reaction rate and conversion. A mixture of linear and branched short chain alcohols that mimics the residual of ethanol distillation (fusel oil) was successfully used for oleic acid ester synthesis. These compounds are important in biodiesel mixtures since they improve low temperature properties.     

Trypanocidal activity of (-)-cubebin derivatives against free amastigote forms of Trypanosoma cruzi

Five (-)-cubebin derivative compounds, (-)-O-acetyl cubebin (3), (-)-O-benzyl cubebin (4), (-)-O-(N,N-dimethylaminoethyl)-cubebin (5), (-)-hinokinin (6) and (-)-6,6'-dinitrohinokinin (7), previously synthesised by our research group, were evaluated on in vitro assay against free amastigote forms of Trypanosoma cruzi, the asogic agent of Chagas' disease. It was observed that 6 was the most active compound (IC(50)=0.7 microM), and that 4 and 5 displayed moderate activity against the parasite, giving IC(50) values of 5.7 and 4.7 microM, respectively. In contrast, it was observed that compound 3 was inactive and that 7 displayed low activity with IC(50) values of congruent with 1.5 x 10(4) and 95.3 microM, respectively.     

Improved synthesis of daunomycin conjugates with triplex-forming oligonucleotides. The polypurine tract of HIV-1 as a target

Triple helix-forming oligonucleotides (TFOs) are promising agents for the control of gene expression, as they can selectively bind to a chosen oligopyrimidine.oligopurine region of a gene of interest thus interfering with its expression. The stability of the triplex formed by the TFO and the duplex is often too poor for successful applications of TFOs in vivo and the conjugation of a DNA intercalating moiety to the TFO is a common way to enhance the TFO affinity for its target. In a previous work, we investigated the properties of daunomycin conjugated TFO (dauno-TFO) and found that this class of compounds showed a higher degree of affinity than native oligonucleotides for an oligopyrimidine.oligopurine duplex target and that the presence of the amino sugar increases such stability. Here, we report a significantly improved synthetic procedure for the preparation of the conjugates, based on the protection of the daunosamine moiety by N-trifluoroacetylation. This protecting group is removed as a final step from the conjugation product by mild basic hydrolysis to give the desired dauno-TFO. Compared to the previous synthetic procedure, the improvement is important. The synthesis is now more reproducible and no side products are formed. Moreover, the thus protected daunomycin derivative is very stable, up to at least one year. Two dauno-TFOs, differing by the length of the oligonucleotide moiety, were prepared to target the polypurine tract (PPT) of HIV-1. Triplex formation by these compounds with model duplexes was studied by UV spectroscopy, thermal gradient gel electrophoresis (TGGE) and gel electrophoretic mobility shift. The experimental results demonstrate that dauno-TFOs bind to the PPT of HIV-1 more strongly than the unconjugated TFOs.     

Direct effect of a gonadotropin-releasing hormone agonist on the growth of canine mammary tumour cells

Gonadotropin-releasing hormone (GnRH) agonist exert "in vivo" an inhibitory action on the growth of hormone-dependent canine mammary tumours (Lombardi et al. [1999] J. Vet. Pharmacol Ther. 22(1):56-61). The present experiments have been performed "in vitro" in order to investigate the mechanisms involved in this direct antiproliferative action of GnRH agonists. In particular, the aim was to study whether these compounds might exert their antiproliferative effect by interfering with the stimulatory action of epidermal growth factor (EGF). To this purpose, the effects of GnRH agonist, Goserelin (GnRH-A), on the mitogenic action of EGF, on EGF-activated intracellular signaling mechanisms (intracellular calcium and nitric oxide production) as well as on ATP induced cell proliferation and signalling, and on the binding of EGF receptors have been evaluated in primary culture of canine mammary tumour cells. The results of these "in vitro" studies show that GnRH-A counteracts the mitogenic action of EGF and ATP, decreases the EGF/ATP-induced calcium signalling and reduces EGF binding, probably by means of NO-induced [Ca2+]i downregulation. These data suggest that GnRH agonists may inhibit the proliferation of the tumour cells by interfering with the stimulatory action of EGF.     

Whale (Balaenoptera physalus) haemoglobin: primary structure,functional characterisation and computer modelling studies

The functional properties of haemoglobin from the Mediterranean whale Balaenoptera physalus have been studied as functions of heterotropic effector concentration and temperature. Particular attention has been given to the effect of carbon dioxide and lactate since the animal is specialised for prolonged dives often in cold water. The molecular basis of the functional behaviour and in particular of the weak interaction with 2,3-diphosphoglycerate is discussed in the light of the primary structure and of computer modelling. On these bases, it is suggested that the A2 (Pro-->Ala) substitution observed in the beta chains of whale haemoglobin may be responsible for the displacement of the A helix known to be a key structural feature in haemoglobins that display an altered interaction with 2,3-diphosphoglycerate as compared with human haemoglobin. The functional and structural results, discussed in the light of a previous study on the haemoglobin from the Arctic whale Balaenoptera acutorostrata, give further insights into the regulatory mechanisms of the interactive effects of temperature, carbon dioxide and lactate.