An Autogenously Regulated Expression System for Gene Therapeutic Ocular Applications.

The future of treating inherited and acquired genetic diseases will be defined by our ability to introduce transgenes into cells and restore normal physiology. Here we describe an autogenous transgene regulatory system (ARES), based on the bacterial lac repressor, and demonstrate its utility for controlling the expression of a transgene in bacteria, eukaryotic cells, and in the retina of mice. This ARES system is inducible by the small non-pharmacologic molecule, Isopropyl ß-D-1-thiogalactopyranoside (IPTG) that has no off-target effects in mammals. Following subretinal injection of an adeno-associated virus (AAV) vector encoding ARES, luciferase expression can be reversibly controlled in the murine retina by oral delivery of IPTG over three induction-repression cycles. The ability to induce transgene expression repeatedly via administration of an oral inducer in vivo, suggests that this type of regulatory system holds great promise for applications in human gene therapy.

Enhanced extracellular production of recombinant Bacillus deramificans pullulanase in Escherichia coli through induction mode optimization and a glycine feeding strategy.

Process optimization strategies were developed to improve extracellular production of recombinant Bacillus deramificans pullulanase in Escherichia coli. Cell growth and pullulanase production in shake-flask cultures were investigated as a function of the concentration of added glycine, and the type and concentration of inducer. From the results of these experiments, a fed-batch fermentation strategy for high-cell-density cultivation was applied in a 3-L fermentor. The gradual addition of lactose was utilized for the induction of protein expression. The optimal lactose feeding rate and induction point were 0.4gL(-1)h(-1) and a dry cell weight (DCW) of 15gL(-1), respectively. Furthermore, a glycine feeding strategy was formulated to promote the secretion of recombinant protein. The optimal total and extracellular pullulanase activity were 2523.5 and 1567.9UmL(-1), respectively, which represent 1.2 and 22.6-fold increases compared with those observed under unoptimized conditions.

Evaluation of a recombinant p24 antigen for the detection of feline immunodeficiency virus-specific antibodies / Avaliação do antígeno recombinante p24 para a detecção de anticorpos específicos do vírus da imunodeficiência felina

Feline Immunodeficiency Virus is a worldwide infection and is considered a significant pathogen. The diagnosis of FIV infections is mainly based on commercially available rapid tests that are highly expensive in Brazil, hence it is rarely performed in the country. Furthermore, lentiviruses grow slowly and poorly in tissue cultures, making the production of viral antigen by classic means and thus the establishment of FIV immunodiagnosis impracticable. In order to deal with this, recombinant DNA techniques were adopted to produce the protein p24, a viral capsid antigen. The protein's reactivity evaluation analyzed by Western blot indicated that this recombinant antigen can be a useful tool for the immunodiagnostic of FIV infections.

O vírus da imunodeficiência felina tem distribuição mundial e é considerado um patógeno significativo. No Brasil, a prática diagnóstica é baseada principalmente em teste rápidos, importados e de custo elevado, disponíveis comercialmente. Devido ao seu custo proibitivo em nosso país, o diagnóstico da infecção pelo FIV é raramente realizado. Ademais, os lentivírus se multiplicam lenta e pobremente em cultura de células, o que torna a produção de antígeno por meios clássicos e o estabelecimento do imunodiagnóstico impraticável. Com o objetivo de lidar com esta questão, técnicas de DNA recombinante foram utilizadas para produção de um antígeno do capsídeo viral, a proteína p24. A avaliação da reatividade realizada por Western blot indicou que este antígeno recombinante pode ser útil para o imunodiagnóstico de infecções pelo FIV.

Cloning of dextransucrase gene from Leuconostoc citreum HJ-P4 and its high-level expression in E. coli by low temperature induction.

A dextransucrase (LcDS) gene from Leuconostoc citreum HJ-P4 has been amplified and cloned in E. coli. The LcDS gene consists of 4,431 nucleotides encoding 1,477 amino acid residues sharing 63-98% of amino acid sequence identities with other known dextransucrases from Leuc. mesenteroides. Interestingly, 0.1 mM of IPTG induction at 15 degrees remarkably increased the LcDS productivity to 19,187 U/l culture broth, which was over 330-fold higher than that induced at 37 degrees. Optimal reaction temperature and pH of LcDS were determined as 35 degrees and pH 5.5 in 20 mM sodium acetate buffer, respectively. Meanwhile, 0.1 mM CaCl(2) increased its activity to the maximum of 686 U/mg, which was 2.1-fold higher than that in the absence of calcium ion. Similar to the native Leuconostoc dextransucrase, recombinant LcDS could successfully produce a series of isomaltooligosaccharides from sucrose and maltose, on the basis of its transglycosylation activity.

Anti-sense inhibition of small-heat-shock-protein (HSP27) expression in MCF-7 mammary-carcinoma cells induces their spontaneous acquisition of a secretory phenotype.

This work was aimed at testing the hypothesis (hitherto supported only by indirect evidence) that, besides contributing to resistance to stress, the small heat-shock-protein HSP27 might be involved in the control of growth and differentiation in mammary-tumour cells, where it is known to be oestrogen-regulated. Therefore, MCF-7 cells were transfected with a modulatable human hsp27 anti-sense cDNA. Clones of transfectants (designated alphahsp27) were selected which, upon expression of the anti-sense, exhibited a decline in HSP27 accumulation, associated with a decrease in resistance to heat shock and in proliferation rate, the degree of the latter reflecting their respective reduction in HSP27 content. The effects of anti-sense inhibition of HSP27 production were similar to those exerted on parental cells by phorbol myristate (TPA). Both resulted in growth inhibition, accumulation of lipid droplets in the cytoplasm, formation of secretory microvesicles with internal microvilli and increased release of several proteins, including the isoforms of a 52-kDa protein, which we identified as the oestrogen-regulated protein cathepsin D, all this without noticeable change in actin organization. These data constitute the first direct support for the hypothesis that, at least in some cell types, HSP27 might play a modulatory role in cell differentiation and (perhaps by this) in proliferation. While allowing dissociation of this role from the known action of HSP27 on actin polymerization, they suggest similar modulation of the function of some protein(s) implicated in the acquisition of the secretory phenotype by MCF-7 cells, with HSP27 also exerting an inhibitory action that can be alleviated either by its phosphorylation (as occurs with TPA) or by inhibition of its production.