In pursuit of new developments for gene therapy of human diseases

Gene therapy aims at transferring a therapeutic gene into human somatic cells in order to treat a disease. Originally addressed to hereditary genetic disorders, gene therapy has found therapeutic applications in cancer, infectious diseases and degenerative disorders, particularly those of the nervous system. Although gene transfer into humans has been demonstrated in several clinical trials, with more than 300 currently underway worldwide, there is still no single outcome that undoubtedly showed a consistent benefit for the patient. Nevertheless, the expectations for gene therapy are still high, and the prospects of future clinical success are increasing together with the growing of the field. The development of better delivery systems specifically tailored to individual diseases, with sustained expression of the therapeutic gene in the appropriate cells, will in the end make possible true therapeutic applications of human gene transfer.     

Structural characterization of transglutaminase-catalyzed cross-linking between glyceraldehyde 3-phosphate dehydrogenase and polyglutamine repeats

The accumulation of abnormal polyglutamine-containing protein aggregates within the cytosol and nuclei of affected neurons is a hallmark of the progressive neurodegenerative disorders caused by an elongated (CAG)(n) repeat in the genome. The polyglutamine domains are excellent substrates for the enzyme transglutaminase type 2 (tissue), resulting in the formation of cross-links with polypeptides containing lysyl groups. Enzymatic activity toward the Q(n) domains increases greatly upon lengthening of such Q(n) stretches (n > 40). Among the possible amine donors, the glycolytic enzyme glyceraldehyde-3-phosphate-dehydrogenase was shown to tightly bind several proteins involved in polyglutamine expansion diseases. Recently, the authors have shown that K191, K268, and K331, out of the 26 lysines present in glyceraldehyde-3-phosphate-dehydrogenase, are the reactive amine-donor sites forming cross-links with substance P, which bears the simplest Q(n) domain (n = 2). The present study reports that synthetic peptides of both pathological and nonpathological length (n = 43 and 17, respectively) form cross-links with the same K residues located in the C-terminal region of glyceraldehyde-3-phosphate-dehydrogenase. In addition, it is shown that extra K residues present in the C termini of glyceraldehyde-3-phosphate-dehydrogenase are susceptible to cross-linking in the presence of transglutaminase. The present results indicate a possible modulating effect of Q(n) stretches on tissue transglutaminase substrate specificity and mechanism of recognition.     

Distribution and diversity of cry genes in native strains of Bacillus thuringiensis obtained from different ecosystems from Colombia

Colombia is a tropical country located at the north of South America. It is considered to be one of the most important countries in terms of its biodiversity worldwide. One hundred and eight soil samples obtained from agricultural crops and wild ecosystems were evaluated in terms of the presence of Bacillus thuringiensis (Bt) native strains. One hundred and eight different Bt strains were isolated and characterized by the presence of crystal proteins by SDS-PAGE and a multiplex PCR with general and specific primers for cry1 and cry3, cry7, and cry8 gene detection. Most of the Bt strains (73%) reacted with the cry1 general primers; 27.8% of the Bt strains reacted with cry3, cry7, and cry8 general primers and 17.8% of strains did not react with any of these two sets of primers. Thirty different PCR profiles were found in the strains with cry1 genes when they were analyzed with specific primers (cry1A to cry1F). A high frequency of joint occurrence was observed for cry1Aa/cry1Ab, cry1Aa/cry1Ac, cry1Ab/cry1Ac, and cry1C/cry1D genes with a Pearson coefficient of 0.88, 0.74, 0.76, and 0.87, respectively. Other distinctive characteristics were found in the Colombian collection as the presence of 22.2% of native strains which presented, at the same time, lepidopteran and coleopteran active genes. Interesting relations were found as well between the cry gene distribution and the geographical areas sampled. Finally, some strains with moderate to high biopesticide activity against Spodoptera frugiperda (Lepidoptera) and Premnotrypes vorax (Coleoptera) insects were identified, this being important to explore future microbial strategies for the control of these crop pests in the region.     

Erythroid differentiation and regulatory gene expression are modulated by adenosine derivatives interfering with S-adenosylmethionine metabolic pathway

The differentiation of murine erythroleukemia cells and the expression of SCL, Id1 and c-myc regulatory genes were studied. The first gene is a positive regulator of differentiation, while the other two are both negative regulators of differentiation and positive regulators of proliferation. Accordingly, our data show that when differentiation is stimulated SCL is upregulated while Id1 and c-myc are, coordinately, downregulated. The cultures were treated with two adenosine derivatives, 3-deazaadenosine and 3-deazaaristeromycin, known to act on the metabolic pathway of the methyl donor S-adenosylmethionin, in order to assess the possibility of a coordinated modulation, by these drugs, of regulatory gene expression and erythroid cell differentiation. 3-Deazaaristeromycin caused the simultaneous downregulation of Id1 and c-myc, whereas 3-deazaadenosine caused their upregulation; both drugs produced a transient increase in SCL expression. The use of these drugs evidenced a predominant regulatory effect of negative regulators in the control of erythroid differentiation. The distinct effects of the two drugs on regulatory gene expression led to an increased differentiation induced by 3-deazaaristeromycin and to a reduced differentiation induced by 3-deazaadenosine, if compared with controls. Southern analysis of DNA digested with methylation-specific restriction endonucleases showed that the administration of 3-deazaaristeromycin resulted in hypomethylation of SCL and c-myc, thus evidencing, in these cells, a clear correlation between DNA hypomethylation and differentiation but no straightforward correlation between DNA methylation and gene expression.     

High levels of exogenous C2-ceramide promote morphological and biochemical evidences of necrotic features in thyroid follicular cells

CD95 and ceramide are known to be involved in the apoptotic mechanism. The triggering of CD95 induces a cascade of metabolic events that progressively and dramatically modifies the cell shape by intense membrane blebbing, leading to apoptotic bodies production. Although the CD95 pathway has been abundantly described in normal thyrocytes, the effects of cell permeable synthetic ceramide at morphological and biochemical levels are not fully known. In the present study, we show that thyroid follicular cells (TFC) exposed to 20 microM of C(2)-ceramide for 4 h are characterized by morphological features of necrosis, such as electron-lucent cytoplasm, mitochondrial swelling, and loss of plasma membrane integrity without drastic morphological changes in the nuclei. By contrast, TFC treated with 2 microM of C(2)-ceramide for 4 h are able to accumulate GD3, activate caspases cascade, and induce apoptosis. Furthermore, we provide evidence that 20 microM of C(2)-ceramide determine the destruction of mitochondria and are not able to induce PARP cleavage and internucleosomal DNA fragmentation, suggesting that the apoptotic program is not activated during the death process and nuclear DNA is randomly cleaved as the consequence of cellular degeneration. Pretreatment with 30 microM of zVAD-fmk rescued TFC from 2 microM of C(2)-ceramide-induced apoptosis, whereas, 20 microM of C(2)-ceramide exposure induced necrotic features. Deltapsi(m) was obviously altered in cells treated with 20 microM of C(2)-ceramide for 4 h (75% +/- 3.5%) compared with the low percentage (12.5% +/- 0.4%) of cells with altered Deltapsi(m) exposed to 2 microM of C(2)-ceramide. Whereas, only 20% +/- 1.1% of cells treated with anti-CD95 for 1 h showed altered Deltapsi(m). Additionally, Bax and Bak, two pro-apoptotic members, seem to be not oligomerized in the mitochondrial membrane following ceramide exposure. These results imply that high levels of exogenous ceramide contribute to the necrotic process in TFC, and may provide key molecular basis to the understanding of thyroid signaling pathways that might promote the apoptotic mechanism in thyroid tumoral cells.     

Mannan changes induced by 3-methyl-5-aminoisoxazole-4-thiocyanate,a new azole derivative,on Epidermophyton floccosum

The antifungal activity of 3-methyl-5-aminoisoxazole-4-thiocyanate, a new azole derivative, was studied on the dermatophyte Epidermophyton floccosum. The compound strongly inhibited the in vitro growth of two different strains of the fungus and even induced profound morphogenetic anomalies. Optical and electron microscopy showed that such treatment targets the endomembrane system, particularly the plasmalemma, causing abnormal extrusion of the wall mannans. This results in improper arrangement of the different parietal materials; the walls are thus weak and subject to subapical rupture which terminates cell growth and elongation of the hypha. The morphological results and the preliminary biochemical data on fungal sterols suggest that this compound employs an action mechanism similar to that of other azoles used in therapy.     

Influence of a low background radiation environment on biochemical and biological responses in V79 cells

We present the results of an experiment aimed at comparing the effects of different background radiation environments on metabolism and responses to gamma-rays and cycloheximide of cultured mammalian cells. Chinese hamster V79 cells were maintained in exponential growth in parallel for up to 9 months at the Istituto Superiore di Sanità (ISS) and at the INFN-Gran Sasso underground Laboratory (LNGS) where exposure due to gamma-rays and to radon was reduced by factors of about 70 and 25, respectively. After 9 months the cells grown at the LNGS (cumulative gamma dose about 30 microGy, average radon concentration around 5 Bq/m(3)), compared to the cells grown at the ISS (cumulative gamma-ray dose about 2 mGy, average radon concentration around 120 Bq/m(3)), exhibited i). a significant increase of the cell density at confluence, ii). a significantly higher capacity to scavenge organic and inorganic hydroperoxides but a reduced scavenging capacity towards superoxide anions and iii). an increase in both the basal hprt mutation frequency and sensitivity to the mutagenic effect of gamma-rays. The cells grown at the LNGS also showed a greater apoptotic sensitivity starting at the third month of culture, that was no longer detected after 9 months. Overall, these data suggest a role of background ionizing radiation in determining an adaptive response, although they cannot be considered conclusive.     

Cultivation of explants of the marine sponge Crambe crambe in closed systems

Explants of the sponge Crambe crambe were cultured in natural seawater, with or without marine microalga (Phaeodactylum tricornutum) in discontinuous flow through systems and in continuous flow-through systems (DFTHS and CFTHS, respectively). Growth was measured as the increase in underwater weight. In the experiment carried out in the CFTHS, the explants average underwater weight increased by up to 1380% of the initial weight in 22-45 days. Growth in DFTHS was much slower producing a gain of up to an average value of 322% of the initial weight in 100-210 days. Growth kinetics varied considerably for different explants. Explants grew fastest in the first 10-days of subculture. The sponges grew better in CFTHS compared with the DFTHS. The high growth rates observed in CFTHS suggest that this technique is a promising method for culturing C. crambe in closed systems.     

Effect of deamidation on folding of ribonuclease A

The folding of ribonuclease A (RNase A) has been extensively studied by characterizing the disulfide containing intermediates using different experimental conditions and analytical techniques. So far, some aspects still remain unclear such as the role of the loop 65-72 in the folding pathway. We have studied the oxidative folding of a RNase A derivative containing at position 67 the substitution Asn --> isoAsp where the local structure of the loop 65-72 has been modified keeping intact the C65-C72 disulfide bond. By comparing the folding behavior of this mutant to that of the wild-type protein, we found that the deamidation significantly decreases the folding rate and alters the folding pathway of RNase A. Results presented here shed light on the role of the 65-72 region in the folding process of RNase A and also clarifies the effect of the deamidation on the folding/unfolding processes. On a more general ground, this study represents the first characterization of the intermediates produced along the folding of a deamidated protein.     

Genomic prediction in CIMMYT maize and wheat breeding programs

Genomic selection (GS) has been implemented in animal and plant species, and is regarded as a useful tool for accelerating genetic gains. Varying levels of genomic prediction accuracy have been obtained in plants, depending on the prediction problem assessed and on several other factors, such as trait heritability, the relationship between the individuals to be predicted and those used to train the models for prediction, number of markers, sample size and genotype × environment interaction (GE). The main objective of this article is to describe the results of genomic prediction in International Maize and Wheat Improvement Center''s (CIMMYT''s) maize and wheat breeding programs, from the initial assessment of the predictive ability of different models using pedigree and marker information to the present, when methods for implementing GS in practical global maize and wheat breeding programs are being studied and investigated. Results show that pedigree (population structure) accounts for a sizeable proportion of the prediction accuracy when a global population is the prediction problem to be assessed. However, when the prediction uses unrelated populations to train the prediction equations, prediction accuracy becomes negligible. When genomic prediction includes modeling GE, an increase in prediction accuracy can be achieved by borrowing information from correlated environments. Several questions on how to incorporate GS into CIMMYT''s maize and wheat programs remain unanswered and subject to further investigation, for example, prediction within and between related bi-parental crosses. Further research on the quantification of breeding value components for GS in plant breeding populations is required.