Development of a non-viral gene delivery vector based on the dynein light chain Rp3 and the TAT peptide.

Gene therapy and DNA vaccination trials are limited by the lack of gene delivery vectors that combine efficiency and safety. Hence, the development of modular recombinant proteins able to mimic mechanisms used by viruses for intracellular trafficking and nuclear delivery is an important strategy. We designed a modular protein (named T-Rp3) composed of the recombinant human dynein light chain Rp3 fused to an N-terminal DNA-binding domain and a C-terminal membrane active peptide, TAT. The T-Rp3 protein was successfully expressed in Escherichia coli and interacted with the dynein intermediate chain in vitro. It was also proven to efficiently interact and condense plasmid DNA, forming a stable, small (∼100nm) and positively charged (+28.6mV) complex. Transfection of HeLa cells using T-Rp3 revealed that the vector is highly dependent on microtubule polarization, being 400 times more efficient than protamine, and only 13 times less efficient than Lipofectamine 2000™, but with a lower cytotoxicity. Confocal laser scanning microcopy studies revealed perinuclear accumulation of the vector, most likely as a result of transport via microtubules. This study contributes to the development of more efficient and less cytotoxic proteins for non-viral gene delivery.

Small-angle X-ray scattering and in silico modeling approaches for the accurate functional annotation of an LysR-type transcriptional regulator.

Xylella fastidiosa is a xylem-limited, Gram-negative phytopathogen responsible for economically relevant crop diseases. Its genome was thus sequenced in an effort to characterize and understand its metabolism and pathogenic mechanisms. However, the assignment of the proper functions to the identified open reading frames (ORFs) of this pathogen was impaired due to a lack of sequence similarity in the databases. In the present work, we used small-angle X-ray scattering and in silico modeling approaches to characterize and assign a function to a predicted LysR-type transcriptional regulator in the X. fastidiosa (XfLysRL) genome. XfLysRL was predicted to be a homologue of BenM, which is a transcriptional regulator involved in the degradation pathway of aromatic compounds. Further functional assays confirmed the structural prediction because we observed that XfLysRL interacts with benzoate and cis,cis-muconic acid (also known as 2E,4E-hexa-2,4-dienedioic acid; hereafter named muconate), both of which are co-factors of BenM. In addition, we showed that the XfLysRL protein is differentially expressed during the different stages of X. fastidiosa biofilm formation and planktonic cell growth, which indicates that its expression responds to a cellular signal that is likely related to the aromatic compound degradation pathway. The assignment of the proper function to a protein is a key step toward understanding the cellular metabolic pathways and pathogenic mechanisms. In the context of X. fastidiosa, the characterization of the predicted ORFs may lead to a better understanding of the cellular pathways that are linked to its bacterial pathogenicity.

Characterization of an oxidative stress response regulator, homologous to Escherichia coli OxyR, from the phytopathogen Xylella fastidiosa.

The OxyR oxidative stress transcriptional regulator is a DNA-binding protein that belongs to the LysR-type transcriptional regulators (LTTR) family. It has the ability to sense oxidative species inside the cell and to trigger the cell's response, activating the transcription of genes involved in scavenging oxidative species. In the present study, we have overexpressed, purified and characterized the predicted OxyR homologue (orf xf1273) of the phytopathogen Xylella fastidiosa. This bacterium is the causal agent of citrus variegated chlorosis (CVC) disease caused by the 9a5c strain, resulting in economic and social losses. The secondary structure of the recombinant protein was analyzed by circular dichroism. Gel filtration showed that XfoxyR is a dimer in solution. Gel shift assays indicated that it does bind to its own predicted promoter under in vitro conditions. However, considering our control experiment we cannot state that this interaction occurs in vivo. Functional complementation assays indicated that xfoxyR is able to restore the oxidative stress response in an oxyr knockout Escherichia coli strain. These results show that the predicted orfxf1273 codes for a transcriptional regulator, homologous to E. coli OxyR, involved in the oxidative stress response. This may be important for X. fastidiosa to overcome the defense mechanisms of its host during the infection and colonization processes.

Ultraestrutura do Paracoccidioides brasiliensis. I - Na fase filamentosa. / Ultrastructure of Paracoccidioides brasiliensis. I - In the mycelial phase

Sao estudados alguns aspectos ultraestruturais do Paracoccidioides brasiliensis em cultura na fase filamentosa, dando-se enfase as celulas que constituem as hifas.Alem dos constituintes citoplasmaticos, sao descritos em detalhes o envoltorio celular, constituido pela membrana plasmatica, membrana basal e parede celular, e o septo que e a zona de contacto entre as celulas que se encontram alinhadas ao longo das hifas. Baseando-se nos aspectos ultraestruturais destas celulas e em seu grau de interrelacionamento, e discutida a possibilidade das mesmas constituirem-se em clamidosporos em potencial

Ultraestrutura do Paracoccidioides brasiliensis. II - Na fase leveduriforme. / Ultrastructure of Paracoccidioides brasiliensis. II - In the yeast phase

Sao estudados alguns aspectos ultraestruturais do Paracoccidioides brasiliensis em cultura na fase leveduriforme, dando se enfase ao envoltorio celular encontrado nesta fase: na fase leveduriforme, aparentemente nao ocorre membrana basal, estrutura facilmente identificavel na fase filamentosa; a parede celular e pouco eletron densa e nao apresenta aspecto laminado tal como observado na fase filamentosa e a membrana plasmatica e de facil identificacao. O microrganismo na fase leveduriforme e unicelular e apresenta nucleos, cromatina e nucleolos bem evidentes. Sua divisao se processa por brotamentos, sendo as gemulas facilmente identificaveis pela microscopia eletronica