Desigualdades sociales en la mortalidad por VIH y tumores malignos en municipios del departamento de Valle del Cauca (Colombia), según indicadores económicos 2009-2013 / Social inequalities in HIV mortality and malignant tumours inmunicipalities in the Department of Valle del Cauca (Colombia), accordingto economic indicators 2009-2013

Objetivo: identificar las desigualdades sociales en la mortalidad por VIH y tumor maligno de próstata y útero en municipios de Valle del Cauca según variables socioeconómicas: necesidades básicas insatisfechas, NBI, y valor agregado municipal, VAM, en el periodo 2009 2013. Materiales y Métodos: estudio ecológico para medir desigualdades sociales en las mortalidades por causas de la lista de mortalidad 6/67 de la OMS, VIH tumor maligno de útero, y tumor maligno de próstata, en municipios del departamento de Valle del Cauca, en relación con las variables socio económicas NBI, y VAM. La medición de las desigualdades se realizó con el software Epidat 4.1 se calcularon tasas crudas y tasas ajustadas por sexo y edad con el método directo; y los índices de desigualdad acotado e índice de concentración. Resultados: la tasa más alta en relación a VIH fue de 1,627 en el año 2011 y en mujeres de 2,304 en el año 2012, en tumor maligno de próstata fue de 1,197 en el año 2009 y tumor maligno de útero 1,139 en 2010. Conclusión: los municipios con peores indicadores socioeconómicos tuvieron mayor desigualdad en la mortalidad por VIH y tumor maligno de próstata..(AU)

Objective: to identify the social inequalities in the mortality by HIV and malignant tumour of prostate and uterus in municipalities of Valle del Cauca according to socioeconomic variables: unsatisfied basic necessities, NBI, and municipal added value, VAM, in the period 2009-2013. Materials and methods: ecological study to measure social inequalities in mortalities due to the WHO mortality list 6/67, HIV malignant tumor of the uterus, and malignant prostate tumour, in municipalities in the Department of Valle del Cauca, in relation to the Socioeconomic variables NBI, and VAM. The measurement of inequalities was carried out withthe software Epidat 4.1 crude rates and rates adjusted by sex and age were calculated using the direct method; and the indices of delimited inequality and index of concentration. Results: the highest rate in relation to HIV was 1.627 in theyear 2011 and in females of 2.304 in the year 2012; in malignant prostate tumor was 1.197 in the year 2009 and malignant tumor of the uterus 1.139 in 2010 Conclusion: municipalities with the worst socioeconomic indicators had greater inequality in HIV mortality and malignant prostate tumor..(AU)

Effects of HIV-1 tat on secretion of TNF-α and IL-1β by U87 cells in AIDS patients with or without AIDS dementia complex / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To explore the role of HIV-1 tat gene variations in AIDS dementia complex (ADC) pathogenesis.</p><p><b>METHODS</b>HIV-1 tat genes derived from peripheral spleen and central basal ganglia of an AIDS patient with ADC and an AIDS patient without ADC were cloned for sequence analysis. HIV-1 tat gene sequence alignment was performed by using CLUSTAL W and the phylogentic analysis was conducted by using Neighbor-joining with MEGA4 software. All tat genes were used to construct recombinant retroviral expressing vector MSCV-IRES-GFP/tat. The MSCV-IRES-GFP/tat was cotransfected into 293T cells with pCMV-VSV-G and pUMVC vectors to assemble the recombinant retrovirus. After infection of gliomas U87 cells with equal amount of the recombinant retrovirus, TNF-α, and IL-1β concentrations in the supernatant of U87 cells were determined with ELISA.</p><p><b>RESULTS</b>HIV-1 tat genes derived from peripheral spleen and central basal ganglia of the AIDS patient with ADC and the other one without ADC exhibited genetic variations. Tat variations and amino acid mutation sites existed mainly at Tat protein core functional area (38-47aa). All Tat proteins could induce U87 cells to produce TNF-α and IL-1β, but the level of IL-1β production was different among Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen. The level of Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen were obviously higher than that from the non-ADC patient's basal ganglia.</p><p><b>CONCLUSION</b>Tat protein core functional area (38-47aa) may serve as the key area of enhancing the secretion of IL-1β. This may be related with the neurotoxicity of HIV-1 Tat.</p>

Inhibition of HIV-1 in vitro by combination of vpr and tat specific short hairpin RNA via lentiviral vectors / 病毒学报

Gene therapy offers the promise of curing the HIV-infected patients. Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. To reduce the probability of viral escape mutants, in this study, we constructed lentiviral vector containing vpr and tat shRNA, respectively, furthermore the bispecific lentiviral vector harboring vpr and tat shRNA expression cassettes from U6 promotor and H1 promotor was cotransfected with recombinant plasmid expressing the vpr and tat gene. The result showed that the bispecific lentiviral vector plvx-vpr-tatshRNA could inhibit the vpr and tat effectively,with ratios of 89.20% and 62.00% respectively. When cotransfected with pNL4-3 in 293T cell, plvx-vpr-tatshRNA showed higher efficacy in down regulating the HIV NL4-3 packaging production than the plvx-vprshRNA or plvx-tatshRNA individually. MT4 cell clones transduced with recombinant lentiviral vectors were screened and challenged with HIV NL4-3. P24 ELISA test showed that MT4 transduced with the combinational lentiviral vector could inhibit virus replication efficiently.

Research on Tat peptide-polyethylene glycol modified gelatin-siloxane nanoparticles across the blood-brain barrier / 生物医学工程学杂志

Gelatin-siloxane nanoparticles (GS NPs) have been considered to be good gene carrier candidate in vitro, since they have several advantages such as low toxicity, easy preparation and surface modification. In this study, the Tat-PEG-GS NPs were synthesized by the gelatin-siloxane, surface-modified with the polyethylene glycol (H2 N-PEG-COOH) and Tat peptide (KYGRRRQRRKKRGC) and thus constructed a delivery system which can cross BBB (Blood-brain barrier). The morphology, diameter, and zeta potential of Tat-PEG-GS NPs carrier system were characterized with transmission electron microscopy (TEM) and Nano-ZS zetasizer dynamic light scattering Detector. The organ distribution and dynamic evolution localized in the brain parenchyma of Tat-PEG-GS NPs in vivo was investigated with Cri in vivo imaging system and TEM. The obtained Tat-PEG-GS NPs were approximately spherical in shape with average particle size of 150-200 nm and zeta potentials of (32.27 +/- 2.47) mV. In vivo imaging results showed that the accumulation of Tat-PEG-GS NPs was higher in the brain than the accumulation of PEG-GS NPs, but the accumulation of Tat-PEG-GS NPs was lower in the liver than the accumulation of PEG-GS NPs. These differences are statistically significant. The nanocomplex could cross the BBB and reach the neural tissues tested with TEM. The Tat-PEG-GS NPs could cross the BBB and escape the arrest of the reticuloendothelial system (RES), and it would be potential nano-carrier systems for central delivery.

Protective effects of transduced Tat-DJ-1 protein against oxidative stress and ischemic brain injury

Reactive oxygen species (ROS) contribute to the development of a number of neuronal diseases including ischemia. DJ-1, also known to PARK7, plays an important role in transcriptional regulation, acting as molecular chaperone and antioxidant. In the present study, we investigated whether DJ-1 protein shows a protective effect against oxidative stress-induced neuronal cell death in vitro and in ischemic animal models in vivo. To explore DJ-1 protein's potential role in protecting against ischemic cell death, we constructed cell permeable Tat-DJ-1 fusion proteins. Tat-DJ-1 protein efficiently transduced into neuronal cells in a dose- and time-dependent manner. Transduced Tat-DJ-1 protein increased cell survival against hydrogen peroxide (H2O2) toxicity and also reduced intracellular ROS. In addition, Tat-DJ-1 protein inhibited DNA fragmentation induced by H2O2. Furthermore, in animal models, immunohistochemical analysis revealed that Tat-DJ-1 protein prevented neuronal cell death induced by transient forebrain ischemia in the CA1 region of the hippocampus. These results demonstrate that transduced Tat-DJ-1 protein protects against cell death in vitro and in vivo, suggesting that the transduction of Tat-DJ-1 may be useful as a therapeutic agent for ischemic injuries related to oxidative stress.

Constructing a phage-displayed random mutation library of HIV-1 Tat38-61 at the sites of 51 and 55 amino acids in basic region / 生物工程学报

We constructed a phage-displayed random mutation library of Tat38-61(51N/55N), for studying the molecular evolution screening of HIV-1 Tat38-61 epitope. We used primers containing the random nucleotide sequences, and introduced the random mutations at the sites of 51 and 55 amino acids coding sequences into full-length Tat sequences by overlapping PCR. With the randomly mutated full-length Tat as template, the Tat38-61(51N/55N) mutants which contained recognition sequences for the Xba I in both ends were amplified by PCR using the designed primers. The mutants were cloned into Xba I site in the phagemid vector pCANTAB5S, then the recombinants were transformed into E. coli TG1, a phage-displayed the random mutation library of Tat38-61(51N/55N) was constructed by the rescue of help virus M13KO7. The results showed that the library consisted of about 5.0 x 10(6) colonies and the phage library titer was 2.65 x 10(12) TU/mL. More than 56.50% colonies in the library were positive for insertion. Sequence analysis showed that the nucleotides encoding amino acids at the sites of 51 and 55 distributed randomly. The constructed mutation library could meet the requirements for the following molecular evolution screening, and might prepare the Tat mutants for the further study of new Tat vaccine candidates.

Analysis of genetic diversity and amino acid sequence of HIV-1tat in CNS and peripheral tissue of a patient with ADC and a patient with non-ADC / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the diversity of HIV-1 tat gene in CNS and peripheral tissue of a patient with ADC and a patient with non-ADC, so as to research HIV evolution, the mechanism of CNS invasion and the pathogenesis of ADC.</p><p><b>METHODS</b>The tat gene was amplified with nested PCR from genomic DNA which was extracted from spleen and basal ganglia of one non-ADC patient with a wide range of cerebral artery atherosclerosis and one ADC patient. PCR products were cloned into the PGEM-T vector, after transformation and selection by ampicillin and blue/white spotting. Five of positive clones were sequenced. HIV-1 tat sequences were processed with BioEdit and MEGA4. With the softwares, neighbor-joining tree, p-distances, values of ds/dn, and analysis of amino acid motifs were all done, so as to research the diversity of HIV-1 tat gene in CNS and peripheral tissue.</p><p><b>RESULTS</b>Gene mutation of HIV-1 tat exist in the two patients, the mutation process of tat isolated from ADC patient suffered more compartmentalization than tat isolated from non-ADC patient, the differences of tat genes between CNS and peripheral tissue in ADC patient were greater than the non-ADC patient. Ds/dn showed that the virus gene mutation played a major role, the body intend to remove harmful non-synonymous mutations.</p><p><b>CONCLUSIONS</b>The compartmentation of tat gene in CNS and peripheral tissue of the two patients was different, the reason may be related to the pathway of HIV into the CNS, the relationship between HIV gene mutation in CNS and ADC still need more investigation.</p>

Design, synthesis and evaluation of novel 2H-1, 4-benzodiazepine-2-ones as inhibitors of HIV-1 transcription / 药学学报

HIV-1 trans-activator of transcription (Tat) plays a critical role in HIV-1 transcription. Based on the beta-turn motif present in HIV-1 Tat, a series of novel benzodiazepine analogs were designed as beta-turn mimetics and prepared from p-chloro-nitrobenzene/2-phenylacetonitrile, p-toluidine/benzoyl chloride, or (Z)-7-nitro-5-phenyl-1H-benzo[e][1, 4]diazepin-2(3H)-one (nitrazepam) through different synthetic routes. Preliminary biological evaluation indicated that compound 30 exhibited inhibitory activity on HIV-1 tat-mediated LTR transcription with EC50 of 25.0 micromol x L(-1) and showed no obvious cytotoxic effects on TZM-BI cells under the concentration of 100 micromol x L(-1).

Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro

BACKGROUND: Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. METHODS: To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-gamma ELISPOT assay, cytotoxicity assay and tetramer staining. RESULTS: DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of CD8+ and CD4+ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. CONCLUSION: Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.

Factores de riesgo para la infección por VIH/SIDA en adolescentes y jóvenes colombianos / Risk factors in the infection by HIV/AIDS in Colombian adolescents and young people / Fatores de risco de infecção por VIH/SIDA em adolescentes e jovens colombianos

Aunque las cifras de personas infectadas con el Virus de Inmunodeficiencia Humana (VIH) y Síndrome de Inmunodeficiencia Adquirida (SIDA) se han estabilizado en el último año, la población adolescente sigue siendo foco de atención debido a la vulnerabilidad y frecuencia de conductas de riesgo en salud sexual y reproductiva que presentan. Esta situación también se evidencia en Colombia, por lo cual se da la necesidad de conocer la situación actual de los adolescentes en esta área, con el fin de generar estrategias de promoción y prevención, que disminuyan los riesgos frente a la infección por VIH/Sida, enfocadas a las características propias de la población. El propósito del siguiente estudio fue determinar los conocimientos, actitudes, susceptibilidad y autoeficacia en adolescentes y jóvenes ente los 10 y 23 años de instituciones educativas públicas de diferentes ciudades de Colombia. Se evaluó una muestra de 978 adolescentes de 6 a 11 grados de educación básica secundaria. El estudio es de carácter no experimental descriptivo transversal. Se les aplicó la Escala VIH/Sida-65 y la Escala de Autoeficacia (SEA-27). De los adolescentes encuestados, más del 50% había recibido información sobre transmisión y prevención del VIH/Sida, sin embargo los conocimientos adquiridos no se reflejaban en las prácticas y continuaban presentando ideas erróneas sobre el tema; también se encontró que a medida en que aumenta la edad, disminuyen los conocimientos, actitudes, susceptibilidad y autoeficacia frente al VIH/Sida. Es necesario que en los adolescentes se desarrollen intervenciones orientadas hacia el cambio de comportamiento y que sean específicas de acuerdo con la edad, género, nivel de escolaridad y nivel socioeconómico.

Although the number of people infected by the Human Immunodeficiency Virus (HIV) and the Acquired Immune Deficiency Syndrome (AIDS) has stabilized during the past year, the adolescent population continues to be the focus of attention due to its vulnerability and the frequency of risk behaviors that occur in sexual and reproductive health. There is evidence that this too is happening in Colombia and therefore it is necessary to understand the present situation of adolescents in this area and focus on the characteristics of this population in order to reduce the risk of infection by HIV/AIDS. The purpose of this study was to establish the level of knowledge, attitudes, susceptibility and self-efficiency among adolescents and young people between the ages of 10 and 23 in state schools of different Colombian cities. A sample of 987 high-school adolescents between 6th and 11th grades was assessed. The study was non-experimental, descriptive, and cross sectional. The VIH/Sida-65 and Self-Efficiency (SEA-27) scales were administered. Among the adolescents interviewed, more that 50% had received information about the transmission and prevention of HIV/AIDS. However, the knowledge acquired was not reflected in their sexual practices, and there were still misconceptions on the subject. It was also found that the levels of knowledge, attitudes, susceptibility and self-efficiency related in HIV/AIDS diminished as the age of those interviewed increased. It is necessary to carry out interventions aimed at changes in behavior, specifically addressed to the adolescents according to their age, gender, academic level and socio-economic status.

Embora o número de pessoas infectadas com o vírus da imunodeficiência humana (VIH) e a síndrome da imunodeficiência adquirida (sida) ter-se estabilizado ao longo do ano passado, a população adolescente continua sendo um foco por causa da vulnerabilidade e da freqüência de comportamentos de risco em saúde sexual e reprodutiva. Como esta situação é também evidente na Colômbia, é necessário saber o estado atual de adolescentes nessa área, a fim de gerar estratégias de promoção e de prevenção que reduzam a exposição ao VIH /sida, focalizadas nas características da população. O objetivo deste estudo é determinar os conhecimentos, as atitudes, a sensibilidade e a auto-eficácia em adolescentes e jovens entre 10 e 23 anos, de instituições públicas de ensino de diversas cidades da Colômbia. Este estudo descritivo, não-experimental transversal, avaliou uma amostra de 978 adolescentes de 6 a 11 graus de ensino secundário. Se lhes aplicaram as escalas VIH/sida-65 e Auto-Eficácia (SEA-27). Dos adolescentes entrevistados, 50% receberam informações sobre transmissão e prevenção do VIH/sida, mas o conhecimento adquirido não repercutia nas práticas e apresentava ainda idéias equivocadas sobre o assunto. O estudo revelou também que quando a idade aumenta, se reduzem os conhecimentos, as atitudes, a sensibilidade e a auto-eficácia contra o VIH/sida. É necessário que os adolescentes desenvolver intervenções para a mudança de comportamento, específicas para a idade, o sexo, a escolaridade e o nível socioeconômico.

Effect of fusion protein TAT and heme oxygenase-1 on liver sinusoidal endothelial cells apoptosis during preservation injury / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Proteins or peptides can be directly transferred into cells when covalently linked to protein transduction domains (PTDs). TAT is one of the most widely studied PTDs. The effect of fusion protein TAT and heme oxygenase-1 (HO-1) on liver sinusoidal endothelial cells (SECs) apoptosis during cold storage is unknown. The present study aimed to determine whether fusion protein TAT-HO-1 would transduce efficiently into liver during cold storage, and, if so, to determine whether TAT-HO-1 would attenuate SECs apoptosis during preservation injury in rat.</p><p><b>METHODS</b>Livers of Sprague-Dawley rats were harvested and randomly assigned to group 1 (HTK solution) and group 2 (HTK solution containing TAT-HO-1 fusion protein) according to the type of the preservation solution. The transduction efficiency of TAT-HO-1 was examined and the impairment of SECs was assessed during the period of cold storage followed by 1 hour of reperfusion.</p><p><b>RESULTS</b>TAT-HO-1 can transduce efficiently into liver during cold storage. A significantly lower apoptotic index of SECs was observed in group 2, at 6, 12 and 18 hours of cold storage after 1 hour reperfusion, when compared with group 1. TAT-HO-1 reduced HA and ET levels in liver at each time point. Both Bcl-2 and Bax protein were expressed in hepatocytes and SECs at the periphery of the sinusoidal space. Moreover, higher Bcl-2 expression and lower Bax expression were observed in group 2.</p><p><b>CONCLUSIONS</b>TAT-HO-1 can transduce efficiently into rat livers and shows a protective effect on SECs by attenuating apoptosis during cold ischemia/reperfusion injury. Protein transduction will be a novel therapeutic strategy to reduce the risk of preservation injury in liver transplantation.</p>

Action of protein phosphatase-1 on Tat-dependent HIV-1 transcription and its related inhibitors / 药学学报

Host cell protein phosphatase-1 (PP1) is an important regulator of human immunodeficiency virus-1 (HIV-1) transcription. PP1 is involved in the regulation of HIV-1 transcription, and dephosphorylates RNA polymerase II C-terminal domain (RNAPII CTD) or CycT1-dependent kinase 9 (CDK9) to increase Tat-dependent HIV-1 transcription. In this review, we discuss the action of PP1 in Tat-induced HIV-1 transcription and related to PP1 inhibitors.

Peptide TAT modified polyethylenimine-beta-cyclodextrin for gene delivery / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop a novel gene delivery vector TAT-PEI-beta-CyD.</p><p><b>METHODS</b>beta-cyclodextrin (beta-CyD) was linked by low molecular weight (PEI 600) via 1, 1-carbonyldiimidazole (CDI), and TAT peptide (RRRQRRKKRC) was coupled to PEI 600 by [N-succinimidy-3-(2-pyridyldithio) propionate, SPDP]. The copolymer was characterized by (1)H-NMR and FT-IR. Physiochemical characteristics of TAT-PEI-beta-CyD/DNA complexes were tested by agarose gel electrophoresis and particle size measurements. Cell viability and transfection efficiency were evaluated in A293 and B16 cells using PEI 25 kDa as a control.</p><p><b>RESULT</b>TAT peptide was successfully coupled to PEI-beta-CyD. The result of gel electrophoresis showed that the TAT-PEI-beta-CyD was able to condense DNA efficiently at N/P ratio of 4. The particle size of TAT-PEI-beta-CyD/DNA complexes was around 100 nm. The cytotoxicity of TAT-PEI-beta-CyD was lower than that of PEI 25 kDa. The transfection efficiency of TAT-PEI-beta-CyD was higher than that of PEI 25 kDa in A293 and B16 cells at N/P ratio of 30.</p><p><b>CONCLUSION</b>The novel vector TAT-PEI-beta-CyD has been developed successfully with low cytotoxicity and high transfection efficiency.</p>

Extracellular HIV-1 Tat up-regulates expression of matrix metalloproteinase-9 via a MAPK-NF-kappaB dependent pathway in human astrocytes

The infiltration of monocytes into the CNS represents one of the early steps to inflammatory events in AIDS-related encephalitis and dementia. Increased activity of selected matrix metalloproteinases (MMPs) such as MMP-9 impairs the integrity of blood-brain barrier leading to enhanced monocyte infiltration into the CNS. In this study, we examined the effect of HIV-1 Tat on the expression of MMP-9 in CRT-MG human astroglioma cells. Treatment of CRT-MG cells with HIV-1 Tat protein significantly increased protein levels of MMP-9, as measured by Western blot analysis, zymography and an ELISA. Treatment of CRT-MG cells with HIV-1 Tat protein markedly increased mRNA levels of MMP-9, as analyzed by RT-PCR. Pretreatment of CRT-MG cells with NF-kappaB inhibitors led to decrease in Tat-induced protein and mRNA expression of MMP-9. Pretreatment of CRT-MG cells with MAPK inhibitors suppressed Tat-induced MMP-9 expression. Furthermore, HIV-1 Tat-induced expression of MMP-9 was significantly inhibited by neutralization of TNF-alpha, but not IL-1beta and IL-6. Taken together, our results indicate that HIV-1 Tat can up-regulate expression of MMP-9 via MAPK-NF-kappaB-dependent mechanisms as well as Tat-induced TNF-alpha production in astrocytes.

Expression and purification of TAT-HBX-EGFP fusion protein and its transmembrane distribution in mouse / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To highly express TAT-HBX-EGFP fusion protein and study its distribution in mouse liver.</p><p><b>METHODS</b>TAT-HBX-EGFP recombinant vector was constructed and fusion protein was induced by IPTG and expression in BL21; fusion protein was purified by Ni-NTA argarose, then injected into the peritoneal cavity of the mice. Distribution of fusion protein was observed by immunofluorescence.</p><p><b>RESULTS</b>TAT-HBX-EGFP was highly expression in E. coli; HBX could be induced into mouse liver by TAT.</p><p><b>CONCLUSION</b>HBX protein could be induced into mouse liver by TAT induced peptide.</p>

Cloning of PTD-NPY fusion gene and its secretory expression in Pichia pastoris / 生物工程学报

The PTD-NPY fusion gene derived from HIV-1 TAT protein transduction domain and rat neuropeptide Y was amplified by overlap extension PCR, digested and subcloned into yeast expression vector pPICZ alpha A to construct recombinant expression plasmid pPICZ alpha-PTD-NPY. The cloned PTD-NPY fusion gene was identified by PCR and restriction enzyme digestion and sequenced. The exact recombinant plasmid was linearized by Sac I and integrated by electrotransformation into the genome of Pichia pastoris GS115 cells. Then, these positive recombinant yeast cells were induced by 10 mL/L methanol to express soluble PTD-NPY fusion protein. After 120 h of methanol induction, the SDS-PAGE electrophoresis result indicated PTD-NPY fusion protein was efficiently secreted into the medium. Western blotting analysis proved that the expressed fusion protein had specific NPY binding activity. The successful expression of PTD-NPY fusion protein in Pichia pastoris provided basis for its further application study.

Human immunodeficiency virus-1 Tat protein: immunological facets of a transcriptional activator.

Human immunodeficiency virus-1 (HIV-1) infection is characterized by chronic immune activation and progressive loss of CD4+ T cells, leading to a wide array of immune dysfunction, particularly involving immune response directed against viral antigens. HIV-1 encodes for fifteen proteins, which might serve as a target for immune recognition. Immune response to the envelope proteins have been studied more due to their presence on the surface of the virus. Recent studies on HIV vaccine development have focused on the Gag and Pol proteins. The transactivator Tat and Rev proteins have also been the focus of immunization studies due to their potent regulatory activity. The Tat (transactivator of transcription) protein although being nuclear in localization is also released from infected cells and acts on uninfected cells. Extracellular Tat seems to play an important role in AIDS pathogenesis. Furthermore, a correlation has been found between anti-Tat immune response and slow progression of the disease. Although several studies have shown Tat as a potential vaccine candidate with encouraging results, there are also reports raising doubt about its efficacy in multi-component HIV vaccine strategy. Here, we have addressed the issue of immune response to the most indispensable HIV-1 regulatory protein Tat.

Reconstruction of an intracellular transduction system based on HIV-1 TAT protein transduction domain / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the reasons for the low intracellular transduction efficiency of a previously constructed His-TAT-Flag recombinant protein and establish a more efficient transduction system.</p><p><b>METHODS</b>The Flag tag of pET14b-His- Tat-Flag vector was deleted with PCR mutant kit, and enhanced green fluorescent protein (EGFP) coding sequence was inserted into the new pET14b-His-TAT recombinant vector. Enzyme digestion and DNA sequencing were performed for identification of pET14b-His-TAT-EGFP vector, which was then transformed into E. coli BL21(DE(3)). After IPTG induction, the recombinant protein of His-TAT-EGFP was isolated and analyzed with SDS-PAGE. Purified His-TAT-EGFP recombinant protein was added to ECV304 cells and the fluorescence was observed to evaluate the transduction efficiency.</p><p><b>RESULTS</b>pET14b-His-TAT vector and pET14b-His-TAT- EGFP vector were successfully constructed, which was identified with enzyme digestion and DNA sequencing. His-TAT-EGFP fusion protein was expressed and purified successfully and showed cellular transduction activity.</p><p><b>CONCLUSION</b>The prokaryotic expression vector has been successfully constructed by modifying pET14b-His-TAT-Flag, and the expressed and purified recombinant protein of His-TAT-EGFP possesses high efficiency of intracellular transduction activity.</p>

Preparation of recombinant HIV-TATm-survivin (T34A) protein and its pro-apoptosis activity to cancer cells in vitro / 生物工程学报

As a novel member of the IAP (Inhibitor of apoptosis protein) family, survivin was observed to be expressed in most human cancerous cells. Fusion protein TATm-survivin (T34A) has drawn considerable attention because it is a potential anti-tumor protein that can be transduced into cancer cell with the help of HIV-TAT domain. In this study, the cDNA encoding survivin was cloned by RT-PCR from human breast cancer cell lines B-Cap-37. An expression vector of pRSET-B-HIV-tatm-survivin (T34A) was constructed by PCR after survivin (T34A) was mutated by site-directed mutagenesis. Subsequently, the resultant plasmid was transformed into E. coli BL21 (DE3). Recombinant HIV-TATm-Survivin (T34A) protein was expressed efficiently with 0.5mM IPTG as inducer, reaching a yield of 650mg/liter (as inclusion body) in fermentation culture. The inclusion bodies were solubilized, refolded and purified to a purity of 96% by ion exchange chromatograghy and size-exclusion chromatography. Remarkable effects of the purified recombinant HIV-TATm-Survivin (T34A) on the morphology of cell line SW1990 and B-Cap-37 were observed after being administrated for 4h. MTT assay showed recombinant HIV-TATm-survivin (T34A) protein could inhibit significantly cell proliferation of SW1990 and B-Cap-37 and SSMC-7721 in vitro. Apoptosis rate and cell circle of SW1990 and B-Cap-37 that had been treated with target protein (final concentration 30 microg/mL) were detected with flow cytometry. Results revealed that more than 65% cancer cells were arrested at G1 phase. The study suggested that TATm-survivin (T34A) protein was a hopeful protein drug in the treatment of cancers by facilitating apoptosis of cancer cells. Key words recombinant HIV-TATm-Survivin (T34A), expression and purification, pro-apoptosis bioactivity, SW1990 and B-Cap-37 cancer cell lines