RESUMEN
Rev, an important accessory protein of equine infectious anaemia virus (EIAV), induces the nuclear export of incompletely spliced viral mRNAs. Rev is translated from the tat-rev mRNA through leaky scanning of the tat CUG. In this study, the function of the Kozak sequence at the beginning of the rev ORF was investigated. Deletion or attenuation of the Kozak sequence resulted in expression of an N-terminal 11 aa-truncated Rev in addition to WT Rev. Truncated Rev displayed weaker promotion of Gag expression and processing than WT Rev. Furthermore, EIAV rescued from an infectious molecular clone (pEIAVUK3) with Kozak attenuation exhibited decreased viral replication in host cells in vitro. These results provide a new understanding of the relationship between EIAV Rev expression and viral replication.
Asunto(s)
Regulación Viral de la Expresión Génica , Productos del Gen rev/biosíntesis , Productos del Gen tat/biosíntesis , Virus de la Anemia Infecciosa Equina/genética , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Replicación Viral , Línea Celular , Productos del Gen rev/genética , HumanosRESUMEN
Depression and psychostimulant abuse are common comorbidities among humans with immunodeficiency virus (HIV) disease. The HIV regulatory protein TAT is one of multiple HIV-related proteins associated with HIV-induced neurotoxicity. TAT-induced dysfunction of dopamine and serotonin systems in corticolimbic brain areas may result in impaired reward function, thus, contributing to depressive symptoms and psychostimulant abuse. Transgenic mice with doxycycline-induced TAT protein expression in the brain (TAT+, TAT- control) show neuropathology resembling brain abnormalities in HIV+ humans. We evaluated brain reward function in response to TAT expression, nicotine and methamphetamine administration in TAT+ and TAT- mice using the intracranial self-stimulation procedure. We evaluated the brain dopamine and serotonin systems with high-performance liquid chromatography. The effects of TAT expression on delay-dependent working memory in TAT+ and TAT- mice using the operant delayed nonmatch-to-position task were also assessed. During doxycycline administration, reward thresholds were elevated by 20% in TAT+ mice compared with TAT- mice. After the termination of doxycycline treatment, thresholds of TAT+ mice remained significantly higher than those of TAT- mice and this was associated with changes in mesolimbic serotonin and dopamine levels. TAT+ mice showed a greater methamphetamine-induced threshold lowering compared with TAT- mice. TAT expression did not alter delay-dependent working memory. These results indicate that TAT expression in mice leads to reward deficits, a core symptom of depression, and a greater sensitivity to methamphetamine-induced reward enhancement. Our findings suggest that the TAT protein may contribute to increased depressive-like symptoms and continued methamphetamine use in HIV-positive individuals.
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Estimulantes del Sistema Nervioso Central/farmacología , Productos del Gen tat/biosíntesis , VIH-1 , Memoria/fisiología , Tiempo de Reacción/fisiología , Recompensa , Animales , Relación Dosis-Respuesta a Droga , Expresión Génica , Productos del Gen tat/genética , Masculino , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Tiempo de Reacción/efectos de los fármacos , Factores de TiempoRESUMEN
INTRODUCTION: The therapeutic potential of acyclic retinoid (ACR), a synthetic retinoid, has been confirmed in experimental and clinical studies. Therapeutic targets include precancerous and cancer stem cells. As ACR is also involved in developmental processes, its effect on normal hepatic stem cells (HpSCs) should be investigated for understanding the underlying mechanisms. Here, we examined effects of the acyclic retinoid peretinoin on fresh isolated murine HpSCs. METHODS: We isolated c-kit-CD29+CD49f+/lowCD45-Ter119- cells from murine fetal livers using flow cytometry. To evaluate the effect of ACR, we traced clonal expansion and analyzed cell differentiation as well as apoptosis during the induction process by immunofluorescent staining and marker gene expression. RESULTS: ACR dose-dependently inhibited HpSCs expansion. Stem cell clonal expansion was markedly inhibited during the culture period. Moreover, ACR showed a significant promotion of HpSC differentiation and induction of cellular apoptosis. The expression of stem cell marker genes, Afp, Cd44, and Dlk, was downregulated, while that of mature hepatocyte genes, Alb and Tat, and apoptosis-related genes, Annexin V and Caspase-3, were upregulated. Flow cytometry showed that the proportion of Annexin V-positive cells increased after ACR incubation compared with the control. Data obtained by immunofluorescent staining for albumin and Caspase-3 corroborated the data on gene expression. Finally, we found that ACR directly regulates the expression of retinoic acid receptors and retinoid X receptors. CONCLUSIONS: These findings indicate that ACR inhibits the clonal expansion of normal HpSCs in vitro and promotes the differentiation of immature cells by regulating receptors of retinoic acid.
Asunto(s)
Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Regeneración Hepática/fisiología , Hígado/citología , Tretinoina/análogos & derivados , Animales , Anexina A5/biosíntesis , Proteínas de Unión al Calcio , Caspasa 3/biosíntesis , Células Cultivadas , Regulación hacia Abajo , Citometría de Flujo , Productos del Gen tat/biosíntesis , Receptores de Hialuranos/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Ratones , Ratones Endogámicos C57BL , Receptores de Ácido Retinoico/metabolismo , Células Madre/citología , Células Madre/metabolismo , Tretinoina/farmacología , Regulación hacia Arriba , alfa-Fetoproteínas/biosíntesisRESUMEN
Excessive reactive oxygen species (ROS) generated from abnormal cellular process lead to various human diseases such as inflammation, ischemia, and Parkinson's disease (PD). Sensitive to apoptosis gene (SAG), a RING-FINGER protein, has anti-apoptotic activity and anti-oxidant activity. In this study, we investigate whether Tat-SAG, fused with a Tat domain, could protect SH-SY5Y neuroblastoma cells against 1-methyl-4-phenylpyridinium (MPP(+)) and dopaminergic (DA) neurons in the substantia nigra (SN) against 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) toxicity. Western blot and immunohistochemical analysis showed that, unlike SAG, Tat-SAG transduced efficiently into SH-SY5Y cells and into the brain, respectively. Tat-SAG remarkably suppressed ROS generation, DNA damage, and the progression of apoptosis, caused by MPP(+) in SH-SY5Y cells. Also, immunohistochemical data using a tyrosine hydroxylase antibody and cresyl violet staining demonstrated that Tat-SAG obviously protected DA neurons in the SN against MPTP toxicity in a PD mouse model. Tat-SAG-treated mice showed significant enhanced motor activities, compared to SAG- or Tat-treated mice. Therefore, our results suggest that Tat-SAG has potential as a therapeutic agent against ROS-related diseases such as PD.
Asunto(s)
Neuronas Dopaminérgicas/patología , Enfermedad de Parkinson Secundaria/patología , Proteínas Recombinantes de Fusión/biosíntesis , Ubiquitina-Proteína Ligasas/biosíntesis , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , Apoptosis , Línea Celular Tumoral , Productos del Gen tat/biosíntesis , Productos del Gen tat/genética , Humanos , Masculino , Ratones Endogámicos C57BL , Degeneración Nerviosa , Enfermedad de Parkinson Secundaria/inducido químicamente , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes de Fusión/genética , Sustancia Negra/metabolismo , Sustancia Negra/patología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Tat (Trans-activator of transcription) is implicated in the neuropathogenesis of HIV-1 infection and known to contribute to neuronal damage and learning and memory impairments. However, direct neuroanatomical demonstration of Tat pathobiology is limited. GT-tg bigenic mice with a doxycycline (Dox)-inducible and brain-selective tat gene were used to test the hypothesis that conditional induction of Tat activity in brain can induce gray matter density abnormalities. Ultra high spatial resolution ex vivo magnetic resonance imaging (MRI) combined with a voxel based morphometry (VBM) analysis revealed gray matter density reductions in the sublenticular extended amygdala, the amygdala, the amygdala-hippocampal area, piriform and peri-/entorhinal cortices, and hypothalamus, in Tat-expressing GT-tg mice compared to Dox-treated C57Bl/6J mice. These neuroanatomical abnormalities are consistent with regions expected to be abnormal based on behavioral deficits exhibited by Tat-expressing mice (Carey et al., 2012). These experiments provide the first neuroimaging evidence that conditional Tat protein expression in the GT-tg bigenic mouse model alters brain structure. The findings warrant future studies to further characterize effects of conditional Tat expression on brain structure. Such studies may improve our understanding of the neurological underpinnings of neuroAIDS and the neurodegeneration associated with HIV-1 infection, potentially leading to new treatments.
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Química Encefálica/fisiología , Encéfalo/patología , Productos del Gen tat/biosíntesis , Complejo SIDA Demencia/psicología , Animales , Animales Modificados Genéticamente , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Química Encefálica/efectos de los fármacos , Doxiciclina/farmacología , Proteína Ácida Fibrilar de la Glía/metabolismo , Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/efectos de los fármacos , Tetraciclina/farmacologíaRESUMEN
Opiate abuse reportedly can exaggerate complications of human immunodeficiency virus type-1 (HIV-1) infection in the central nervous system (CNS), while opiate drugs are often indicated in the treatment of HIV-1-related neuropathic pain. Despite this quandary, few studies have assessed the relationship between the duration or extent of HIV-1 infection and the intrinsic neurobehavioral responsiveness to opioids. To address this problem, doxycycline (DOX)-inducible HIV-Tat(1-86) transgenic mice were used as a model for HIV-1-associated neurocognitive disorders, which permitted the regulation of Tat exposure and duration. The effects of continuous Tat induction on the activity of morphine were examined at weekly intervals using standard behavioral assays for nociception and motor function. In the spinal cord, Tat mRNA levels did not increase until the second and third weeks following induction, which corresponded to a significant loss of morphine antinociception as assessed in the tail-flick test. Alternatively, in the striatum, sustained increases in Tat mRNA expression during the second week of induction coincided with significant decreases in rotarod performance and interactions with morphine. Importantly, the behavioral effects of morphine differed depending on the timing and location of Tat expression, with increases in Tat transcript levels in the spinal cord and striatum corresponding to significant alterations in morphine-dependent nociception and rotarod performance, respectively. Assuming Tat levels contribute to the clinical manifestations of HIV-1, the results suggest that regional differences in viral load and opioid phenotype might influence the nature and degree that opiate responsiveness is altered in HIV-1-infected individuals.
Asunto(s)
Productos del Gen tat/biosíntesis , Productos del Gen tat/genética , VIH-1 , Morfina/farmacología , Dimensión del Dolor/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Productos del Gen tat/fisiología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , VIH-1/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfina/uso terapéutico , Dimensión del Dolor/métodos , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Resultado del TratamientoRESUMEN
OBJECTIVE: To identify the dural activities of neuroprotection and penetrating blood-brain barrier (BBB) for TAT-BDNF (transactivating-brain-derived neurotrophic factor) fusion protein to explore an alternative treatment for the injury of central nerve system (CNS). METHODS: With molecular cloning techniques, a recombinant vector termed pTAT-BDNF was constructed to encode both TAT protein transduction domain and human BDNF. Purified TAT-BDNF fusion protein was generated from Escherichia coli BL21 (DE3). The injury model was established with in vitro cultured cortical neurons of neonatal rats. To observe the neuroprotective effects of TAT-BDNF fusion protein on glutamate-mediated excitotoxic insults, the contents of lactate dehydrogenase (LDH) were measured by spectrophotometry. Immunofluorescence and Hoechst 33342 analyses were used to observe the morphological changes. Immunocytochemical and Nissl stain analysis of TAT-BDNF content in CNS tissue were performed after an intravenous injection of TAT-BDNF fusion protein in normal or spinal cord injured rats. RESULTS: During the study of glutamate-induced excitotoxic insults, as compared with the control group, TAT-BDNF could decrease the apoptotic ratio, reduce the leakage of LDH and enhance the survival of neurons (P < 0.05 ). As demonstrated by immunohistochemistry, TAT-BDNF fusion protein was efficiently delivered into rat brain and spinal cord tissues at 4 h post-injection. At Day 7 post-injury, Nissl stain show that the number and morphology of neurons in the TAT-BDNF group were better than those in the control group. CONCLUSION: The synthetic neotype TAT-BDNF possess the dual biological effects of neuroprotection and penetrating BBB.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Productos del Gen tat/biosíntesis , Animales , Factor Neurotrófico Derivado del Encéfalo/farmacología , Productos del Gen tat/farmacología , Masculino , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
HIV-2 infection is associated with a slower rate of disease progression with limited impact on the survival of the majority of infected adults, and much lower plasma viral load than HIV-1. In spite of the major differences in viremia, the quantitative assessment of HIV-2 proviral load documented levels similar to those observed in HIV-1 infected individuals, suggesting an equivalent number of circulating infected cells in both infections. It remains unclear whether this apparent paradox results from a contribution of latent/quiescent viruses or from transcriptional and/or post-transcriptional control of HIV-2 replication. In order to investigate these possibilities, a one-step and two-step reverse transcription quantitative real-time PCR based methods (RT-qPCR) for gag and tat mRNA HIV-2 transcripts were developed. These methods were validated and compared to assess the expression of HIV-2 gag and tat transcripts in parallel with proviral DNA and viral production. The results suggest that the two-step approach may allow a better detection of low level gag and tat mRNA HIV-2 transcripts.
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VIH-2/genética , Empalme del ARN/genética , ARN Mensajero/análisis , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Células Cultivadas , Productos del Gen gag/biosíntesis , Productos del Gen gag/genética , Productos del Gen tat/biosíntesis , Productos del Gen tat/genética , Humanos , Procesamiento Postranscripcional del ARN , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , Transcripción GenéticaRESUMEN
In the study, a gene encoding Tat protein N terminal 1- 21 amino acid residues-deleted mutant (Tat22-101) was amplified by PCR from a full length Tat gene of human immunodeficiency virus type 1, and the prokaryotic expression plasmid pET32a-Tat22-101 was constructed. After identification by digestion with endonucleases and sequencing, the recombinant plasmid pET32a-Tat22-101 was transformed into E. coli BL21(DE3) and expressed with IPTG induction. The mutant fusion protein with deleted Tat N terminal was purified by an affinity chromatography column Ni(2+)-NTA and subsequently identified by SDS-PAGE and Western blotting. The results showed that the molecular weight of the mutant protein was approximately 26.9kD. Furthermore, BALB/c mice were immunized with the mutant protein and the anti-sera were collected. ELISA results showed that the mutant protein preserved its immunogenicity, particularly it could improve the production of antibodies to other epitopes in addition to the N terminal epitope of Tat protein, which might provide some valuable information for the study of Tat functions as well as for development of potential novel HIV Tat vaccine.
Asunto(s)
Productos del Gen tat/genética , VIH-1/genética , VIH-1/inmunología , Proteínas Mutantes/genética , Proteínas Recombinantes de Fusión/genética , Animales , Femenino , Productos del Gen tat/biosíntesis , Productos del Gen tat/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Mutantes/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) Tat protein is a major viral transactivator required for HIV-1 replication. In the nucleus Tat greatly stimulates the synthesis of full-length transcripts from the HIV-1 promoter by causing efficient transcriptional elongation. Tat induces elongation by directly interacting with the bulge of the transactivation response (TAR) RNA, a hairpin-loop located at the 5'-end of all nascent viral transcripts, and by recruiting cellular transcriptional co-activators. In the cytoplasm, Tat is thought to act as a translational activator of HIV-1 mRNAs. Thus, Tat plays a central role in the regulation of HIV-1 gene expression both at the level of mRNA and protein synthesis. The requirement of Tat in these processes poses an essential question on how sufficient amounts of Tat can be made early on in HIV-1 infected cells to sustain its own synthesis. To address this issue we studied translation of the Tat mRNA in vitro and in human cells using recombinant monocistronic and dicistronic RNAs containing the 5' untranslated region (5'-UTR) of Tat RNA. RESULTS: This study shows that the Tat mRNA can be efficiently translated both in vitro and in cells. Furthermore, our data suggest that translation initiation from the Tat mRNA probably occurs by a internal ribosome entry site (IRES) mechanism. Finally, we show that Tat protein can strongly stimulate translation from its cognate mRNA in a TAR dependent fashion. CONCLUSION: These results indicate that Tat mRNA translation is efficient and benefits from a feedback stimulation by the Tat protein. This translational control mechanism would ensure that minute amounts of Tat mRNA are sufficient to generate enough Tat protein required to stimulate HIV-1 replication.
Asunto(s)
Productos del Gen tat/biosíntesis , VIH-1/fisiología , Biosíntesis de Proteínas , ARN Viral/metabolismo , Replicación Viral , Fusión Artificial Génica , Línea Celular , Genes Reporteros , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , ARN Viral/genética , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
In order to improve the transduction efficiency of insect baculovirus in mammalian cells, we constructed two recombinant baculoviruses, AcRed-tat and AcRed. Both viruses expressed red fluorescence protein gene (dsRed) as a reporter in mammalian cell lines. AcRed-tat also contained the coding sequence of HIV-1 Tat transduction peptide fused with viral major capsid protein gene vp39 and enhanced green fluorescence gene (egfp) driven by virus polyhedrin promoter. It expressed the Tat fusion protein in infected insect cells, which was incorporated into the nucleocapsids of progeny virus. As a control, AcRed had the fusion gene of vp39 and egfp driven by polyhedrin promoter. Flow cytometry analysis demonstrated that although similar level of red fluorescence was produced in HEK23 cells transduced by the two recombinant viruses, significantly higher red fluorescence level was seen in CHO and Vero cells transduced by AcRed-tat than that by AcRed. These results suggested that Tat transduction peptide might improve the baculovirus-mediated gene expression in some mammalian cells. Our work provided a new approach to improve baculovirus as a gene delivery vector for mammalian cells.
Asunto(s)
Baculoviridae/metabolismo , Productos del Gen tat/biosíntesis , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Virales/biosíntesis , Animales , Baculoviridae/genética , Células CHO , Chlorocebus aethiops , Cricetinae , Cricetulus , Productos del Gen tat/genética , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Nucleocápside/genética , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Transducción Genética/métodos , Células Vero , Proteínas Virales/genéticaRESUMEN
HIV encephalitis (HIVE) is accompanied by brain inflammation, leukocyte infiltration, and glial activation, and HIV patients who abuse opiates are more likely to develop HIVE. To better understand how opiates could alter HIV-related brain inflammation, the expression of astrocyte (GFAP immunoreactivity) and macrophage/microglial (F4/80 or Mac1 immunoreactivity) markers in the striatum, and the percentage of 3-nitrotyrosine (3-NT) positive macrophages/microglia, was determined following a 2-day exposure to morphine (5 mg/kg/day via time-release, subcutaneous implant) and doxycycline in GFAP-driven, doxycycline-inducible HIV-1 Tat transgenic mice. Data show that both morphine and Tat induction via doxycycline increased astrocyte activation, with significant additive increases achieved with combined morphine and doxycycline exposure. By contrast, combined Tat induction and morphine exposure, but neither manipulation alone, significantly increased the proportion of macrophages/microglia present in the striatum of transgenic mice, although morphine exposure was necessary to elevate 3-NT co-detection in Mac1-positive macrophages/microglia. Finally, Tat induction increased the percentage of neurons expressing active caspase-3, and this was even more significantly elevated by co-administration of morphine. In spite of elevations in caspase-3, neuronal TUNEL reactivity was unchanged in all groups, even after 10 days of Tat induction. Importantly, co-administration of naltrexone completely antagonized the effects of morphine. These findings indicate that morphine rapidly and significantly increases the activation of astrocytes and macrophages/microglia in the brains of inducible Tat transgenic mice, supporting the theory that early inflammatory changes in glia could underlie the development of HIVE in opiate-abusing AIDS patients.
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Cuerpo Estriado/fisiología , Productos del Gen tat/biosíntesis , VIH-1/efectos de los fármacos , VIH-1/metabolismo , Morfina/toxicidad , Neuroglía/fisiología , Neuronas/patología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/patología , Femenino , Productos del Gen tat/genética , Productos del Gen tat/fisiología , VIH-1/genética , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuroglía/efectos de los fármacos , Neuroglía/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/biosíntesisRESUMEN
A number of gene therapy approaches have been developed for protecting neurons from necrotic neurological insults. Such therapies are limited by the need for transcription and translation of the protective protein, delaying therapeutic impact. As an alternative, we explore the neuroprotective potential of protein therapy, using a fusion protein comprised of the death-suppressing BH4 domain of the Bcl-xL protein and the protein transduction domain of the human immunodeficiency virus Tat protein. This fusion protein decreased neurotoxicity caused by the excitotoxins glutamate and kainic acid in primary hippocampal cultures, and decreased hippocampal damage in vivo in an excitotoxic seizure model.
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Apoptosis/fisiología , Productos del Gen tat/uso terapéutico , Síndromes de Neurotoxicidad/tratamiento farmacológico , Proteínas Recombinantes/uso terapéutico , Proteína bcl-X/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Embrión de Mamíferos , Productos del Gen tat/biosíntesis , Ácido Glutámico/toxicidad , Hipocampo/citología , Ácido Kaínico/toxicidad , Masculino , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Síndromes de Neurotoxicidad/etiología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/biosíntesis , Proteína bcl-X/metabolismoRESUMEN
Human acidic fibroblast growth factor (haFGF) stimulates repair and regeneration of central and peripheral nerves after various injuries. However, it is unable to cross the blood-brain barrier (BBB). To produce a therapeutic haFGF with cell-permeable activity, we fused the haFGF(19-154) gene with Tat-PTD. After its construction by a single-step insertion of a polymerase chain reaction (PCR)-amplified coding sequence, the vector pTat-haFGF(19-154)-His was expressed in Escherichia coli BL21 (DE3) cells. The optimal expression level of the soluble fusion protein was up to 36.7% of the total cellular protein. The recombinant Tat-haFGF(19-154)-His was purified by a combination of Ni-NTA affinity, Sephadex G-25, and heparin affinity chromatography to 95% as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final yield was 171 mg/l culture. Purified Tat-haFGF(19-154)-His had distinct mitogenic activity in Balb/c 3T3 cells, as measured by methylthiazoletetrazolium (MTT) assay and its ED(50) was 3.931 x 10(-4) micromol/l. Tat-haFGF(19-154)-His protein intravenously injected at the dose of 10 mg/kg could be detected in the pallium and hippocampi.
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Factores de Crecimiento de Fibroblastos/genética , Productos del Gen tat/genética , Proteínas Recombinantes de Fusión/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Animales , Células 3T3 BALB , Proliferación Celular , Corteza Cerebral/química , Cromatografía de Afinidad , Cromatografía en Gel , Clonación Molecular , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Factores de Crecimiento de Fibroblastos/biosíntesis , Factores de Crecimiento de Fibroblastos/aislamiento & purificación , Factores de Crecimiento de Fibroblastos/farmacocinética , Expresión Génica , Productos del Gen tat/biosíntesis , Productos del Gen tat/aislamiento & purificación , Productos del Gen tat/farmacocinética , Hipocampo/química , Humanos , Ratones , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacocinéticaRESUMEN
Kaposi's sarcoma (KS), a neoplasm often associated with iatrogenic and acquired immunosuppression, is characterized by prominent angiogenesis. Angiogenic factors released from KS and host cells and HIV viral products-the protein Tat are reported to be involved in angiogenesis. Mounting evidence further suggests that multiple angiogenic activities of Tat contribute to AIDS-associated Kaposi's sarcoma (AIDS-KS). Herein, we report that sulfated polymannuroguluronate (SPMG), a novel anti-AIDS drug candidate now undergoing phase II clinical trial, significantly eliminated Tat-induced angiogenesis in SLK cells both in vitro and in vivo. SPMG significantly and dose-dependently inhibits proliferation, migration, and tube formation by SLK cells. SPMG also dramatically arrested Tat-driven KDR phosphorylation and blocked the interaction between Tat and integrin beta1, thus inhibiting the phosphorylation of the downstream kinases of FAK, paxillin and MAPKs. In addition, SPMG was noted to block the release of bFGF and VEGF from ECM. All these collectively favor an issue that SPMG functions as a promising therapeutic against Tat-induced angiogenesis and pathologic events relevant to AIDS-KS, which adds novel mechanistic profiling to the anti-AIDS action of SPMG.
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Fármacos Anti-VIH/farmacología , Productos del Gen tat/farmacología , VIH-1/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Polisacáridos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Sarcoma de Kaposi , Animales , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/uso terapéutico , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colágeno , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Escherichia coli/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Productos del Gen tat/biosíntesis , Glutatión Transferasa/metabolismo , Humanos , Laminina , Masculino , Ratones , Neovascularización Patológica/metabolismo , Polisacáridos/administración & dosificación , Polisacáridos/uso terapéutico , Proteoglicanos , Proteínas Recombinantes de Fusión/biosíntesis , Sarcoma de Kaposi/irrigación sanguínea , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The positive transcription elongation factor b (P-TEFb) is an essential regulator of viral gene expression during the life cycle of human immunodeficiency virus type 1 (HIV-1). Its cyclin T1 subunit forms a ternary complex with the viral transcriptional transactivator (Tat) protein and the transactivation response (TAR) RNA element thereby activating cyclin dependent kinase 9 (Cdk9), which stimulates transcription at the level of chain elongation. We report the structure of the cyclin box domain of human cyclin T1 at a resolution of 2.67 A. The structure was obtained by crystallographic analysis of a fusion protein composed of cyclin T1 linked to the transactivator protein Tat from equine infectious anemia virus (EIAV), which is functionally and structurally related to HIV-1 Tat. The conserved cyclin box domain of cyclin T1 exhibits structural features for interaction with physiological binding partners such as Cdk9. A recognition site for Cdk/Cyclin substrates is partly covered by a cyclin T-specific insert, suggesting specific interactions with regulatory factors. The previously identified Tat/TAR recognition motif (TRM) forms a C-terminal helix that is partly occluded in the cyclin box repeat interface, while cysteine 261 is accessible to form an intermolecular zinc finger with Tat. Residues of the TRM contribute to a positively charged groove that may directly attract RNA molecules. The EIAV Tat protein instead appeared undefined from the electron density map suggesting that it is highly disordered. Functional experiments confirmed the TAR binding properties of the fusion protein and suggested residues on the second cyclin box repeat to contribute to Tat stimulated transcription.
Asunto(s)
Ciclinas/química , Productos del Gen tat/química , Virus de la Anemia Infecciosa Equina/genética , Modelos Moleculares , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Ciclina T , Quinasa 9 Dependiente de la Ciclina/química , Ciclinas/biosíntesis , Ciclinas/genética , Ensayo de Cambio de Movilidad Electroforética , Productos del Gen tat/biosíntesis , Productos del Gen tat/genética , Humanos , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Conformación Proteica , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/química , Subunidades de Proteína/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Distal symmetric polyneuropathy (DSP) has emerged as the most common complication of human immunodeficiency virus (HIV) infection, which is associated with neuronal injury in the dorsal root ganglion (DRG). With the advent of highly active antiretroviral therapy, especially nucleoside analogs, patients are living longer. Some of the antiretroviral drugs used to treat HIV infection have been associated with neuropathies. The pathogenesis of these neuropathies remains poorly understood. Utilizing a human fetal DRG model of predominantly nociceptive fibers, the authors investigated the effects of HIV gp120 and Tat(1-72), alone or in combination with nucleoside analogs on both morphological and ultra-structural changes in DRG neurons. Nucleoside analogs and HIV proteins both caused a significant decrease in the mean axonal length. However, ddI was the most potent, followed by ddC, d4T, and AZT. Despite the combined exposure to toxic dosages of HIV proteins and nucleoside analogs, there appeared to be a ceiling effect on the amount of axonal retraction, indicating that the proximal and distal axon are differentially regulated. In conclusion, both HIV proteins and nucleoside reverse transcriptase inhibitors (NRTIs) cause axonal damage by inducing mitochondrial injury and rearrangement of microtubules.
Asunto(s)
Productos del Gen tat/toxicidad , Proteína gp120 de Envoltorio del VIH/toxicidad , Inhibidores de la Transcriptasa Inversa/toxicidad , Axones/patología , Células Cultivadas , Ganglios Espinales/patología , Ganglios Espinales/ultraestructura , Productos del Gen tat/biosíntesis , Humanos , Microscopía Electrónica , Microtúbulos/patología , Mitocondrias/patología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/toxicidadRESUMEN
Cells harboring infectious, but transcriptionally latent, human immunodeficiency virus type 1 (HIV-1) proviruses currently pose an insurmountable barrier to viral eradication in infected patients. To better understand the molecular basis for HIV-1 latency, we used the J-Lat model of postintegration HIV-1 latency to assess the kinetic relationship between the induction of NF-kappaB and the activation of latent HIV-1 gene expression. Chromatin immunoprecipitation analyses revealed an oscillating pattern of RelA recruitment to the HIV-1 long terminal repeat (LTR) during continuous tumor necrosis factor alpha (TNF-alpha) stimulation. RNA polymerase II (Pol II) recruitment to the HIV-1 LTR closely mirrored RelA binding. Transient stimulation of cells with TNF-alpha for 15 min induced only a single round of RelA and RNA Pol II binding and failed to induce robust expression of latent HIV-1. Efficient formation of elongated HIV-1 transcripts required sustained induction by NF-kappaB, which promoted de novo synthesis of Tat. Cyclin-dependent kinase 9 (CDK9) and serine-2-phosphorylated RNA Pol II were rapidly recruited to the HIV-1 LTR after NF-kappaB induction; however, these elongating polymerase complexes were progressively dephosphorylated in the absence of Tat. Okadaic acid promoted sustained serine-2 phosphorylation of the C-terminal domain of RNA Pol II and stimulated efficient transcriptional elongation and HIV-1 expression in the absence of Tat. These findings underscore important differences between NF-kappaB and Tat stimulation of RNA Pol II elongation. While NF-kappaB binding to the HIV-1 LTR induces serial waves of efficient RNA Pol II initiation, elongation is impaired by the action of an okadaic acid-sensitive phosphatase that dephosphorylates the C-terminal domain of RNA Pol II. Conversely, the action of this phosphatase is overcome in the presence of Tat, promoting very efficient RNA Pol II elongation.
Asunto(s)
Regulación Viral de la Expresión Génica/fisiología , VIH-1/fisiología , FN-kappa B/biosíntesis , Latencia del Virus/fisiología , Línea Celular , Productos del Gen tat/biosíntesis , Productos del Gen tat/genética , Humanos , FN-kappa B/fisiología , Provirus/fisiología , ARN Polimerasa II/fisiología , Factor de Transcripción ReIA/fisiología , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Herpes simplex type-1 virus (HSV-1) based vectors have been widely used in different gene therapy approaches and also as experimental vaccines against HSV-1 infection. Recent advances in the HSV-1 technology do support the use of replication defective HSV-1 as vaccine vectors for delivery of foreign antigens. We have examined the ability of a recombinant replication-defective HSV-1 vector expressing the HIV-1 Tat protein to induce long-term Tat-specific immune responses in the Balb/c murine model. The results showed that vector administration by the subcutaneous route elicits anti-Tat specific T-cell mediated immune responses in mice characterized by the presence of the Tat-specific cytotoxic activity and production of high levels of IFN-gamma.
Asunto(s)
Vacunas contra el SIDA/inmunología , Productos del Gen tat/biosíntesis , Productos del Gen tat/inmunología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/inmunología , Linfocitos T/inmunología , Replicación Viral/genética , Vacunas contra el SIDA/administración & dosificación , Administración Intranasal , Animales , Western Blotting , Proliferación Celular , Chlorocebus aethiops , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Esquemas de Inmunización , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/genética , Inyecciones Subcutáneas , Interferón gamma/inmunología , Ratones , Ratones Endogámicos BALB C , Plásmidos/genética , Bazo/citología , Bazo/inmunología , Vacunas Sintéticas/inmunología , Células VeroRESUMEN
We examined the production and secretion of fusion constructs containing alpha-N-acetylglucosaminidase, the enzyme deficient in Sanfilippo B, and either wildtype TAT or modified TAT in cultured Spodoptera frugiperda cells. All constructs exhibited successful expression of active enzyme, suggesting the future possibility of utilizing TAT/alpha-N-acetylglucosaminidase chimerae in enzyme replacement therapy.