Anti-HIV Agents ; pharmacology ; Ciprofloxacin ; analogs & derivatives ; pharmacology ; Gene Products, tat ; chemistry ; genetics ; Genes, tat ; HIV-1 ; drug effects ; genetics ; Humans ; Oligopeptides ; pharmacology ; Organophosphates ; pharmacology ; Transcription, Genetic ; Transcriptional Activation ; Uridine ; analogs & derivatives ; pharmacology ; tat Gene Products, Human Immunodeficiency Virus

OBJECTIVETo develop a novel gene delivery vector TAT-PEI-beta-CyD.

METHODSbeta-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.

RESULTTAT 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.

CONCLUSIONThe novel vector TAT-PEI-beta-CyD has been developed successfully with low cytotoxicity and high transfection efficiency.

Cell Line ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Humans ; Peptide Fragments ; chemistry ; Polyethyleneimine ; chemistry ; beta-Cyclodextrins ; chemistry ; tat Gene Products, Human Immunodeficiency Virus ; chemistry

In order to detect the protein delivery mediated by the PTD (protein transduction domain) of TAT Protein, a expression vector, named pT7460-GFP, was constructed by insert the PTD DNA Sequence, followed by a GFP (green fluorescent protein) gene fused in-frame, into the pT7450 vector. The TAT-GFP fusion protein was expressed in the E. coli ER2566. Most of the fusion protein was presented in the inclusion body. The protein was purified by Ni2+ affinity chromatography under denature conditions, then by a Sepharose Q column to remove urea. The soluble denatured protein was added directly to medium containing the Myeloma Cell SP2/0. It came out that the fusion protein could be detected delivered into the cells under fluorescent microscope in a short time.
Gene Products, tat ; chemistry ; metabolism ; Green Fluorescent Proteins ; Humans ; Luminescent Proteins ; metabolism ; Microscopy, Fluorescence ; Multiple Myeloma ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; metabolism ; Tumor Cells, Cultured

In our previous work we reported that HIV Tat and 6 cysteine rich peptides of Tat induce tumor necrosis factor-related apoptosis-induced ligand (TRAIL) in human monocytes (Yang et al., 2003). Here our results showed that HIV Tat and Tat cysteine rich peptide increase CCR5 expression in human monocytes, and this activity is inhibited by rabbit anti-Tat. Boiled Tat does not increase CCR5 expression in monocytes. These results provide insight into a new mechanism by which HIV Tat plays a key role in the pathogenesis of HIV-1 infection.
Amino Acid Sequence ; Cells, Cultured ; Cysteine ; chemistry ; Dose-Response Relationship, Drug ; Extracellular Fluid ; chemistry ; Gene Expression Regulation ; drug effects ; physiology ; Gene Products, tat ; chemistry ; pharmacology ; Humans ; Molecular Sequence Data ; Monocytes ; drug effects ; metabolism ; Peptides ; chemistry

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.
Animals ; Blood-Brain Barrier ; metabolism ; Drug Delivery Systems ; Female ; Gelatin ; administration & dosage ; chemistry ; pharmacokinetics ; Male ; Mice ; Mice, Nude ; Nanoparticles ; chemistry ; Peptide Fragments ; chemistry ; Polyethylene Glycols ; chemistry ; Siloxanes ; administration & dosage ; chemistry ; pharmacokinetics ; tat Gene Products, Human Immunodeficiency Virus ; chemistry

HIV-1 Tat is considered to be one of key players to facilitate monocyte entry into the CNS, which is characteristic feature of AIDS-related encephalitis and dementia. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the HIV-1 Tat-induced signaling pathways leading to NF-kappaB activation, expression of adhesion molecules, and monocyte adhesion in CRT-MG human astroglioma cells by using cell-permeable SOD. When cell-permeable SOD was added to the culture medium of CRT-MG cells, it rapidly entered the cells in dose- and time-dependent manners. Treatment of astrocytes with cell-permeable SOD led to decrease in Tat-induced ROS generation as well as NF-kappaB activation. Cell-permeable SOD inhibited the activation of MAP kinases including ERK, JNK and p38 by HIV-1 Tat. Treatment of CRT-MG cells with cell-permeable SOD significantly inhibited protein and mRNA levels of ICAM-1 and VCAM-1 up-regulated by HIV-1 Tat, as measured by Western blot analysis and RT-PCR. Furthermore, enhanced adhesiveness of monocyte to astrocyte by HIV-1 Tat was significantly abrogated by pretreatment with cell-permeable SOD fusion proteins. These data indicate that SOD has a regulatory function for HIV-1 Tat-induced NF-kappaB activation in astrocytes and suggest that cell-permeable SOD can be used as a feasible therapeutic agent for regulation of ROS-related neurological diseases.
Astrocytes/*enzymology ; Cell Adhesion/*physiology ; Cell Membrane Permeability ; Gene Products, tat/*pharmacology ; HIV Infections/metabolism ; HIV-1/*chemistry ; Humans ; Monocytes/cytology/*drug effects ; Signal Transduction ; Superoxide Dismutase/genetics/*physiology

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.
Anti-HIV Agents ; pharmacology ; Enzyme Inhibitors ; pharmacology ; HIV-1 ; genetics ; Humans ; Okadaic Acid ; pharmacology ; Protein Phosphatase 1 ; antagonists & inhibitors ; chemistry ; physiology ; Pyrans ; pharmacology ; Spiro Compounds ; pharmacology ; Transcription, Genetic ; tat Gene Products, Human Immunodeficiency Virus ; physiology

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).
Benzodiazepinones ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; HIV Long Terminal Repeat ; genetics ; HIV-1 ; genetics ; Humans ; Transcription, Genetic ; drug effects ; tat Gene Products, Human Immunodeficiency Virus ; antagonists & inhibitors

Doxorubicin loaded micelles were prepared by film-hydration method using stearyl sulfadiazine (SA-SD) which is pH sensitive, methoxy (polyethylene glycol)-2000-1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (mPEG-DOPE) and transactivator of transcription (TAT) peptide conjugated PEG-DOPE. Mean diameter of the pH-sensitive micelles was about 20 nm with a (99.1 +/- 2.1) % drug entrapment efficiency at pH 7.4. Flow cytometry studies revealed that the simple TAT micelles was taken up rapidly at the same level at pH 6.8 and pH 7.4. However, the pH-sensitive micelles entered the tumor cell less at pH 7.4 and significantly increase at pH 6.8. After 1 h incubation at pH 6.8, the amount of the pH-sensitive micelles taken up by cancer cell 4T1 was almost similar to simple TAT micelles. The confocal microscopy indicated that the pH-sensitive micelles entered the 4T1 cells at pH 6.8 more than at pH 7.4. It was indicated that the pH-sensitive micelles could shield TAT peptide at normal pH 7.4 and deshield it at pH 6.8. Hence, TAT peptides lead the drug-loaded micelles into the tumor cells and killed them selectively. The pH-sensitive micelle may provide a novel strategy for design of cancer targeting drug delivery system.
Animals ; Antibiotics, Antineoplastic ; administration & dosage ; chemistry ; Cell Line, Tumor ; Cell-Penetrating Peptides ; chemistry ; Doxorubicin ; administration & dosage ; chemistry ; Drug Carriers ; Drug Compounding ; Drug Delivery Systems ; Female ; Gene Products, tat ; chemistry ; Hydrogen-Ion Concentration ; Mammary Neoplasms, Experimental ; pathology ; Mice ; Micelles ; Phosphatidylethanolamines ; chemistry ; Polyethylene Glycols ; chemistry ; Sulfadiazine ; chemistry

OBJECTIVE: We wanted to compare the human neural stem cell (hNSC) labeling efficacy of different superparamagnetic iron oxide nanoparticles (SPIONs), namely, ferumoxides, monocrystalline iron oxide (MION), cross-linked iron oxide (CLIO)-NH2 and tat-CLIO. MATERIALS AND METHODS: The hNSCs (5x105 HB1F3 cells/ml) were incubated for 24 hr in cell culture media that contained 25 microgram/ml of ferumoxides, MION or CLIO-NH2, and with or without poly-L-lysine (PLL) and tat-CLIO. The cellular iron uptake was analyzed qualitatively with using a light microscope and this was quantified via atomic absorption spectrophotometry. The visibility of the labeled cells was assessed with MR imaging. RESULTS: The incorporation of SPIONs into the hNSCs did not affect the cellular proliferations and viabilities. The hNSCs labeled with tat-CLIO showed the longest retention, up to 72 hr, and they contained 2.15+/-0.3 pg iron/cell, which are 59 fold, 430 fold and six fold more incorporated iron than that of the hNSCs labeled with ferumoxides, MION or CLIO-NH2, respectively. However, when PLL was added, the incorporation of ferumoxides, MION or CLIO-NH2 into the hNSCs was comparable to that of tat-CLIO. CONCLUSION: For MR imaging, hNSCs can be efficiently labeled with tat-CLIO alone or with a combination of ferumoxides, MION, CLIO-NH2 and the transfection agent PLL.
Cells, Cultured ; Contrast Media/chemical synthesis/pharmacokinetics ; Cross-Linking Reagents/chemistry ; Ferric Compounds/chemistry/*pharmacokinetics ; Ferrosoferric Oxide/chemical synthesis/pharmacokinetics ; Gene Products, tat/chemistry ; Humans ; Iron/*pharmacokinetics ; Magnetic Resonance Imaging/methods ; Nanoparticles ; Neural Tube ; Oxides/*pharmacokinetics ; Phantoms, Imaging ; Polylysine/pharmacokinetics ; Spectrophotometry, Atomic ; Staining and Labeling/*methods ; Stem Cells/cytology/*drug effects/metabolism ; Time Factors ; Transfection