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Mesenchymal stem cells (MSCs) can differentiate toward various lineages, including the osteogenic lineage, and thus hold great potential for clinic purposes. By using pharmacological inhibitors, protein kinase C (PKC) signaling has been shown to either negatively or positively regulate differentiation of bone, however, due to the low transfection efficiency in MSCs, the role of individual PKC isoforms is still not fully understood. In this study, we established a TAT peptide-mediated transduction system that efficiently delivered PKCα proteins into MSCs in a non-invasive fashion. The increased PKCα protein levels significantly promoted osteogenic differentiation in the murine mesenchymal C3H10T1/2 cells and in primary MSCs from both human and mouse, as demonstrated by the enhanced activity of the osteoblast marker, alkaline phosphatase, and the enhanced expression of the key transcription factor runx2. Mineralization is an important functional indication for bone differentiation. Our results further showed that PKCα promoted expression of the important osteocalcin gene and the accumulation of calcium minerals. Taken together, this study provides direct evidence showing that PKCα positively regulates osteogenic differentiation and demonstrates that the TAT peptide-mediated method enables functional study of specific PKC isoforms in MSCs without using viral infection. This may promote the application of PKCs in therapeutic treatment.

Neurogenin-2 (Ngn2), as a proneural gene that promotes the survival and differentiation of neural precursor cells, is an attractive candidate for therapy against cerebral ischemia-reperfusion injury. However, the delivery approach limits its clinical application. To deliver Ngn2 protein into the cerebral ischemic region and exert a therapeutic effect on injured neurons after ischemia, we here reported that the fusion protein TAT-LBD-Ngn2 was constructed by fusing a transactivator of transcription (TAT) domain and a laminin-binding domain (LBD) to Ngn2. TAT-LBD-Ngn2 promoted the outgrowth of neuronal neurite, increased the survival rate and alleviated apoptosis of hippocampal neurons exposed to oxygen glucose deprivation in vitro. Furthermore, a focal cerebral ischemia model in C57BL/6 mice showed that TAT-LBD-Ngn2 efficiently crossed the blood brain barrier, aggregated in the ischemic zone and was consistently incorporated into neurons. Moreover, TAT-LBD-Ngn2 transduced into brains attenuated neuronal degeneration and apoptosis in the ischemic zone. TAT-LBD-Ngn2 treatment resulted in a reduction of infarct volume that was associated with a parallel improvement in neurological functional outcomes after reperfusion. In conclusion, the targeted delivery of TAT-LBD-Ngn2 into the ischemic zone attenuated cerebral ischemia-reperfusion injury through the inhibition of neuronal degeneration and apoptosis, suggesting that TAT-LBD-Ngn2 is a promising target candidate for the treatment of ischemic stroke., (© 2013 Elsevier Ltd. All rights reserved.)

Background: Protein production as secretory-form is a powerful tool in industrial enzyme production due to the simple purification procedure. Streptomyces lividans is a versatile host for secretory production of useful proteins. In order to expand the amount of secreted protein, signal peptide sequences, which encourage protein secretion from inside cell to extracellular environment, are one of the most significant factors. In this study, we focused on Streptomyces lividans as a host strain to secrete useful proteins, and screened for signal peptides from the biomass-degradation enzymes derived from Thermobifida fusca YX and S. lividans., Results: Three candidate signal peptides were isolated and evaluated for their protein secretion ability using β-glucosidase derived from T. fusca YX, which is a non-secreted protein, as a model protein. Using S. lividans xylanase C signal peptide, the amount of produced the β-glucosidase reached 10 times as much as that when using Streptomyces cinnamoneus phospholipase D signal peptide, which was identified as a versatile signal peptide in our previous report. In addition, the introduction of the β-glucosidase fused to xylanase C signal peptide using two kinds of plasmid, pUC702 and pTYM18, led to further protein secretion, and the maximal level of produced the β-glucosidase increased up to 17 times (1.1 g/l) compared to using only pUC702 carrying the β-glucosidase fused to S. cinnamoneus phospholipase D signal peptide., Conclusion: In the present study, we focused on signal peptide sequences derived from biomass degradation enzymes, which are usually secreted into the culture supernatant, and screened for signal peptides leading to effective protein secretion. Using the signal peptides, the hyper-protein secretion system was successfully demonstrated for the cytoplasmic β-glucosidase.

Objective: To study the dose-dependent manner of HIV-1 Tat-induced effects on viral replication, internalization and spread, and to directly observe these effects on soluble Env immunogens and virus-like particles., Design: In order to determine the manner through which Tat affects viral replication, we incubated cells, virions and soluble Env spikes with Tat at different concentrations, and directly visualized the effects of such incubation., Methods: Cell-based infectivity assays were carried out to assay Tat dose-dependency of viral infectivity. Transmission electron microscopy of virus-like particles and soluble gp140 immunogens incubated with Tat at various concentrations was performed to directly observe Tat-induced effects., Results: Treating virus with exogenous Tat increased infectivity in a dose-dependent manner. In the presence of anti-Tat antibodies, virus replication and spread were repressed, postulating Tat contributions to disease progression. When CXCR4 coreceptors were blocked, Tat treatment overcame the inhibition relative to absence of Tat treatment. Similarly, syncytium formation between chronically infected and uninfected target cells was also increased by exogenous Tat treatment. Inhibiting the CD4 receptor for virus entry abolished syncytium formation and Tat treatment was unable to overcome CD4 dependency. We show that Tat reduces virus infectivity at higher Tat concentrations through Env interactions resulting in viral aggregation., Conclusion: Treating virions or chronically infected cells with exogenous Tat could enhance virus infectivity and spread through coreceptor tropism switch or through another undetermined mechanism. The aggregation potential of Tat suggests a mechanism of negative-feedback regulation of viral replication, providing another regulative function to control viral replication., (© 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins)

Preventing cell entry of human immunodeficiency virus 1 (HIV-1) is of interest for the development of innovative therapies. We previously reported a specific interaction between HIV-1 envelope glycoprotein 120 (gp120) and Tat at the cell surface, which enhances virus attachment and entry. We also identified a gp120-mimicking peptide, CT319, that competes with gp120 for Tat binding, thus inhibiting HIV-1 infection. Here we report a molecular dissection of gp120 regions involved in this mechanism. Our findings identify the V1/V2 loop of gp120 as involved in Tat binding, and define this interaction as functionally relevant for HIV-1 entry into host cells., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Purpose: To create poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), where a drug-encapsulating NP core is covered with polyethylene glycol (PEG) in a normal condition but exposes a cell-interactive TAT-modified surface in an environment rich in matrix metalloproteinases (MMPs)., Methods: PLGA NPs were modified with TAT peptide (PLGA-pDA-TAT NPs) or dual-modified with TAT peptide and a conjugate of PEG and MMP-substrate peptide (peritumorally activatable NPs, PANPs) via dopamine polymerization. Cellular uptake of fluorescently labeled NPs was observed with or without a pre-treatment of MMP-2 by confocal microscopy and flow cytometry. NPs loaded with paclitaxel (PTX) were tested against SKOV-3 ovarian cancer cells to evaluate the contribution of surface modification to cellular delivery of PTX., Results: While the size and morphology did not significantly change due to the modification, NPs modified with dopamine polymerization were recognized by their dark color. TAT-containing NPs (PLGA-pDA-TAT NPs and PANPs) showed changes in surface charge, indicative of effective conjugation of TAT peptide on the surface. PLGA-pDA-TAT NPs and MMP-2-pre-treated PANPs showed relatively good cellular uptake compared to PLGA NPs, MMP-2-non-treated PANPs, and NPs with non-cleavable PEG. After 3 h treatment with cells, PTX loaded in cell-interactive NPs showed greater toxicity than non-interactive ones as the former could enter cells during the incubation period. However, due to the initial burst drug release, the difference was not as clear as microscopic observation., Conclusions: PEGylated polymeric NPs that could expose cell-interactive surface in response to MMP-2 were successfully created by dual modification of PLGA NPs using dopamine polymerization.

TAT peptide is one of the best-characterized cell penetrating peptides derived from the transactivator of transcription protein from the human immunodeficiency virus 1. The aim of this study was to investigate the interaction between TAT peptide and partially negatively-charged phospholipid bilayer by using lamellar neutron diffraction. The main findings are the existence of a contiguous water channel across the bilayer in the presence of TAT peptide. Taken in combination with other observations, including thinning of the lipid bilayer, this unambiguously locates the peptide within the lipid bilayer. The interaction of TAT peptide with anionic lipid bilayer, composed of an 80:20 mixture of DOPC and DOPS, takes place at two locations. One is in the peripheral aqueous phase between adjacent bilayers and the second is below the glycerol backbone region of bilayer. A membrane thinning above a peptide concentration threshold (1mol%) was found, as was a contiguous transbilayer water channel at the highest peptide concentration (10mol%). This evidence leads to the suggestion that the toroidal pore model might be involved in the transmembrane of TAT peptide. We interpret the surface peptide distribution in the peripheral aqueous phase to be a massive exclusion of TAT peptide from its intrinsic location below the glycerol backbone region of the bilayer, due to the electrostatic attraction between the negatively-charged headgroups of phospholipids and the positively charged TAT peptides. Finally, we propose that the role that negatively-charged headgroups of DOPS lipids play in the transmembrane of TAT peptide is less important than previously thought., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Protein delivery vectors can be grouped into two classes, those with specific membrane receptors undergoing conventional endocytosis and cell penetrating peptides (CPP) that have the capacity to cross cell or endosomal membranes. For both forms of vectors, translocation across a membrane is usually an inefficient process. In the current study, a novel vector combining the widely used CPP, Tat and the non-toxic neuronal binding domain of tetanus toxin (fragment C or TTC) was assessed for its capacity to deliver GFP as a test cargo protein to human neural progenitor cells (NPCs). These two functional membrane interacting domains dramatically enhanced internalization of the conjugated cargo protein. Tat-TTC-GFP was found to be bound or internalized at least 83-fold more than Tat-GFP and 33-fold more than TTC-GFP in NPCs by direct fluorimetry, and showed enhanced internalization by quantitative microscopy of 18 - and 14-fold, respectively. This preferential internalization was observed to be specific to neuronal cell types. Photochemical internalization (PCI) was utilized to facilitate escape of the endosome-sequestered proteins. The combined use of the Tat-TTC delivery vector with PCI led to both enhancement of neural cell type specific delivery to an endosomal target, followed by the option of efficient release to the cytosol.

The transcription factors Runx1 and c-Myc have individually been shown to regulate important gene targets as well as to collaborate in oncogenesis. However, it is unknown whether there is a regulatory relationship between the two genes. In this study, we investigated the transcriptional regulation of endogenous c-Myc by Runx1 in the human T cell line Jurkat and murine primary hematopoietic cells. Endogenous Runx1 binds to multiple sites in the c-Myc locus upstream of the c-Myc transcriptional start site. Cells transduced with a C-terminally truncated Runx1 (Runx1.d190), which lacks important cofactor interaction sites and can block C-terminal-dependent functions of all Runx transcription factors, showed increased transcription of c-Myc. In order to monitor c-Myc expression in response to early and transiently-acting Runx1.d190, we generated a cell membrane-permeable TAT-Runx1.d190 fusion protein. Murine splenocytes treated with TAT-Runx1.d190 showed an increase in the transcription of c-Myc within 2 hours, peaking at 4 hours post-treatment and declining thereafter. This effect is dependent on the ability of Runx1.d190 to bind to DNA. The increase in c-Myc transcripts is correlated with increased c-Myc protein levels. Collectively, these data show that Runx1 directly regulates c-Myc transcription in a C-terminal- and DNA-binding-dependent manner.

Nuclear export and translation of intron-containing viral mRNAs are required for HIV-1 gene expression and replication. In this report, we provide evidence to show that DDX3 regulates the translation of HIV-1 mRNAs. We found that knockdown of DDX3 expression effectively inhibited HIV-1 production. Translation of HIV-1 early regulatory proteins, Tat and rev, was impaired in DDX3-depleted cells. All HIV-1 transcripts share a highly structured 5' untranslated region (UTR) with inhibitory elements on translation of viral mRNAs, yet DDX3 promoted translation of reporter mRNAs containing the HIV-1 5' UTR, especially with the transactivation response (TAR) hairpin. Interestingly, DDX3 directly interacts with HIV-1 Tat, a well-characterized transcriptional activator bound to the TAR hairpin. HIV-1 Tat is partially targeted to cytoplasmic stress granules upon DDX3 overexpression or cell stress conditions, suggesting a potential role of Tat/DDX3 complex in translation. We further demonstrated that HIV-1 Tat remains associated with translating mRNAs and facilitates translation of mRNAs containing the HIV-1 5' UTR. Taken together, these findings indicate that DDX3 is recruited to the TAR hairpin by interaction with viral Tat to facilitate HIV-1 mRNA translation.

RNA-binding proteins are an important class of mediators that regulate cell function and differentiation. Methylation of arginine, a post-translational modification (PTM) found in these proteins, can modulate their function. Arginine can be monomethylated or dimethylated, depending on the type of methyl transferases involved. This paper describes a comparative study of the thermodynamics of unmodified and modified Tat peptide interaction with TAR RNA, where the peptide is methylated at epsilon (ɛ) and eta (η) nitrogen atoms of guanidinium group of arginine side chain at position 52 or 53. The results indicate that monomethylation of arginine at epsilon (ɛ) nitrogen atom enhances binding affinity, owing to a more favourable enthalpy component which overrides the less favourable entropy change. In contrast, monomethylation of arginine residue at η nitrogen results in reduced binding affinity originating exclusively from a less favourable enthalpy change leaving entropic component unaffected. However, in case of simultaneous methylation at ɛ and η positions, the binding parameters remain almost unaffected, when compared to the unmodified peptide. In case of symmetric dimethylation at η position the observed enthalpy change of the binding was found to be smaller than the values obtained for the unmodified peptide. Asymmetric dimethylation at η position showed the most reduced binding affinities owing to less favourable enthalpy changes. These results provide insights that enable elucidation of the biological outcome of arginine methylation as PTMs that regulate protein function, and will contribute to our understanding of how these PTMs are established in vitro and in vivo., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

The twin-arginine translocation (Tat) system works to export folded proteins across the cytoplasmic membrane via specific signal peptides harbouring a twin-arginine motif. In Escherichia coli, a functional GFP is exported to the periplasm through the Tat pathway by fusion of the signal peptide of TorA, which is one of the periplasmic proteins exported by the Tat pathway. In this study, we fused the signal peptide of Vibrio alginolyticus TorA (TorASP) to GFP and demonstrate the export of functional GFP to the periplasm of V. alginolyticus. We also made fusions of TorASP-GFP with MotX, MotY and FlgT, which are periplasmic components of the Na(+)-driven flagellar motor. Those fusion proteins were localized to the flagellar motor independent of the Na(+) concentration in the environment.

As a result of its interaction with transcription factors, HIV type 1 (HIV-1) Tat can modulate the expression of both HIV and cellular genes. In antigen-presenting cells Tat induces the expression of a subset of interferon (IFN)-stimulated genes (ISGs) in the absence of IFNs. We investigated the genome-wide Tat association with promoters in immature dendritic cells and in monocyte-derived macrophages. Among others, Tat associated with the MAP2K6, MAP2K3, and IRF7 promoters that are functionally part of IL-1 and p38 mitogen-activated protein kinase (MAPK) signaling pathways. The association correlated with their increased gene expression, increased activation of p38 MAPK and of phosphorylated signal transducer and activator of transcription 1 (STAT1), and consequent induction of ISGs. Probing these pathways with RNA interference, pharmacological p38 MAPK inhibition, and in cell lines lacking STAT1s or the type I IFN receptor chain confirmed the role of MAPKKs and IRF7 in Tat-mediated modulation of ISGs and excluded the involvement of IFNs in this modulation. Tat interaction with the 2 MAPKK and IRF7 promoters in HIV-1-infected cells and the resulting persistent activation of ISGs, which include inflammatory cytokines and chemokines, can contribute to the increased immune activation that characterizes HIV infection.

Neuroinflammation is an integral component of neurodegenerative disorders, CNS infection and trauma. Astroglial chemokines, such as CXCL10, are instrumental in neuroinflammatory signaling as well as neurotoxicity. We have utilized proinflammatory-induced CXCL10 expression in normal human astrocytes (NHA) as a model in which to assess the anti-inflammatory actions of the selective, mu-opioid receptor (MOR) antagonist, β-funaltrexamine (β-FNA). Interferon (IFN)γ+HIV-1 Tat-induced CXCL10 expression (secreted protein and mRNA) was inhibited by co-treatment with β-FNA. Neither the MOR-selective antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2 (CTAP) nor the nonselective opioid receptor antagonist, naltrexone inhibited IFNγ+HIV-1 Tat-induced CXCL10 expression. Furthermore, co-treatment with excess CTAP or naltrexone did not prevent β-FNA mediated inhibition of IFNγ+HIV-1 Tat-induced CXCL10 expression. Additionally, we utilized an inhibitor of NF-κB activation (SN50) to demonstrate that IFNγ+HIV-1 Tat-induced CXCL10 expression is NF-κB-dependent in NHA. Subsequent experiments revealed that β-FNA did not significantly affect NF-κB activation. Interestingly, we discovered that β-FNA inhibited p38 activation as indicated by decreased expression of phospho-p38. Together, these findings suggest that the inhibitory actions of β-FNA are MOR-independent and mediated, in part, via a transcriptional mechanism. These findings add to our understanding of the mechanism by which chemokine expression is inhibited by β-FNA. In conjunction with future investigations, these novel findings are expected to provide insights into the development of safe and effective treatments for neuroinflammation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Polyomavirus JC (JCV) is the aetiological agent of progressive multifocal leukoencephalopathy (PML), a frequently fatal infection of the brain afflicting nearly 4% of AIDS patients in the USA. Human immunodeficiency virus type 1 (HIV-1) Tat, acting together with cellular proteins at the JCV non-coding control region (NCCR), can stimulate JCV DNA transcription and replication. Tat in the brain is secreted by HIV-1-infected cells and incorporated by oligodendroglia, cells capable of infection by JCV. Thus far the effects of Tat on JCV have been studied primarily with protein encoded by the HIV-1 B clade most common in North America. Here, we determine the abilities of Tat from different HIV-1 clades to alter JCV early and late gene transcription and DNA replication initiated at the JCV origin. Tat from all clades tested stimulates both JCV early and late gene promoters, with clade B Tat being significantly most effective. Tat proteins from the HIV-1 clades display parallel patterns of differences in their effects on HIV-1 and JCV transcription, suggesting that Tat effects in both cases are mediated by the same cellular proteins. Clade B Tat is most effective at directing Smad mediators of tumour growth factor beta and cellular partner Purα to the NCCR. Tat proteins from all non-B clades inhibit initiation of JCV DNA replication. The effectiveness of HIV-1 clade B Tat at promoting JCV transcriptional and replicative processes highlights a need for further investigation to determine which molecular aspects of Tat from distinct HIV-1 substrains can contribute to the course of PML development in neuroAIDS.

Background: Human immunodeficiency virus (HIV) associated neurocognitive disorders (HAND), including memory dysfunction, continue to be a major clinical manifestation of HIV type-1 infection. Viral proteins released by infected glia are thought to be the principal triggers of inflammation and bystander neuronal injury and death, thereby driving key symptomatology of HAND., Methods: We used a glial fibrillary acidic protein-driven, doxycycline-inducible HIV type-1 transactivator of transcription (Tat) transgenic mouse model and examined structure-function relationships in hippocampal pyramidal cornu ammonis 1 (CA1) neurons using morphologic, electrophysiological (long-term potentiation [LTP]), and behavioral (Morris water maze, fear-conditioning) approaches., Results: Tat induction caused a variety of different inclusions in astrocytes characteristic of lysosomes, autophagic vacuoles, and lamellar bodies, which were typically present within distal cytoplasmic processes. In pyramidal CA1 neurons, Tat induction reduced the number of apical dendritic spines, while disrupting the distribution of synaptic proteins (synaptotagmin 2 and gephyrin) associated with inhibitory transmission but with minimal dendritic pathology and no evidence of pyramidal neuron death. Electrophysiological assessment of excitatory postsynaptic field potential at Schaffer collateral/commissural fiber-CA1 synapses showed near total suppression of LTP in mice expressing Tat. The loss in LTP coincided with disruptions in learning and memory., Conclusions: Tat expression in the brain results in profound functional changes in synaptic physiology and in behavior that are accompanied by only modest structural changes and minimal pathology. Tat likely contributes to HAND by causing molecular changes that disrupt synaptic organization, with inhibitory presynaptic terminals containing synaptotagmin 2 appearing especially vulnerable., (Published by Elsevier Inc.)

At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles "housekeeping" export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm.

The interaction of the trans-activation responsive (TAR) region of bovine immunodeficiency virus (BIV) RNA with the Tat peptide is known to play important role in viral replication. Despite being thoroughly studied through a structural point of view, the nature of binding between BIV TAR RNA and the BIV Tat peptide requires information related to its thermodynamics and the nature of hydration around the TAR-Tat complex. In this context, we carried out the thermodynamic study of binding of the Tat peptide to the BIV TAR RNA hairpin through different calorimetric and spectroscopic measurements. Fluorescence titration of 2-aminopurine tagged BIV TAR RNA with the Tat peptide gives their binding affinity. The isothermal titration calorimetric experiment reveals the enthalpy of binding between BIV TAR RNA and the Tat peptide to be largely exothermic with the value of -11.7 (SEM 0.2) kcal mol(-1). Solvation dynamics measurements of BIV TAR RNA having 2-AP located at the bulge region have been carried out in the absence and presence of the BIV Tat peptide using the time correlated single photon counting technique. The solvent cage around the Tat binding site of RNA appears to be more rigid in the presence of the Tat peptide as compared to the free RNA. The displacement of solvent and ions on RNA due to peptide binding influences the entropic contributions to the total binding energy.

RNA and protein are potential molecules that can be used to construct functional nanobiomaterials. Recent findings on riboswitches emphasize on the dominative function of RNAs in regulating protein functions through allosteric interactions between RNA and protein. In this study, we demonstrate a simple strategy to obtain RNAs that have a switching ability with respect to protein function in response to specific target molecules. RNA aptamers specific for small ligands and a trans-activation-responsive (TAR)-RNA were connected by random RNA sequences. RNAs that were allosterically bound to a trans-activator of transcription (Tat)-peptide in response to ligands were selected by repeated negative and positive selection in the absence and presence of the ligands, respectively. The selected RNAs interacted with artificially engineered Renilla Luciferase, in which the Tat-peptide was inserted within the Luciferase, in the presence of the specific ligand and triggered the "Lighting-UP" switch of the engineered Luciferase.

Rapid and efficient detection of viral infection is crucial for the prevention of disease spread during an outbreak and for timely clinical management. In this paper, the utility of Tat peptide-modified molecular beacons (MBs) as a rapid diagnostic tool for the detection of virus-infected cells was demonstrated. The rapid intracellular delivery mediated by the Tat peptide enabled the detection of infected cells within 30 s, reaching saturation in signal in 30 min. This rapid detection scheme was coupled with flow cytometry (FC), resulting in an automated, high-throughput method for the identification of virus-infected cells. Because of the 2-order-of-magnitude difference in fluorescence intensity between infected and uninfected cells, as few as 1% infected cells could be detected. Because of its speed and sensitivity, this approach may be adapted for the practical diagnosis of multiple viral infections.

Potent melanin production enhancement of human tyrosinase plasmid (pAH7/Tyr, P) in mouse melanoma cells (B(16)F(10)) by Tat peptide (T) and an entrapment in elastic cationic niosomes (E) was described. The E composed of Tween 61/cholesterol/dodecyl dimethyl ammonium bromide at 1:1:0.5 molar ratio was prepared by freeze-dried emptying liposomes method. PE at P/E ratio of 1:160 w/w and TPE at T/P/E ratio of 0.125:1:160, 0.25:1:160, and 0.5:1:160 w/w/w were prepared. The final concentration of the plasmid in the study was 4 ng/μL. By sulforhodamine B assay, PE and TPE complexes showed slight or no cytotoxic effect. The cells transfected with TPE (0.5:1:160) exhibited the highest enhancement of tyrosinase enzyme activity of 11.82-, 7.67-, 5.07-, and 6.29-folds of control, P, PE, and TP (0.5:1) and melanin production of 13.03-, 8.46-, 5.36-, and 6.58-folds of control, P, PE, and TP (0.5:1), respectively. The elastic cationic niosomes demonstrated an increase in thermal stability of P at 4 ± 2, 25 ± 2, and 45 ± 2 °C. The vesicular size and the zeta potential values of PE and TPE complexes were slightly increased but still in the range of stable dispersion (out of ±30 mV). These results indicated the high potential application of the TPE complexes for further investigation for vitiligo gene therapy., (© 2012 John Wiley & Sons A/S.)

Background: Lysis of endocytic organelles is a necessary step in many cellular delivery methodologies. This is achieved efficiently in the photochemical internalization approach but the cell death that accompanies this process remains a problem., Methods: We investigate the mechanisms of cell death that accompanies photochemical internalization of the fluorescent peptide TMR-TAT., Results: TMR-TAT kills cells after endocytosis and light irradiation. The lysis of endocytic organelles by TMR-TAT causes a rapid increase in the concentration of calcium in the cytosol. TMR-TAT co-localizes with endocytic organelles containing calcium prior to irradiation and photochemical internalization leads to the release of the lumenal content of these organelles. Ruthenium red and cyclosporin A, inhibitors of calcium import in mitochondria and of the mitochondria permeability transition pore, inhibit cell death., Conclusions: TMR-TAT mediated photochemical internalization leads to a disruption of calcium homeostasis. The subsequent import of calcium in mitochondria is a causative factor of the cell death that accompanies photochemical internalization. General significance Understanding how the lysis of endocytic organelles affects cellular physiology and causes cell death is crucial to the development of optimal delivery methodologies., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The twin arginine translocation (Tat) pathway exports folded proteins from the cytoplasm to the periplasm of bacteria. The targeting of the exported proteins to the Tat pathway relies on a specific amino-terminal signal sequence, which is cleaved after exportation. In the phytopathogen Dickeya dadantii, the pectin lyase homologue PnlH is exported by the Tat pathway without cleavage of its signal sequence, which anchors PnlH into the outer membrane. In proteobacteria, the vast majority of outer membrane proteins consists of β-barrel proteins and lipoproteins. Thus, PnlH represents a new kind of outer membrane protein. In Escherichia coli, periplasmic chaperones SurA, Skp, and DegP work together with the β-barrel assembly machinery (Bam) to target and insert β-barrel proteins into the outer membrane. In this work, we showed that SurA is required for an efficient targeting of PnlH to the outer membrane. Moreover, we were able to detect an in vitro interaction between SurA and the PnlH signal sequence. Since the PnlH signal sequence contains a highly hydrophobic region, we propose that SurA protects it from the hydrophobic periplasm during targeting of PnlH to the outer membrane. We also studied the nature of the information carried by the PnlH signal sequence responsible for its targeting to the outer membrane after exportation by the Tat system.

p55PIK, a regulatory subunit of phosphatidylinositol 3-kinases, promotes cell cycle progression by interacting with cell cycle modulators such as retinoblastoma protein (Rb) via its unique amino-terminal 24 amino-acid residue (N24). Overexpression of N24 specifically inhibits these interactions and leads to cell cycle arrest. Herein, we describe the generation of a fusion protein (Tat transactivator protein (TAT)-N24) that contains the protein transduction domain and N24, and examined its effects on the proliferation and differentiation of leukemia cells. TAT-N24 not only blocks cell proliferation but remarkably induces differentiation of leukemia cells in vitro and in vivo. Systemically administered TAT-N24 also significantly decreases growth of leukemia cell tumors in animal models. Furthermore, overexpression of p55PIK in leukemia cells leads to increased proliferation; however, TAT-N24 blocks this effect and concomitantly induces differentiation. There is significant upregulation of p55PIK mRNA and protein expression in leukemia cells from patients. TAT-N24 inhibits cell cycle progression and induces differentiation of bone marrow cells derived from patients with several different types of leukemia. These results show that cell-permeable N24 peptide induces leukemia cell differentiation and suggest that p55PIK may be a novel drug target for the treatment of hematopoetic malignancies.

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

Fluorescent quantum dots have great potential to act as labeling tools in biological research, especially cellular tracking and imaging. Tat peptide conjugated quantum dots were introduced into living human hematopoietic stem cells (HSCs). The internalized quantum dots were assessed by laser confocal microscope and flow cytometer. The quantum dots labeled HSCs were injected intravenously into the tail veins of NOD/SCID beta2M null mice. The tissue collections in the major organs were examined with fluorescence microscope to assess the distribution of transplanted stem cells. HSCs with internalized quantum dots offer a promising approach for stem cell transplantation, which will hold a significant impact on stem cell based therapy for several disorders, especially to cure leukemia in current China.

Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic macromolecules. It is widely accepted that they can enter cells directly across the plasma membrane but also gain access through endocytic pathways that are yet to be fully defined. Here we developed siRNA methods in epithelial cell lines, HeLa and A431, to inhibit endocytic pathways regulated by clathrin heavy chain, flotillin-1, caveolin-1, dynamin-2 and Pak-1. In each case, functional uptake assays were developed to characterize the requirement for these proteins, and the pathways they regulate, in the internalisation of defined endocytic probes and also the CPPs octaarginine and HIV-Tat. Peptide uptake was only inhibited in A431 cells depleted of the macropinocytosis regulator Pak-1, but experimental variables including choice of cell line, pharmacological inhibitor, macropinocytic probe and serum starvation significantly influence our ability to assess and assign this pathway as an important route for CPP uptake. Actin disruption with Cytochalasin D inhibited peptide entry in both cell lines but the effects of this agent on dextran uptake was cell line dependent, reducing uptake in HeLa cells and increasing uptake in A431 cells. This was further supported in experiments inducing actin stabilisation by Jasplakinolide, emphasising that the actin cytoskeleton can both promote and hinder endocytosis. Overall the data identify important aspects regarding the comparative mechanisms of CPP uptake and macropinocytosis, and accentuate the significant methodological challenges of studying this pathway as an endocytic portal and an entry route for drug delivery vectors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

HIV cardiomyopathy remains highly prevalent among the estimated 33 million HIV-infected individuals worldwide. This is particularly true in developing countries. Potential mechanisms responsible for myocardial dysfunction following HIV infection include direct effects of HIV proteins. We have previously reported that cardiac myocyte-specific expression of HIV-Tat (Tat) results in a murine cardiomyopathy model. We now report that Tat exhibits decreased myocardial ATP [wild type (WT) vs. Tat transgenic (TG), P < 0.01] and myocyte GSH levels (WT vs. TG, P < 0.01), decreased GSH/GSSG ratio (WT vs. TG, P < 0.01), increased H(2)O(2) levels (WT vs. TG, P < 0.05), and increased catalase (TG vs. WT, P < 0.05) and GPX1 (glutathione peroxidase 1) activities (WT vs. TG, P < 0.05), blunted cardiac myocyte positive inotropy (% peak shortening, WT vs. TG, P < 0.01; +dl/dt, WT vs. TG, P < 0.01) and negative inotropy (-dl/dt, WT vs. TG, P < 0.01), and blunted inotropic responses to Ca(2+) (P < 0.01, for each) and shortened anatomical and functional survival in vitro (P < 0.01). The sulfhydryl donor, N-acetylcysteine (NAC; 10(-4) M), completely reversed both the positive and negative inotropic defects in Tat; increased GSH (P < 0.01) and GSH/GSSG (P < 0.01); reversed H(2)O(2) level (P < 0.05) and GPX1 activity (P < 0.05); and normalized the blunted inotropic response to Ca(2+) (P < 0.01). NAC (10(-7)) M normalized duration of contractile function from <40 min to >120 min (P < 0.01), with no effect on GSH and GSH/GSSG. NAC (10(-4) M) reverses cardiac myocyte dysfunction and markers of oxidative stress. NAC (10(-7) M) enhances myocyte function independent of changes in glutathione. Elucidating the molecular mechanisms involved in the GSH-dependent and GSH-independent salutary effects of NAC should identify novel therapeutic targets for myocardial proteinopathies recently appreciated in human cardiomyopathies.

HIV-1 infection causes profound effects both inside and outside of cells through multiple mechanisms, including those mediated by exosomes. Using the technique of stable isotope labeling by amino acids in cell culture, we compared protein expression patterns in the exosomal compartment of HIV-1-infected and -uninfected lymphocytic H9 cells. Of 770 proteins identified in two independent sets of exosomal samples, 14 proteins were found to be differentially expressed in the exosomal fraction of HIV-1-infected cells versus -uninfected controls. Gene Ontology survey and DAVID analysis revealed that identified proteins were enriched for functional categories such as binding. Of these 14 proteins, three immunomodulatory molecules were reproducibly identified in both replicates and included ADP-ribosyl cyclase 1 (CD38), L-lactate dehydrogenase B chain (LDHB), and Annexin A5 (ANXA5). In addition to previously reported HIV-1 associations with CD38 and LDHB, new interactions were identified and validated for ANXA5, CD38, and LDHB, which were found to bind to HIV-1 p24 and Tat. In summary, our studies reveal that exosomes released from HIV-1-infected cells are composed of a unique and quantitatively different protein signature and harbor regulatory molecules that impact the processes of cellular apoptosis (ANXA5 and LDHB) and proliferation (CD38)., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Parkinson's disease (PD) is a well known neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra pars compact (SN). Although the exact mechanism remains unclear, oxidative stress plays a critical role in the pathogenesis of PD. DJ-1 is a multifunctional protein, a potent antioxidant and chaperone, the loss of function of which is linked to the autosomal recessive early onset of PD. Therefore, we investigated the protective effects of DJ-1 protein against SH-SY5Y cells and in a PD mouse model using a cell permeable Tat-DJ-1 protein. Tat-DJ-1 protein rapidly transduced into the cells and showed a protective effect on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death by reducing the reactive oxygen species (ROS). In addition, we found that Tat-DJ-1 protein protects against dopaminergic neuronal cell death in 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP)-induced PD mouse models. These results suggest that Tat-DJ-1 protein provides a potential therapeutic strategy for against ROS related human diseases including PD.

Introduction: It has been 20 years since the discovery of the membrane-translocating property of the HIV-1 transactivator of transcription (Tat) protein. The Tat protein transduction domain (Tat PTD) is a very promising tool for non-invasive cellular import of cargos and has been successfully applied to in vitro and in vivo delivery of different therapeutic agents for the treatment of many diseases. A growing number of reports on Tat PTD-mediated delivery have extensively revealed the mechanisms involved. Yet, due to the varied conditions used, the reports on the internalization mode of Tat PTD-cargo chimera are often varied., Areas Covered: This article reviews the possible intracellular trafficking mechanisms of Tat PTD including its binding, cellular entry process, and the roles of participants of the cell membrane. The therapeutic applications via local administration, such as those for the treatment of skin, ocular, cardiac and cerebral diseases, are also reviewed. In addition, some novel systems built by different groups are elucidated, which are utilized to overcome the poor targeting efficiency of Tat PTD for the treatment of CNS diseases, cancer and other diseases via systemic administration., Expert Opinion: With the development of targeting factors, such as antibodies, some cell targeting peptides and novel polymers, Tat PTD is expected to play a more efficient and/or better tolerated therapeutic role in the drug delivery field.

HIV infection is an independent risk factor for atherosclerosis development and cardiovascular damage. As vessel wall mesenchymal stem cells (MSCs) are involved in the regulation of vessel structure homeostasis, we investigated the role of Tat, a key factor in HIV replication and pathogenesis, in MSC survival and differentiation. The survival of subconfluent MSCs was impaired when Tat was added at high concentrations (200-1,000 ng/ml), whereas lower Tat concentrations (1-100 ng/ml) did not promote apoptosis. Tat enhanced the differentiation of MSC toward adipogenesis by the transcription and activity upregulation of PPARγ. This Tat-related modulation of adipogenesis was tackled by treatment with antagonists of Tat-specific receptors such as SU5416 and RGD Fc. In contrast, Tat inhibited the differentiation of MSCs to endothelial cells by downregulating the expression of VEGF-induced endothelial markers such as Flt-1, KDR, and vWF. The treatment of MSCs with Tat-derived peptides corresponding to the cysteine-rich, basic, and RGD domains indicated that these Tat regions are involved in the inhibition of endothelial marker expression. The Tat-related impairment of MSC survival and differentiation might play an important role in vessel damage and formation of the atherosclerotic lesions observed in HIV-infected patients., (© 2011 Wiley Periodicals, Inc.)

HIV-Tat protein has been implicated in the pathogenesis of HIV-1 neurological complications (i.e., neuroAIDS), but direct demonstrations of the effects of Tat on behavior are limited. GT-tg mice with a doxycycline (Dox)-inducible and brain-selective tat gene coding for Tat protein were used to test the hypothesis that the activity of Tat in brain is sufficient to impair learning and memory processes. Western blot analysis of GT-tg mouse brains demonstrated an increase in Tat antibody labeling that seemed to be dependent on the dose and duration of Dox pretreatment. Dox-treated GT-tg mice tested in the Barnes maze demonstrated longer latencies to find an escape hole and displayed deficits in probe trial performance versus uninduced GT-tg littermates, suggesting Tat-induced impairments of spatial learning and memory. Reversal learning was also impaired in Tat-induced mice. Tat-induced mice additionally demonstrated long-lasting (up to one month) deficiencies in novel object recognition learning and memory performance. Furthermore, novel object recognition impairment was dependent on the dose and duration of Dox exposure, suggesting that Tat exposure progressively mediated deficits. These experiments provide evidence that Tat protein expression is sufficient to mediate cognitive abnormalities seen in HIV-infected individuals. Moreover, the genetically engineered GT-tg mouse may be useful for improving our understanding of the neurological underpinnings of neuroAIDS-related behaviors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Translation of the full-length messenger RNA (mRNA) of the human immunodeficiency virus type 1 (HIV-1) generates the precursor of the viral enzymes via a programmed -1 ribosomal frameshift. Here, using dual-luciferase reporters, we investigated whether the highly structured 5' untranslated region (UTR) of this mRNA, which interferes with translation initiation, can modulate HIV-1 frameshift efficiency. We showed that, when the 5' UTR of HIV-1 mRNA occupies the 5' end of the reporter mRNA, HIV-1 frameshift efficiency is increased about fourfold in Jurkat T-cells, compared with a control dual-luciferase reporter with a short unstructured 5' UTR. This increase was related to an interference with cap-dependent translation initiation by the TAR-Poly(A) region at the 5' end of the messenger. HIV-1 mRNA 5' UTR also contains an internal ribosome entry site (IRES), but we showed that, when the cap-dependent initiation mode is available, the IRES is not used or is weakly used. However, when the ribosomes have to use the IRES to translate the dual-luciferase reporter, the frameshift efficiency is comparable to that of the control dual-luciferase reporter. The decrease in cap-dependent initiation and the accompanying increase in frameshift efficiency caused by the 5' UTR of HIV-1 mRNA is antagonized, in a dose-dependent way, by the Tat viral protein. Tat also stimulates the IRES-dependent initiation and decreases the corresponding frameshift efficiency. A model is presented that accounts for the variations in frameshift efficiency depending on the 5' UTR and the presence of Tat, and it is proposed that a range of frameshift efficiencies is compatible with the virus replication.

Some short and cationic peptides such as the Tat peptide can cross the cell membrane and function as vectors for intracellular delivery. Here we show that an α-AApeptide is able to penetrate the membranes of living cells from an extracellular environment and enter the endosome and cytoplasm of cells. The efficiency of the cellular uptake is comparable to a Tat peptide (48-57) of the same length and is unexpectedly superior to an α-peptide with identical functional groups. The mechanism of uptake is similar to that of the Tat peptide and is through endocytosis by an energy-dependent pathway. Due to the easy synthesis of the α-AApeptides, their resistance to proteolytic hydrolysis, and their low cytotoxicity, α-AApeptides represent a new class of transporters for the delivery of drugs.

Tat is a viral protein secreted from HIV infected cells and extra cellular Tat is suspected to prevent destruction of HIV infected cells from cells of the cellular immunity. The effect of anti retroviral therapy (ART) on Tat secretion has never been investigated. In this study, we tested for antibody reactivity against Tat variants representative of the main HIV subtypes in HIV positive patients receiving ART with undetectable viral loads ( < 40 copies/mL) over the course of one year with a blood sampling every three months. For each of theses five blood sampling, an average of 50 % of patients had Anti-Tat IgG, it turned out that 86% of patients could recognize Tat at least in one blood sampling during the course of the study. Amazingly, anti-Tat IgG appeared and/or disappeared in 66 % of patients. Only 20% had anti-Tat IgG remaining persistently while 14% were consistently without anti Tat IgG in the five blood sampling. No significant correlation was found between anti-Tat IgG and CD4+ T cell, CD8+ T cell and B cell counts revealing the incapacity of these anti Tat IgG to neutralize extra cellular Tat. Interestingly the absence and then the appearance of anti-Tat IgG in patients suggest the presence of HIV infected cells in the blood that may constitute a significant reservoir of HIV infected cells. As a conclusion antiretroviral therapy does not block the secretion of Tat and may explain why HIV infected cells can survive in spite of an effective ART treatment.

The Tat machinery enables folded proteins to be translocated across biological membranes. In vitro studies have shown that Tat substrates can interact with membranes prior to translocation. In this study we investigated the initial states of this interaction with thylakoid lipid monolayers at the air-water interface by using monolayer techniques combined with infrared reflection-absorption spectroscopy (IRRAS). We used enhanced green fluorescent protein (EGFP) as a model substrate and the signal peptide SP16 from the 16 kDa protein of the spinach oxygen-evolving complex (OEC16). We found that the signal peptide is essential for the interaction of the model substrate with lipid monolayers. IRRA spectroscopy showed an increased amount of α-helical secondary structure elements for the chimeric model substrate i16/EGFP (SP16 fused to EGFP) compared with EGFP; this can be attributed to the signal peptide., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005 ≈ 0.01%) than that of TAT-M13 (0.001 ≈ 0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.

In affinity-based chemoproteomics strategies, the direct immobilization of small bioactive probe molecules to a solid support may pose problems with respect to the preservation of the functional activity toward the target proteins. Typically, immobilized molecules on solid supports exhibit lower affinity for target proteins compared to the free parent molecule. This may lead to a failure to specifically capture the target proteins or to unacceptable losses during the washing steps. To circumvent these shortcomings, we have devised small molecule-peptide conjugates (SMPCs), which enable wide-ranging experimental strategies for the capturing of protein targets of small molecules from cells or tissues. With the possibilities of synthesizing peptides of tailored biochemical and biophysical properties, SMPCs enable the identification of protein targets of small molecules from cell-lysates and intact cells. Moreover, labeling of these conjugates with fluorophores can provide information on the cellular localization and distribution of the target.

Human immunodeficiency virus-1 (HIV) infection of the central nervous system may cause a neurological syndrome termed HIV-associated neurocognitive disorder (HAND) which includes minor neurocognitive disorders or a more severe form of motor and cognitive impairments. Although treatment with highly active antiretroviral agents decreases the load of HIV in the brain, the prevalence of mild forms of HAND is actually increased due to longer life. Therefore, adjunctive and combined therapies must be developed to prevent and perhaps reverse the neurologic deficits observed in individuals with HAND. Key to developing effective therapies is a better understanding of the molecular and cellular mechanisms by which the virus causes this disorder. A number of HIV proteins has been shown to be released from HIV-infected cells. Moreover, these proteins have been shown to possess neurotoxic properties. This review describes new evidence of a direct interaction of the HIV protein gp120 with neurons, which might play a role in the etiopathology of HAND.

Phosphatidylinositol 3-kinase (PI3K)/Akt inhibitors were isolated from the rhizome of Polygala tenuifolia WILLD (PT, Polygalaceae), which has been used in traditional Chinese medicine for inflammation, dementia, amnesia, neurasthenia and cancer, by activity-guided fractionation. For the assay of PI3K/Akt pathway, cytoprotective Tat-transduced CHME5 cells, which are the cytoprotective phenotype against lypopolysaccharide (LPS)/cycloheximide (CHX), were used. We isolated 4 anti-cytoprotective compounds, clionasterol (1), ethyl cholestan-22-enol (2), 3-O-β-D-glucosyl ethyl cholestan-22-enol (3), and 3-O-β-D-glucopyranosyl clionasterol (4) from EtOAc fraction of PT against Tat-transduced CHME5 cells. Of them, (1) and (2) most potently abolished cytoprotective effect of Tat-transduced CHME5 cells. These constituents (1) and (2) inhibited the activation of 3-phosphoinositide-dependent kinase 1 (PDK1) and its downstream molecules, Akt/glycogen synthase kinase (GSK)3β, in PI3K/Akt cell survival signaling pathway, but did not suppress the activation of PI3K. Based on these finding, (1) and (2) may abolish the cytoprotective phenotype of Tat-transduced CHME5 cells by inhibiting PDK1 phosphorylation in PI3K/Akt pathway.

TAT-SOD is a recombinant protein of superoxide dismutase fused with TAT peptide. By pure accident, we discovered that topical application of TAT-SOD to acupoints could result in acupuncture-like action. This study aimed to validate the accidental discovery by investigating the effect on simple obesity of the topical application of TAT-SOD to acupoints in comparison with acupuncture. 90 subjects were divided into 3 groups for 12-week treatments. Regular hospital acupuncture treatment was given to Acupuncture Group 3 times a week. TAT-SOD Group were instructed first to locate acupoints and apply 0.1ml of 5000u SOD/ml TAT-SOD cream in an area of 1cm(2) to each of the same set of acupoints, which they then conducted at home three times daily. Placebo Group applied the vehicle cream the same manner as TAT-SOD Group. Both TAT-SOD and acupuncture treatments decreased adiposity with overall clinical effective rates of 60.0% and 76.7%, respectively. The placebo group showed no improvement. The results validate that the enzymatic removal of the intracellular superoxide at acupoints could generate acupuncture-like effects, and indicate a possibility of the new method as a simple substitute to acupuncture and an insight of superoxide modulation along meridians for acupuncture mechanism., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We investigated whether silk fibroin peptide derived from the silkworm, Bombyx mori, could inhibit inflammation and enhance the anti-inflammatory activity of Tat-superoxide dismutase (Tat-SOD), which was previously reported to effectively penetrate various cells and tissues and exert anti-oxidative activity in a mouse model of inflammation. Inflammation was induced by topical treatment of mouse ears with 12-O-tetradecanoylphorbol-13-acetate (TPA). Histological, Western blot, and reverse transcription-polymerase chain reaction data demonstrated that silk fibroin peptide or Tat-SOD alone could suppress elevated levels of cyclooxygenase-2, interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha induced by TPA. Moreover, silk fibroin peptide significantly enhanced the anti-inflammatory activity of Tat-SOD, although it had no influence on in vitro and in vivo transduction of Tat-SOD. Silk fibroin peptide exhibited anti- inflammatory activity in a mice model of inflammation. Therefore, silk fibroin peptide alone or in combination with Tat-SOD might be used as a therapeutic agent for various inflammatory diseases.

The delivery enhancements of green fluorescent protein (GFP), a model reporter protein into/transepithelial colon adenocarcinoma (HT-29) cells and excised rat skin by coincubation, by simple mixing or as fusion protein with HIV1-trans-activating transcriptional (Tat), a cell-penetrating peptide, have been described. By simple mixing, Tat/GFP mixture could increase the cellular uptake of GFP into HT-29 cells by 4.25-fold and 1.79-fold of GFP and Tat-GFP fusion protein, respectively. The incubation time showed no effect on the cellular uptake of Tat/GFP and Tat-GFP. In transepithelial study, Tat-GFP demonstrated the highest ability to penetrate through the HT-29 cells of about 1.3-fold and 1.2-fold of GFP and Tat/GFP, respectively. Only Tat/GFP gave lower cytotoxicity than Tat or GFP alone. In transdermal delivery study, Tat/GFP showed better transdermal delivery profile with higher cumulative amount than Tat-GFP in stratum corneum (SC), viable epidermis and dermis, and the receiver compartment of the diffusion cell with the highest fluxes of 7.42 and 35.6; 8.87-fold and 5.57-fold of GFP and Tat-GFP in SC and receiver compartment, respectively. This study demonstrated an efficient enhancement of GFP uptake into cells and through excised rat skin by simple mixing with Tat peptide, which can be further applied for the development of protein drug delivery systems., (Copyright © 2011 Wiley-Liss, Inc.)

Background: Reactive oxygen species are considered an important cause of the death of pancreatic β cells, thereby triggering the development of type 2 diabetes as well as failure of islet transplantation. The biological properties of metallothionein (MT) and superoxide dismutase (SOD) are likely to be related to their antioxidant and free-radical scavenging abilities, but their access across biological membranes is limited., Methods: We investigated whether Tat-MT and Tat-SOD fusion protein could be introduced into islets by a novel protein transduction technology and protect them from oxidative damage. We used 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and Annexin V/propidium iodide assays to analyse cell viability, and assessed expression of apoptosis marker proteins by Western blotting. We examined the protective effect of Tat-MT and Tat-SOD on the development of diabetes and on graft failure after syngeneic islet transplantation into Otsuka Long Evans Tokushima Fatty (OLETF) rats and Imprinting Control Region (ICR) mice, respectively., Results: Tat-MT and Tat-SOD were successfully delivered into the rat islets, and reactive oxygen species, nitric oxide, glucolipotoxicity-induced cell death, cytokine injury, and DNA fragmentation due to ischaemia-reperfusion in pancreatic β cells were significantly reduced. In addition Tat-MT and Tat-SOD treatment protected OLETF rats from developing diabetes, and enhanced the survival of antioxidant-treated islets transplanted into the renal capsules of diabetic mice., Conclusions: Transduction of Tat-MT and Tat-SOD proteins offers a new strategy for protecting against the development of diabetes by relieving oxidative stress., (Copyright © 2011 John Wiley & Sons, Ltd.)

Metallothioneins (MTs) are intracellular low-molecular-weight, cysteine-rich proteins with potent metal-binding and redox functions, but with limited membrane permeativity. The aim of this study was to investigate whether we could enhance delivery of MT-1 to pancreatic islets or β cells in vitro and in vivo. The second goal was to determine whether increased MT-1 could prevent cellular toxicity induced by high glucose and free fatty acids in vitro (glucolipotoxicity) and ameliorate the development of diabetes induced by streptozotocin in mice or delay the development of diabetes by improving insulin secretion and resistance in the OLETF rat model of type 2 diabetes. Expression of HIV-1 Tat-MT-1 enabled efficient delivery of MT into both INS-1 cells and rat islets. Intracellular MT activity increased in parallel with the amount of protein delivered to cells. The formation of reactive oxygen species, glucolipotoxicity, and DNA fragmentation due to streptozotocin decreased after treating pancreatic β cells with Tat-MT in vitro. Importantly, in vivo, intraperitoneal injection resulted in delivery of the Tat-MT protein to the pancreas as well as liver, muscle, and white adipose tissues. Multiple injections increased radical-scavenging activity, decreased apoptosis, and reduced endoplasmic reticulum stress in the pancreas. Treatment with Tat-MT fusion protein delayed the development of diabetes in streptozotocin-induced mice and improved insulin secretion and resistance in OLETF rats. These results suggest that in vivo transduction of Tat-MT may offer a new strategy to protect pancreatic β cells from glucolipotoxicity, may improve insulin resistance in type 2 diabetes, and may have a protective effect in preventing islet destruction in type 1 diabetes., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The study aims to design a new gene delivery method utilizing the complementary strengths of baculovirus, such as relatively high transduction efficiency and easy scale-up, and non-viral nanodelivery systems, such as low immunogenicity. This formulation was developed by generating a self assembled binary complex of negatively charged baculovirus (Bac) and positively charged endosomolytic histidine rich Tat peptide/DNA nanoparticles (NP). The synergistic effect of this hybrid (Bac-NP) system to induce myocardial angiogenesis in acute myocardial infarction (AMI) model has been explored in this study, using Angiopoietin-1 (Ang-1) as the transgene carried by both vector components. Under optimal transduction conditions, Bac-NP(Ang1) showed 1.75 times higher and sustained Ang-1 expression in cardiomyocytes than Bac(Ang1), with significantly high angiogenic potential as confirmed by functional assays. For in vivo analysis, we intramyocardially delivered Bac-NP(Ang1) to AMI rat model. 3 weeks post AMI, data showed increase in capillary density (p < 0.01) and reduction in infarct sizes (p < 0.05) in Bac-NP(Ang1) compared to Bac(Ang1), NP(Ang1) and control groups due to enhanced myocardial Ang-1 expression at peri-infarct regions (1.65 times higher than Bac(Ang1)). Furthermore, the Bac-NP(Ang1) group showed significantly higher cardiac performance in echocardiography than Bac(Ang1) (44.2 ± 4.77% vs 37.46 ± 5.2%, p < 0.01), NP(Ang1) and the control group (32.26 ± 2.49% and 31.58 ± 2.26%). Collectively, this data demonstrates hybrid Bac-NP as a new and improved gene delivery system for therapeutic applications., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

Chronic human immunodeficiency virus infection is associated with higher incidence of pulmonary complications including hypertension, vasculopathy, lymphocytic alveolitis, and interstitial pneumonitis not attributed to either opportunistic infections or presence of the virus. The Tat (transactivator of transcription) protein, a required transactivator for expression of full-length viral genes, is pleiotropic and influences expression of cellular inflammatory genes. Tat-dependent transactivation of cellular genes requires specific mediators, including NF-κB, widely recognized as sensitive to changes in cellular oxidant burden. We hypothesized that overproduction of Tat leads to increased oxidant burden and to alterations in basal inflammatory status as measured by NF-κB activation. We engineered transgenic mouse lines that express Tat (86-amino-acid isoform) in the lung under the control of the surfactant protein C promoter. Tat-transgenic mice exhibit increased pulmonary cellular infiltration, increased nitrotyrosine and carbonyl protein modifications, and increased levels of NF-κB, MnSOD, and thioredoxin-interacting protein. These data indicate that Tat increases oxidant burden and resets the threshold for inflammation, which may increase susceptibility to secondary injuries., (Published by Elsevier Inc.)

The AAA+ family in eukaryotes has many members in various cellular compartments with a role in protein unfolding and degradation. We show that the mitochondrial AAA-ATPase Bcs1 has an unusual function in protein translocation. Bcs1 mediates topogenesis of the Rieske protein, Rip1, a component of respiratory chains in bacteria, mitochondria, and chloroplasts. The oligomeric AAA-ATPase Bcs1 is involved in export of the folded Fe-S domain of Rip1 across the inner membrane and insertion of its transmembrane segment into an assembly intermediate of the cytochrome bc(1) complex, thus revealing an unexpected mechanistical concept of protein translocation across membranes. Furthermore, we describe structural elements of Rip1 required for recognition and export by as well as ATP-dependent lateral release from the AAA-ATPase. In bacteria and chloroplasts Rip1 uses the Tat machinery for topogenesis; however, mitochondria have lost this machinery during evolution and a member of the AAA-ATPase family has taken over its function., (Copyright © 2011 Elsevier Inc. All rights reserved.)