Cu-NirK from Haloferax mediterranei as an example of metalloprotein maturation and exportation via Tat system.

The green Cu-NirK from Haloferax mediterranei (Cu-NirK) has been expressed, refolded and retrieved as a trimeric enzyme using an expression method developed for halophilic Archaea. This method utilizes Haloferax volcanii as a halophilic host and an expression vector with a constitutive and strong promoter. The enzymatic activity of recombinant Cu-NirK was detected in both cellular fractions (cytoplasmic fraction and membranes) and in the culture media. The characterization of the enzyme isolated from the cytoplasmic fraction as well as the culture media revealed important differences in the primary structure of both forms indicating that Hfx. mediterranei could carry out a maturation and exportation process within the cell before the protein is exported to the S-layer. Several conserved signals found in Cu-NirK from Hfx. mediterranei sequence indicate that these processes are closely related to the Tat system. Furthermore, the N-terminal sequence of the two Cu-NirK subunits constituting different isoforms revealed that translation of this protein could begin at two different points, identifying two possible start codons. The hypothesis proposed in this work for halophilic Cu-NirK processing and exportation via the Tat system represents the first approximation of this mechanism in the Halobacteriaceae family and in Prokarya in general.

Chronic central nervous system expression of HIV-1 Tat leads to accelerated rarefaction of neocortical capillaries and loss of red blood cell velocity heterogeneity.

OBJECTIVES: HIV-1 infection of the CNS is associated with impairment of CBF and neurocognitive function, and accelerated signs of aging. As normal aging is associated with rarefaction of the cerebral vasculature, we set out to examine chronic viral effects on the cerebral vasculature. METHODS: DOX-inducible HIV-1 Tat-tg and WT control mice were used. Animals were treated with DOX for three weeks or five to seven months. Cerebral vessel density and capillary segment length were determined from quantitative image analyses of sectioned cortical tissue. In addition, movement of red blood cells in individual capillaries was imaged in vivo using multiphoton microscopy, to determine RBCV and flux. RESULTS: Mean RBCV was not different between Tat-tg mice and age-matched WT controls. However, cortical capillaries from Tat-tg mice showed a significant loss of RBCV heterogeneity and increased RBCF that was attributed to a marked decrease in total cortical capillary length (35-40%) compared to WT mice. CONCLUSIONS: Cerebrovascular rarefaction is accelerated in HIV-1 Tat-transgenic mice, and this is associated with alterations in red cell blood velocity. These changes may have relevance to the pathogenesis of HIV-associated neurocognitive disorders in an aging HIV-positive population.

Latent HIV-1 can be reactivated by cellular superinfection in a Tat-dependent manner, which can lead to the emergence of multidrug-resistant recombinant viruses.

The HIV-1 latent reservoir represents an important source of genetic diversity that could contribute to viral evolution and multidrug resistance following latent virus reactivation. This could occur by superinfection of a latently infected cell. We asked whether latent viruses might be reactivated when their host cells are superinfected, and if so, whether they could contribute to the generation of recombinant viruses. Using populations of latently infected Jurkat cells, we found that latent viruses were efficiently reactivated upon superinfection. Pathways leading to latent virus reactivation via superinfection might include gp120-CD4/CXCR4-induced signaling, modulation of the cellular environment by Nef, and/or the activity of Tat produced upon superinfection. Using a range of antiviral compounds and genetic approaches, we show that gp120 and Nef are not required for latent virus reactivation by superinfection, but this process depends on production of functional Tat by the superinfecting virus. In a primary cell model of latency in unstimulated CD4 T cells, superinfection also led to latent virus reactivation. Drug-resistant latent viruses were also reactivated following superinfection in Jurkat cells and were able to undergo recombination with the superinfecting virus. Under drug-selective pressure, this generated multidrug-resistant recombinants that were identified by unique restriction digestion band patterns and by population-level sequencing. During conditions of poor drug adherence, treatment interruption or treatment failure, or in drug-impermeable sanctuary sites, reactivation of latent viruses by superinfection or other means could provide for the emergence or spread of replicatively fit viruses in the face of strong selective pressures.

Effects of HIV-1 tat on secretion of TNF-α and IL-1ß by U87 cells in AIDS patients with or without AIDS dementia complex.

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

Comparative characterization and cytotoxicity study of TAT-peptide as potential vectors for siRNA and Dicer-substrate siRNA.

Recently, a newly discovered Dicer-substrate siRNA (DsiRNA) demonstrates higher potency in gene silencing than siRNA but both suffer from rapid degradation, poor cellular uptake and chemical instability. Therefore, Tat-peptide was exploited to protect and facilitate their delivery into cells. In this study, Tat-peptide was complexed with siRNA or DsiRNA through simple complexation. The physicochemical properties (particle size, surface charge and morphology) of the complexes formed were then characterized. The ability of Tat-peptide to carry and protect siRNA or DsiRNA was determined by UV-Vis spectrophotometry and serum protection assay, respectively. Cytotoxicity effect of these complexes was assessed in V79 cell line. siRNA-Tat complexes had particle size ranged from 186 ± 17.8 to 375 ± 8.3 nm with surface charge ranged from -9.3 ± 1.0 to +13.5 ± 1.0 mV, depending on the Tat-to-siRNA concentration ratio. As for DsiRNA-Tat complexes, the particle size was smaller than the ones complexed with siRNA, ranging from 176 ± 8.6 to 458 ± 14.7 nm. Their surface charge was in the range of +27.1 ± 3.6 to +38.1 ± 0.9 mV. Both oligonucleotide (ON) species bound strongly to Tat-peptide, forming stable complexes with loading efficiency of more than 86%. These complexes were relatively non cytotoxic as the cell viability of ∼90% was achieved. In conclusion, Tat-peptide has a great potential as siRNA and DsiRNA vector due to the formation of stable complexes with desirable physical characteristics, low toxicity and able to carry high amount of siRNA or DsiRNA.

Identification and characterization of a novel tyrosine aminotransferase gene (SmTAT3-2) promotes the biosynthesis of phenolic acids in Salvia miltiorrhiza Bunge.

Rosmarinic acid (RA) and salvianolic acid B (SAB) are main phenolic acids in Salvia miltiorrhiza Bunge have been widely used in the treatment of cardiovascular and cerebrovascular diseases due to their excellent pharmacological activity. RA is a precursor of SAB, and tyrosine transaminase (TAT, EC 2.6.1.5) is a crucial rate-limiting enzyme in their metabolism pathway. This study identified a novel TAT gene, SmTAT3-2, and found that it is a new transcript derived from unconventional splicing of SmTAT3. We used different substrates for enzymatic reaction with SmTAT1, SmTAT3 and SmTAT3-2. Subcellular localization of SmTAT1 and SmTAT3-2 was completed based on submicroscopic techniques. In addition, they were overexpressed and CRISPR/Cas9 gene edited in hairy roots of S. miltiorrhiza. Revealed SmTAT3-2 and SmTAT1 showed a stronger affinity for L-tyrosine than SmTAT3, localized in the cytoplasm, and promoted the synthesis of phenolic acid. In overexpressed SmTAT3-2 hairy roots, the content of RA and SAB was significantly increased by 2.53 and 3.38 fold, respectively, which was significantly higher than that of overexpressed SmTAT1 strain compared with EV strain. These findings provide a valuable key enzyme gene for the phenolic acids metabolism pathway and offer a theoretical basis for the clinical application.

Viral CTL escape mutants are generated in lymph nodes and subsequently become fixed in plasma and rectal mucosa during acute SIV infection of macaques.

SIV(mac239) infection of rhesus macaques (RMs) results in AIDS despite the generation of a strong antiviral cytotoxic T lymphocyte (CTL) response, possibly due to the emergence of viral escape mutants that prevent recognition of infected cells by CTLs. To determine the anatomic origin of these SIV mutants, we longitudinally assessed the presence of CTL escape variants in two MamuA*01-restricted immunodominant epitopes (Tat-SL8 and Gag-CM9) in the plasma, PBMCs, lymph nodes (LN), and rectal biopsies (RB) of fifteen SIV(mac239)-infected RMs. As expected, Gag-CM9 did not exhibit signs of escape before day 84 post infection. In contrast, Tat-SL8 escape mutants were apparent in all tissues by day 14 post infection. Interestingly LNs and plasma exhibited the highest level of escape at day 14 and day 28 post infection, respectively, with the rate of escape in the RB remaining lower throughout the acute infection. The possibility that CTL escape occurs in LNs before RBs is confirmed by the observation that the specific mutants found at high frequency in LNs at day 14 post infection became dominant at day 28 post infection in plasma, PBMC, and RB. Finally, the frequency of escape mutants in plasma at day 28 post infection correlated strongly with the level Tat-SL8-specific CD8 T cells in the LN and PBMC at day 14 post infection. These results indicate that LNs represent the primary source of CTL escape mutants during the acute phase of SIV(mac239) infection, suggesting that LNs are the main anatomic sites of virus replication and/or the tissues in which CTL pressure is most effective in selecting SIV escape variants.

TAT gene polymorphism and its relationship with production traits in Muscovy ducks (Cairina Moschata).

In a previous study, the laying pattern of Muscovy duck was explored by macro-fitting the laying curve of Muscovy duck, and transcriptome sequencing technique of the ovarian tissues was used to screen the egg-related gene "TAT." Moreover, recent results have shown that TAT is expressed in organs such as oviduct, ovary, and testis. The objective of this study is to examine the effect of TAT gene on egg production traits of Muscovy ducks. First, the expression levels of TAT gene in highest producing (HP) and lowest producing (LP) in 3 tissues related to reproduction were examined, and the results indicated that the expression of TAT gene in hypothalamus was significantly different between HP and LP groups. Then, 6 SNP loci (g. 120G>T, g, 122G>A, g, 254G> A, g. 270C >T, g, 312G>A, and g. 341C>A) were detected in TAT gene. Further, association analysis between the six SNP loci of TAT gene and egg production traits of 652 individual Muscovy ducks was done. The results showed that g. 254G>A and g. 270C>T were significantly correlated (P < 0.05 or 0.001) with the egg production traits of Muscovy ducks. This study elucidated the molecular mechanism that TAT gene might be regulating the egg production traits of Muscovy ducks.

Genetic stability of SIV Gag/Tat gene inserted into Del-II in modified vaccinia virus ankara after serial passage of recombinant vector in pCEFs cells.

Modified vaccinia virus Ankara (MVA) is an attenuated vaccinia virus with restricted replication in human cells. The virus serves as an ideal vaccine vector suitable for safe use even in immune-compromised individuals. With its inherently large packaging capacity, expression cassettes encoding bulky genes can be inserted into deletion regions within the MVA genome. These deletion sites develop during the process of the attenuation of the virus by passage in Chicken Embryo Fibroblasts (pCEFs). Transgene stability in MVA is important to assure immunogenicity and efficacy. In the present study, we assessed the effect of substantial passage of recombinant MVA vectors on the stability of expression cassette encoding SIV Gag/Tat genes inserted at the Del-II site, as part of generating a vaccine to protect from HIV. Our data indicated that after 15 passages there was a significant loss or mutation of the inserted genes.

Serum amyloid P component facilitates DNA clearance and inhibits plasmid transfection: implications for human DNA vaccine.

The demonstration that naked plasmid DNA can induce strong immune responses in mice has attracted considerable attention in the vaccine community. However, similar immunizations have been less/not effective in clinical trials during the past decade, and the underlying mechanisms remain unknown. In this study, we hypothesized that some DNA-binding proteins in human serum may serve as host barriers, responsible for the low efficiency of plasmids' transfection in vivo. Using proteomics, we showed that human serum amyloid P component (hSAP) is specifically present in human DNA-protein complexes. Further analysis indicated that hSAP effectively binds plasmid DNA, inhibits DNA transfection into somatic cells and facilitates the endocytosis of DNA by macrophages, whereas mouse SAP (mSAP) has similar, but much weaker, activities. In the presence of hSAP, the plasmid DNA expression in vivo and plasmid DNA-induced immune responses also significantly decreased. Therefore, our results suggest that hSAP contributes to extracellular DNA clearance and the inhibition of plasmid DNA transfection in vivo. This mechanism may be partly responsible for the insufficient immune responses to DNA vaccination in human beings; therefore, it may serve as a novel target for the improvement of DNA vaccines and DNA-based gene therapy.

CTGC motifs within the HIV core promoter specify Tat-responsive pre-initiation complexes.

BACKGROUND: HIV latency is an obstacle for the eradication of HIV from infected individuals. Stable post-integration latency is controlled principally at the level of transcription. The HIV trans-activating protein, Tat, plays a key function in enhancing HIV transcriptional elongation. The HIV core promoter is specifically required for Tat-mediated trans-activation of HIV transcription. In addition, the HIV core promoter has been shown to be a potential anti-HIV drug target. Despite the pivotal role of the HIV core promoter in the control of HIV gene expression, the molecular mechanisms that couple Tat function specifically to the HIV core promoter remain unknown. RESULTS: Using electrophoretic mobility shift assays (EMSAs), the TATA box and adjacent sequences of HIV essential for Tat trans-activation were shown to form specific complexes with nuclear extracts from peripheral blood mononuclear cells, as well as from HeLa cells. These complexes, termed pre-initiation complexes of HIV (PICH), were distinct in composition and DNA binding specificity from those of prototypical eukaryotic TATA box regions such as Adenovirus major late promoter (AdMLP) or the hsp70 promoter. PICH contained basal transcription factors including TATA-binding protein and TFIIA. A mutational analysis revealed that CTGC motifs flanking the HIV TATA box are required for Tat trans-activation in living cells and correct PICH formation in vitro. The binding of known core promoter binding proteins AP-4 and USF-1 was found to be dispensable for Tat function. TAR RNA prevented stable binding of PICH-2, a complex that contains the general transcription factor TFIIA, to the HIV core promoter. The impact of TAR on PICH-2 specifically required its bulge sequence that is also known to interact with Tat. CONCLUSION: Our data reveal that CTGC DNA motifs flanking the HIV TATA box are required for correct formation of specific pre-initiation complexes in vitro and that these motifs are also required for Tat trans-activation in living cells. The impact of TAR RNA on PICH-2 stability provides a mechanistic link by which pre-initiation complex dynamics could be coupled to the formation of the nascent transcript by the elongating transcription complex. Together, these findings shed new light on the mechanisms by which the HIV core promoter specifically responds to Tat to activate HIV gene expression.

A gene therapy approach to eliminate HIV-1-infected cells.

The ideal therapy for HIV infection requires a method to eliminate all HIV-harboring cells in the infected individual. The authors are developing an HIV-specific promoter to drive the expression of suicide genes that would induce cell death specifically in HIV-infected cells. The authors constructed a promoter that is 100-fold more responsive to the HIV transcriptional activator, Tat, than cellular transcription factors, using a plasmid expressing luciferase under the control of the mutated LTR promoter.

Genetic and functional heterogeneity of CNS-derived tat alleles from patients with HIV-associated dementia.

Human immunodeficiency virus type 1 (HIV-1) demonstrates a high degree of viral diversity which has an impact on viral fitness. Genetic compartmentalization of HIV-1 proteins between central nervous system (CNS) and lymphoid tissues is well established and reflects altered requirements for HIV-1 replication in macrophages/microglia, brain-specific immune selection pressures and possibly the timing of virus invasion of the CNS. Tat-encoding mRNA has been detected in the CNS of HIV-1 infected individuals and its neurotoxic effects in the CNS are well documented. However, while CNS-derived tat sequences have demonstrated significant diversity, the effect of this molecular diversity on transcriptional regulation and its impact on the pathogenesis of HIV-associated dementia (HAD) remains unclear. In this study, we cloned and characterised 44 unique tat alleles from brain, cerebral spinal fluid, spinal cord and blood/lymphoid tissue-derived HIV-1 isolates from five subjects with HAD. While phylogenetic analyses revealed tissue-specific compartmentalization of Tat variants for two patients, broad compartmentalization across the panel of tissue-derived viruses was not observed. Despite the lack of consistent tissue-specific compartmentalization, sequence variations within patients segregated CNS and non-CNS tat alleles. These amino acid alterations predominated within the transactivation domain of Tat and could account for alterations in the ability of particular Tat proteins to transactivate the LTR. Although a subset of patients demonstrated reduced transactivation capacity among CNS-derived Tat proteins compared to those from matched lymphoid tissues, overall Tat proteins from the CNS to lymphoid compartments maintained similar levels of transactivation function. Together, these data suggest that despite the observed heterogeneity in tat alleles isolated from matched lymphoid to CNS compartments, Tat function is maintained, highlighting the importance of Tat function in HIV-1 neuropathogenesis.

Human papillomavirus infection and its association with HIV.

Human papillomavirus (HPV) can infect oral mucosa, causing asymptomatic infection or warty lesions. Several case-control studies have confirmed HPV as an independent risk factor for squamous cell carcinoma. HPV-related cancers seem to have better prognoses and different risk factors than do HPV-negative ones. HIV-infected patients are known to be at increased risk for persistent genital and anal high-risk HPV infections and intraepithelial neoplasm. Since the era of highly active antiretroviral therapy, the prevalence and persistence of warty lesions in oral mucosa have increased. Oral squamous cell carcinoma was recently added in the case definitions for common HIV-related oral mucosa lesions. The increased risk of HPV infection in HIV patients has been associated with impaired immune response to HPV, highly active antiretroviral therapy, aging of the HIV-infected patients, and direct interaction between the 2 viruses. HPV32 seems to be much more prevalent in asymptomatic HPV infections and warts among those infected with HIV than among those in the general population. Regarding HIV genes, there is evidence of an interaction between HPV and tat, rev, and vpr. HIV might play a role in HPV-associated pathogenesis by exhorting oncogenic stimuli via tat and rev or visa versa.

The expression of the interleukin 6 gene is induced by the human immunodeficiency virus 1 TAT protein.

Human immunodeficiency virus 1 (HIV1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin 6 (IL-6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL-6 secretion of HIV1-infected cells may include transactivation of the IL-6 gene by HIV1. To test this hypothesis, we used the pIL6Pr-chloramphenicol acetyltransferase (CAT) plasmid, an IL-6 promoter-CAT construct, as a target of the transactivating function of the HIV1 TAT protein. By cotransfecting the pIL6Pr-CAT and the tat-expressing pSVT8 plasmid in MC3 B-lymphoblastoid or in HeLa epithelial cells, we observed that TAT transactivates the human IL-6 promoter. These results were confirmed when pIL6Pr-CAT was transfected in MC3 or HeLa cells that constitutively expressed the tat gene in a sense (pSVT8 cells) or antisense (pSVT10 cells) orientation. 5' deletion plasmids of pIL6Pr-CAT, in which regions at -658, -287, and -172 were inserted 5' to the cat gene, were transiently transfected in pSVT10 and pSVT8 cells and showed that TAT-induced activation of the IL-6 promoter required a minimal region located between -287 and -54 bp. Moreover, experiments with plasmids carrying the -658, -287, and -172 bp regions of the IL-6 promoter inserted downstream to a TAR-deleted HIV1-LTR identified the sequence of -172 to -54 as the minimal region of the IL-6 promoter required for TAT to transactivate the TAR-deleted HIV1-LTR. By DNA-protein binding experiments, tat-transfected cells expressed a consistent increase in kappa B and nuclear factor (NF)-IL-6 binding activity. Accordingly, the pDRCAT and IL-1REK9CAT, carrying tandem repeats of NF-kappa B or NF-IL6 binding motifs, respectively, were activated in TAT-expressing cells. The biological relevance of the TAT-induced IL-6 secretion was addressed by generating 7TD1 cells, an IL-6-dependent mouse cell line, stably expressing the tat gene. These tat-positive cells expressed the endogenous IL-6 gene, secreted high amounts of murine IL-6, and grew efficiently in the absence of exogenous IL-6. Moreover, the tat-positive 7TD1 cells sustained the growth of parental 7TD1 cells and showed a dramatic increase in their tumorigenic potency. These results suggest that TAT protein may play a role in the pathogenesis of some HIV1-associated diseases by modulating the expression of host cellular genes.

Determinants of human immunodeficiency virus type 1 escape from the primary CD8+ cytotoxic T lymphocyte response.

CD8+ cytotoxic T lymphocytes (CTLs) play an important role in containment of virus replication in primary human immunodeficiency virus (HIV) infection. HIV's ability to mutate to escape from CTL pressure is increasingly recognized; but comprehensive studies of escape from the CD8 T cell response in primary HIV infection are currently lacking. Here, we have fully characterized the primary CTL response to autologous virus Env, Gag, and Tat proteins in three patients, and investigated the extent, kinetics, and mechanisms of viral escape from epitope-specific components of the response. In all three individuals, we observed variation beginning within weeks of infection at epitope-containing sites in the viral quasispecies, which conferred escape by mechanisms including altered peptide presentation/recognition and altered antigen processing. The number of epitope-containing regions exhibiting evidence of early CTL escape ranged from 1 out of 21 in a subject who controlled viral replication effectively to 5 out of 7 in a subject who did not. Evaluation of the extent and kinetics of HIV-1 escape from >40 different epitope-specific CD8 T cell responses enabled analysis of factors determining escape and suggested that escape is restricted by costs to intrinsic viral fitness and by broad, codominant distribution of CTL-mediated pressure on viral replication.

Alpha-galactosidase A-Tat fusion enhances storage reduction in hearts and kidneys of Fabry mice.

The protein transduction domain from human immunodeficiency virus (HIV) Tat allows proteins to penetrate the cell membrane. Enhanced cellular uptake of therapeutic proteins could benefit a number of disorders. This is especially true for lysosomal storage disorders (LSDs) where enzyme replacement therapy (ERT) and gene therapy have been developed. We developed a novel recombinant lentiviral vector (LV) that engineers expression of alpha-galactosidase A (alpha-gal A)-Tat fusion protein for correction of Fabry disease, the second-most prevalent LSD with manifestations in the brain, kidney and heart. In vitro experiments confirmed mannose-6-phosphate independent uptake of the fusion factor. Next, concentrated therapeutic LV was injected into neonatal Fabry mice. Analysis of tissues at 26 wks demonstrated similar alpha-gal A enzyme activities but enhanced globotriaosylceramide (Gb3) reduction in hearts and kidneys compared with the alpha-gal A LV control. This strategy might advance not only gene therapy for Fabry disease and other LSDs, but also ERT, especially for cardiac Fabry disease.

Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.

HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.

Gene delivery into ischemic myocardium by double-targeted lipoplexes with anti-myosin antibody and TAT peptide.

The treatment of myocardial ischemia using gene therapy is a rather novel but promising approach. Gene delivery to target cells may be enhanced by using double-targeted delivery systems simultaneously capable of extracellular accumulation and intracellular penetration. With this in mind, we have used low cationic liposomes-plasmid DNA complexes (lipoplexes) modified with cell-penetrating transactivating transcriptional activator (TAT) peptide (TATp) and/or with monoclonal anti-myosin monoclonal antibody 2G4 (mAb 2G4) specific toward cardiac myosin, for targeted gene delivery to ischemic myocardium. In vitro transfection of both normoxic and hypoxic cardiomyocytes was enhanced by the presence of TATp as determined by fluorescence microscopy and ELISA. The in vitro transfection was further enhanced by the additional modification with mAb 2G4 antibody in the case of hypoxic, but not normoxic cardiomyocytes. However, we did not observe a synergism between TATp and mAb 2G4 ligands under our experimental condition. In in vivo experiments, we have clearly demonstrated an increased accumulation of mAb 2G4-modified TATp lipoplexes in the ischemic rat myocardium and significantly enhanced transfection of cardiomyocytes in the ischemic zone. Thus, the genetic transformation of normoxic and hypoxic cardiomyocytes can be enhanced by using lipoplexes modified with TATp and/or mAb 2G4. Such complexes also demonstrate an increased accumulation in the ischemic myocardium and effective transfection of hypoxic cardiomyocytes in vivo.

A high mobility group B-1 box A peptide combined with an artery wall binding peptide targets delivery of nucleic acids to smooth muscle cells.

The TAT-high mobility group box-1 A box peptide (TAT-HMGB1A) has been reported previously to be able to deliver DNA into cells without cytotoxicity. In this study, an artery wall smooth muscle cell-targeting carrier was developed using TAT-HMGB1A combined with an artery wall binding peptide (ABP). For the production of ABP linked TAT-HMGB1A (TAT-HMGB1A-ABP), pET15b-TAT-HMGB1A-ABP was constructed by inserting the ABP cDNA into pET15b-TAT-HMGB1A. TAT-HMGB1A-ABP was expressed in E. coli and purified by Nickel chelate chromatography. Gel retardation assays showed that TAT-HMGB1A-ABP formed a complex with the plasmid at or above a 5:1 weight ratio (peptide:plasmid). At a 20:1 weight ratio, the zeta-potential was approximately 25 mV and the particle size was approximately 120 nm. TAT-HMGB1A-ABP had the highest transfection efficiency in A7R5 smooth muscle cells at a weight ratio of 20:1. TAT-HMGB1A-ABP exhibited higher transfection efficiency in A7R5 cells than PLL or TAT-HMGB1A, while TAT-HMGB1A-ABP had lower transfection efficiencies in Hep3B hepatoma, 293 kidney, NIH3T3 fibroblast, and Raw264.7 macrophage cells compared with PLL. Together, these results suggest that the ABP moiety of the peptide increased transfection efficiency specifically in smooth muscle cells. In a competition assay, the transfection efficiency of TAT-HMGB1A-ABP in A7R5 cells was reduced by the addition of free ABP. MTT assays showed that TAT-HMGB1A-ABP did not produce any cytotoxicity in A7R5 cells. Therefore, TAT-HMGB1A-ABP may be useful for a targeting gene delivery to smooth muscle cells.