Human Immunodeficiency Virus Tat Protein Research Articles

Overexpression of C-terminal fragment of glutamate receptor 6 prevents neuronal injury in kainate-induced seizure via disassembly of GluR6-PSD-95-MLK3 signaling module.

Our previous study showed that when glutamate receptor (GluR)6 C terminus-containing peptide conjugated with the human immunodeficiency virus Tat protein (GluR6)-9c is delivered into hippocampal neurons in a brain ischemic model, the activation of mixed lineage kinase 3 (MLK3) and c-Jun NH2-terminal kinase (JNK) is inhibited via GluR6-postsynaptic density protein 95 (PSD95). In the present study, we investigated whether the recombinant adenovirus (Ad) carrying GluR6c could suppress the assembly of the GluR6-PSD95-MLK3 signaling module and decrease neuronal cell death induced by kainate in hippocampal CA1 subregion. A seizure model in Sprague-Dawley rats was induced by intraperitoneal injections of kainate. The effect of Ad-Glur6-9c on the phosphorylation of JNK, MLK3 and mitogen-activated kinase kinase 7 (MKK7) was observed with western immunoblots and immunohistochemistry. Our findings revealed that overexpression of GluR6c inhibited the interaction of GluR6 with PSD95 and prevented the kainate-induced activation of JNK, MLK3 and MKK7. Furthermore, kainate-mediated neuronal cell death was significantly suppressed by GluR6c. Taken together, GluR6 may play a pivotal role in neuronal cell death.

NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter

Human immunodeficiency virus-1 (HIV-1) Tat protein plays an essential role in HIV gene transcription from the HIV-1 long terminal repeat (LTR) and replication. Transcriptional activity of Tat is modulated by several host factors, but the mechanism responsible for Tat regulation by host factors is not understood fully. Using a yeast two-hybrid screening system, we identified Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) as a novel Tat-interacting partner. Here, we report its function as a positive regulator of Tat. In a coimmunoprecipitation assay, HIV-1 Tat interacted sufficiently with both endogenous and ectopically expressed NUCKS1. In a reporter assay, ectopic expression of NUCKS1 significantly increased Tat-mediated transcription of the HIV-1 LTR, whereas knockdown of NUCKS1 by small interfering RNA diminished Tat-mediated transcription of the HIV-1 LTR. We also investigated which mechanism contributes to NUCKS1-mediated Tat activation. In a chromatin immunoprecipitation assay (ChIP), knockdown of NUCKS1 interrupted the accumulation of Tat in the transactivation-responsive (TAR) region on the LTR, which then led to suppression of viral replication. However, NUCKS1 expression did not increase Tat nuclear localization and interaction with Cyclin T1. Interestingly, the NUCKS1 expression level was lower in latently HIV-1-infected cells than in uninfected parent cells. Besides, expression level of NUCKS1 was markedly induced, which then facilitated HIV-1 reactivation in latently infected cells. Taken together, our data demonstrate clearly that NUCKS1 is a novel Tat coactivator that is required for Tat-mediated HIV-1 transcription and replication, and that it may contribute to HIV-1 reactivation in latently HIV-1 infected cells.

Human Immunodeficiency Virus-1 Tat Protein Increases the Number of Inhibitory Synapses between Hippocampal Neurons in Culture

Synaptodendritic damage correlates with cognitive decline in many neurodegenerative diseases, including human immunodeficiency virus-1 (HIV-1)-associated neurocognitive disorders (HAND). Because HIV-1 does not infect neurons, viral-mediated toxicity is indirect, resulting from released neurotoxins such as the HIV-1 protein transactivator of transcription (Tat). We compared the effects of Tat on inhibitory and excitatory synaptic connections between rat hippocampal neurons using an imaging-based assay that quantified clusters of the scaffolding proteins gephyrin or PSD95 fused to GFP. Tat (24 h) increased the number of GFP-gephyrin puncta and decreased the number of PSD95-GFP puncta. The effects of Tat on inhibitory and excitatory synapse number were mediated via the low-density lipoprotein receptor-related protein and subsequent Ca(2+) influx through GluN2A-containing NMDA receptors (NMDARs). The effects of Tat on synapse number required cell-autonomous activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Ca(2+) buffering experiments suggested that loss of excitatory synapses required activation of CaMKII in close apposition to the NMDAR, whereas the increase in inhibitory synapses required Ca(2+) diffusion to a more distal site. The increase in inhibitory synapses was prevented by inhibiting the insertion of GABAA receptors into the membrane. Synaptic changes induced by Tat (16 h) were reversed by blocking either GluN2B-containing NMDARs or neuronal nitric oxide synthase, indicating changing roles for pathways activated by NMDAR subtypes during the neurotoxic process. Compensatory changes in the number of inhibitory and excitatory synapses may serve as a novel mechanism to reduce network excitability in the presence of HIV-1 neurotoxins; these changes may inform the development of treatments for HAND.

Exercise and the brain: a slap on the HAND

Exercise and the brain: a slap on the HAND

Chondrogenic effect of precartilaginous stem cells following NLS-TAT cell penetrating peptide-assisted transfection of eukaryotic hTGFβ3

Cell penetrating peptides (CPPs) are a series of promising carriers for delivering exogenous DNA to living cells. Among them, the combination of the human immunodeficiency virus TAT protein (TAT) with the SV40 large T protein nuclear localization signal (NLS) to form NLS-TAT performs well. In the present study, we took advantage of this new carrier to deliver transforming growth factor-beta 3 (TGFβ3) genes. TGFβ3 was expressed by the pEGFP-N1 vector following transfection of rat precartilaginous stem cells (PSCs), which promoted hTGFβ3 protein self-expression. At 24, 48, 72, and 120 h after transfection, the expression levels of hTGFβ3 were found to be elevated as compared with the control. The expression of hTGFβ3 was found to mediate the chondrogenic effect of PSCs. Thus, we determined the expression of the chondrogenesis-related genes type II collagen, Sox 9, and aggrecan in PSCs at 24, 48, 72, and 120 h after transfection. We found that their transcription and translation was augmented, which indicated a trend of active chondrogenesis in the PSCs. Our results demonstrated that NLS-TAT had the ability to deliver exogenous DNA into rat PSCs and could be actively expressed. This process successfully promoted PSC chondrogenesis. Additionally, PSC, may represent a new type of stem cells, and thus show great potential in regenerative repair following cartilage injury.

A Cationic Peptide, TAT-Cd0, Inhibits Herpes Simplex Virus Type 1 Ocular Infection In Vivo

To test the in vivo activity of a peptide derived from the protein transducing domain of the human immunodeficiency virus (HIV) Tat protein, TAT-Cd°, in a murine herpes simplex type 1 (HSV-1) keratitis model. the efficacy of TAT-CD° was assessed in a postinfection treatment model with different concentrations (1 mg/mL, 0.1 mg/mL, 0.01 mg/mL) of the peptide in one of four delivery vehicles: artificial tears, PBS, methylcellulose, and aquaphor cream. Treatment began within 4 or 24 hours postinfection. Viral titers in the tear film were determined by plaque assay. TAT-Cd° reduced the severity of keratitis in all of the delivery vehicles tested when treatment started, 4 hours postinfection. Peptide in the tears or PBS delivery vehicle had the most significant reduction in disease severity and delayed the onset of vascularization and stromal keratitis. The percentage of mice presenting with disease was also significantly reduced and viral titers were reduced by 1 log at 24 hours postinfection in mice treated with 1 mg/mL TAT-Cd°, suggesting that inhibiting replication early is sufficient to achieve clinical effects. Lower concentrations were not effective and delaying treatment by 24 hours was also not effective. This study shows that TAT-Cd° is an effective antiviral against HSV-1 strain KOS when applied shortly postinfection and that aqueous-based formulations are more suitable.

LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein

BackgroundHuman Immunodeficiency Virus-1 (HIV-1) associated neurocognitive disorders (HANDs) are accompanied by significant morbidity, which persists despite the use of combined antiretroviral therapy (cART). While activated microglia play a role in pathogenesis, changes in their immune effector functions, including phagocytosis and proinflammatory signaling pathways, are not well understood. We have identified leucine-rich repeat kinase 2 (LRRK2) as a novel regulator of microglial phagocytosis and activation in an in vitro model of HANDs, and hypothesize that LRRK2 kinase inhibition will attenuate microglial activation during HANDs.MethodsWe treated BV-2 immortalized mouse microglia cells with the HIV-1 trans activator of transcription (Tat) protein in the absence or presence of LRRK2 kinase inhibitor (LRRK2i). We used Western blot, qRT-PCR, immunocytochemistry and latex bead engulfment assays to analyze LRRK2 protein levels, proinflammatory cytokine and phagocytosis receptor expression, LRRK2 cellular distribution and phagocytosis, respectively. Finally, we utilized ex vivo microfluidic chambers containing primary hippocampal neurons and BV-2 microglia cells to investigate microglial phagocytosis of neuronal axons.ResultsWe found that Tat-treatment of BV-2 cells induced kinase activity associated phosphorylation of serine 935 on LRRK2 and caused the formation of cytoplasmic LRRK2 inclusions. LRRK2i decreased Tat-induced phosphorylation of serine 935 on LRRK2 and inhibited the formation of Tat-induced cytoplasmic LRRK2 inclusions. LRRK2i also decreased Tat-induced process extension in BV-2 cells. Furthermore, LRRK2i attenuated Tat-induced cytokine expression and latex bead engulfment. We examined relevant cellular targets in microfluidic chambers and found that Tat-treated BV-2 microglia cells cleared axonal arbor and engulfed neuronal elements, whereas saline treated controls did not. LRRK2i was found to protect axons in the presence of Tat-activated microglia, as well as AnnexinV, a phosphatidylserine-binding protein. In addition, LRRK2i decreased brain-specific angiogenesis inhibitor 1 (BAI1) receptor expression on BV-2 cells after Tat-treatment, a key receptor in phosphatidylserine-mediated phagocytosis.ConclusionTaken together, these results implicate LRRK2 as a key player in microglial inflammation and, in particular, in the phagocytosis of neuronal elements. These studies show that LRRK2 kinase inhibition may prove an effective therapeutic strategy for HANDs, as well as other neuroinflammatory conditions.

Inhibition of Transmitter Release and Attenuation of Anti-retroviral-associated and Tibial Nerve Injury-related Painful Peripheral Neuropathy by Novel Synthetic Ca2+ Channel Peptides

N-type Ca(2+) channels (CaV2.2) are a nidus for neurotransmitter release and nociceptive transmission. However, the use of CaV2.2 blockers in pain therapeutics is limited by side effects resulting from inhibition of the physiological functions of CaV2.2 within the CNS. We identified an anti-nociceptive peptide (Brittain, J. M., Duarte, D. B., Wilson, S. M., Zhu, W., Ballard, C., Johnson, P. L., Liu, N., Xiong, W., Ripsch, M. S., Wang, Y., Fehrenbacher, J. C., Fitz, S. D., Khanna, M., Park, C. K., Schmutzler, B. S., Cheon, B. M., Due, M. R., Brustovetsky, T., Ashpole, N. M., Hudmon, A., Meroueh, S. O., Hingtgen, C. M., Brustovetsky, N., Ji, R. R., Hurley, J. H., Jin, X., Shekhar, A., Xu, X. M., Oxford, G. S., Vasko, M. R., White, F. A., and Khanna, R. (2011) Suppression of inflammatory and neuropathic pain by uncoupling CRMP2 from the presynaptic Ca(2+) channel complex. Nat. Med. 17, 822-829) derived from the axonal collapsin response mediator protein 2 (CRMP2), a protein known to bind and enhance CaV2.2 activity. Using a peptide tiling array, we identified novel peptides within the first intracellular loop (CaV2.2(388-402), "L1") and the distal C terminus (CaV1.2(2014-2028) "Ct-dis") that bound CRMP2. Microscale thermophoresis demonstrated micromolar and nanomolar binding affinities between recombinant CRMP2 and synthetic L1 and Ct-dis peptides, respectively. Co-immunoprecipitation experiments showed that CRMP2 association with CaV2.2 was inhibited by L1 and Ct-dis peptides. L1 and Ct-dis, rendered cell-penetrant by fusion with the protein transduction domain of the human immunodeficiency virus TAT protein, were tested in in vitro and in vivo experiments. Depolarization-induced calcium influx in dorsal root ganglion (DRG) neurons was inhibited by both peptides. Ct-dis, but not L1, peptide inhibited depolarization-stimulated release of the neuropeptide transmitter calcitonin gene-related peptide in mouse DRG neurons. Similar results were obtained in DRGs from mice with a heterozygous mutation of Nf1 linked to neurofibromatosis type 1. Ct-dis peptide, administered intraperitoneally, exhibited antinociception in a zalcitabine (2'-3'-dideoxycytidine) model of AIDS therapy-induced and tibial nerve injury-related peripheral neuropathy. This study suggests that CaV peptides, by perturbing interactions with the neuromodulator CRMP2, contribute to suppression of neuronal hypersensitivity and nociception.

The TAT peptide endows PACAP with an enhanced ability to traverse bio-barriers

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potential therapeutic neuropeptide. The 11-amino acid human immunodeficiency virus TAT protein transduction domain is able to deliver protein cargoes across the cell membrane and the blood–brain barrier. A novel fusion protein PACAP-TAT, containing TAT at the C-terminus of PACAP was therefore produced and studied for the ability to cross blood barriers. The gene encoding PACAP-TAT was cloned into the expression vector pKYB, and the target peptide PACAP-TAT was purified using the Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) system. The results of cell assays showed that PACAP-TAT stimulated the cell viability of PAC1-CHO cells with the same potency as PACAP, which indicated that the fusion of TAT did not affect the ability of PACAP-TAT to activate the PACAP-specific receptor PAC1. The transfer efficiencies of PACAP-TAT and PACAP across the blood–brain barrier (BBB), blood–air barrier (BAB) and blood–testis barrier (BTB) were assayed using peptides labeled with fluorescein isothiocyanate (FITC). The results showed that PACAP-TAT traversed blood barriers with an efficiency approximately 2.5-fold greater than PACAP. Fluorescence microscopic examination showed that PACAP-TAT traversed the BBB significantly more efficiently than PACAP. Furthermore, intraperitoneal (i.p.) injection of PACAP-TAT induced a stronger inhibitory effect on food intake than PACAP (p<0.01, PACAP-TAT vs. PACAP), which indicated that TAT helped to increase the localization of PACAP-TAT in the brain. Preparation of PACAP-TAT with the enhanced ability to cross biological barriers will improve its route of administration and expand its scope of application.

The Tat protein of human immunodeficiency virus-1 enhances hepatitis C virus replication through interferon gamma-inducible protein-10

BackgroundCo-infection with human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) is associated with faster progression of liver disease and an increase in HCV persistence. However, the mechanism by which HIV-1 accelerates the progression of HCV liver disease remains unknown.ResultsHIV-1/HCV co-infection is associated with increased expression of interferon gamma-induced protein-10 (IP-10) mRNA in peripheral blood mononuclear cells (PBMCs). HCV RNA levels were higher in PBMCs of patients with HIV-1/HCV co-infection than in patients with HCV mono-infection. HIV-1 Tat and IP-10 activated HCV replication in a time-dependent manner, and HIV-1 Tat induced IP-10 production. In addition, the effect of HIV-1 Tat on HCV replication was blocked by anti-IP-10 monoclonal antibody, demonstrating that the effect of HIV-1 Tat on HCV replication depends on IP-10. Taken together, these results suggest that HIV-1 Tat protein activates HCV replication by upregulating IP-10 production.ConclusionsHIV-1/HCV co-infection is associated with increased expression of IP-10 mRNA and replication of HCV RNA. Furthermore, both HIV-1 Tat and IP-10 activate HCV replication. HIV-1 Tat activates HCV replication by upregulating IP-10 production. These results expand our understanding of HIV-1 in HCV replication and the mechanism involved in the regulation of HCV replication mediated by HIV-1 during co-infection.

Antiretroviral Therapy Does Not Block the Secretion of the Human Immunodeficiency Virus Tat Protein

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.

HIV-1 Replication and Latency Are Regulated by Translational Control of Cyclin T1

Human immunodeficiency virus (HIV) exploits cellular proteins during its replicative cycle and latent infection. The positive transcription elongation factor b (P-TEFb) is a key cellular transcription factor critical for these viral processes and is a drug target. During viral replication, P-TEFb is recruited via interactions of its cyclin T1 subunit with the HIV Tat (transactivator of transcription) protein and TAR (transactivation response) element. Through RNA silencing and over-expression experiments, we discovered that nuclear factor 90 (NF90), a cellular RNA binding protein, regulates P-TEFb expression. NF90 depletion reduced cyclin T1 protein levels by inhibiting translation initiation. Regulation was mediated by the 3′ untranslated region of cyclin T1 mRNA independently of microRNAs. Cyclin T1 induction is involved in the escape of HIV-1 from latency. We show that the activation of viral replication by phorbol ester in latently infected monocytic cells requires the posttranscriptional induction of NF90 and cyclin T1, implicating NF90 in protein kinase C signaling pathways. This investigation reveals a novel mechanism of cyclin T1 regulation and establishes NF90 as a regulator of HIV-1 replication during both productive infection and induction from latency.

Induction of monocyte chemoattractant protein-1 (MCP-1/CCL2) gene expression by human immunodeficiency virus-1 Tat in human astrocytes is CDK9 dependent

Human immunodeficiency virus-1 (HIV-1) invades the brain early in infection and may cause HIV-associated dementia (HAD), which is characterized by reactive astrocytes, and macrophage and T-cell infiltrates. HIV-1 Tat protein is thought to contribute to HAD by transactivating host genes, such as that encoding monocyte chemoattractant protein-1 (MCP-1/CCL2), although its mechanisms of action are not fully understood. We investigated the molecular pathways involved in Tat-induced MCP-1/CCL2 gene expression in human astrocytes. We found that Tat induced MCP-1/CCL2 synthesis in human astrocytes infected with a lentivirus carrying the gene encoding Tat or treated with a biologically active synthetic Tat protein. The induction of MCP-1/CCL2 was independent of the nuclear factor kappaB (NF-kappaB) classical pathway, but was significantly inhibited by specific cyclin-dependent kinase 9 (cdk9) inhibitors, such as a dominant-negative mutant or siRNA. By contrast, broader-spectrum cdk inhibitors, such as roscovitine, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), and flavopiridol, inhibited MCP-1/CCL2 induction by Tat. We also analyzed the effects of roscovitine, DRB, and flavopiridol on Tat-induced HIV-1 long terminal repeat (LTR) expression following the infection of astrocytes and HeLa cells. Astrocytes showed no inhibition by roscovitine, 59% inhibition by DRB, and 80% inhibition by flavopiridol. In control HeLa cells, high levels of inhibition were observed with roscovitine, DRB, and flavopiridol. We have ascertained the direct implication of cdk9 in Tat-induced MCP-1 expression by performing ChIP assay. These results demonstrate that cdk9 is involved in Tat-induced HIV-1 LTR, MCP-1/CCL2 gene expression.

Revised Role of Glycosaminoglycans in TAT Protein Transduction Domain-mediated Cellular Transduction

Cellular uptake of the human immunodeficiency virus TAT protein transduction domain (PTD), or cell-penetrating peptide, has previously been surmised to occur in a manner dependent on the presence of heparan sulfate proteoglycans that are expressed ubiquitously on the cell surface. These acidic polysaccharides form a large pool of negative charge on the cell surface that TAT PTD binds avidly. Additionally, sulfated glycans have been proposed to aid in the interaction of TAT PTD and other arginine-rich PTDs with the cell membrane, perhaps aiding their translocation across the membrane. Surprisingly, however, TAT PTD-mediated induction of macropinocytosis and cellular transduction occurs in the absence of heparan sulfate and sialic acid. Using labeled TAT PTD peptides and fusion proteins, in addition to TAT PTD-Cre recombination-based phenotypic assays, we show that transduction occurs efficiently in mutant Chinese hamster ovary cell lines deficient in glycosaminoglycans and sialic acids. Similar results were obtained in cells where glycans were enzymatically removed. In contrast, enzymatic removal of proteins from the cell surface completely ablated TAT PTD-mediated transduction. Our findings support the hypothesis that acidic glycans form a pool of charge that TAT PTD binds on the cell surface, but this binding is independent of the PTD-mediated transduction mechanism and the induction of macropinocytotic uptake by TAT PTD.

The BRCA‐1 binding protein BRAP2 is a novel, negative regulator of nuclear import of viral proteins, dependent on phosphorylation flanking the nuclear localization signal

This study describes for the first time the ability of the novel BRCA1-binding protein 2 (BRAP2) to inhibit the nuclear import of specific viral proteins dependent on phosphorylation. Ectopic expression of BRAP2 in transfected African green monkey kidney COS-7 cells was found to significantly reduce nuclear localization signal (NLS)-dependent nuclear accumulation of either simian virus SV40 large-tumor antigen (T-ag) or human cytomegalovirus DNA polymerase processivity factor ppUL44; this was also observed in HL-60 human promyelocytic leukemia cells on induction of BRAP2 expression by vitamin D3 treatment. BRAP2 inhibition of nuclear accumulation was dependent on phosphorylation sites flanking the respective NLSs, where substitution of the cyclin-dependent kinase site T124 of T-ag with Ala or Asp prevented or enhanced BRAP2 inhibition of nuclear import, respectively. Substitution of T427 within the NLS of ppUL44 gave similar results, whereas no effect of BRAP2 was observed on nuclear targeting of other viral proteins, such as herpes simplex virus-1 pUL30, which lacks a phosphorylation site near its NLS, and the human immunodeficiency virus-1 Tat protein. Pulldowns/AlphaScreen assays indicated direct, high-affinity binding of BRAP2(442-592) to T-ag(111-135), strictly dependent on negative charge at T124 and the NLS. All results are consistent with BRAP2 being a novel, phosphorylation-regulated negative regulator of nuclear import, with potential as an antiviral agent.

The Tat-conjugated N-terminal region of mucin antigen 1 (MUC1) induces protective immunity against MUC1-expressing tumours

Mucin antigen 1 (MUC1) is overexpressed on various human adenocarcinomas and haematological malignancies and has long been used as a target antigen for cancer immunotherapy. Most of the preclinical and clinical studies using MUC1 have used the tandem repeat region of MUC1, which could be presented by only a limited set of major histocompatibility complex haplotypes. Here, we evaluated N-terminal region (2-147 amino acids) of MUC1 (MUC1-N) for dendritic cell (DC)-based cancer immunotherapy. We used Esherichia coli-derived MUC1-N that was fused to the protein transduction domain of human immunodeficiency virus Tat protein for three reasons. First, mature DCs do not phagocytose soluble protein antigens. Secondly, tumour cells express underglycosylated MUC1, which can generate epitopes repertoire that differs from normal cells, which express hyperglycosylated MUC1. Finally, aberrantly glycosylated MUC1 has been known to impair DC function. In our study, Tat-MUC1-N-loaded DCs induced type 1 T cell responses as well as cytotoxic T lymphocytes efficiently. Furthermore, they could break tolerance in the transgenic breast tumour mouse model, where MUC1-positive breast cancers grow spontaneously. Compared with DCs pulsed with unconjugated MUC1-N, DCs loaded with Tat-conjugated MUC1-N could delay tumour growth more effectively in the transgenic tumour model as well as in the tumour injection model. These results suggest that the recombinant N-terminal part of MUC1, which may provide a diverse epitope repertoire, could be utilized as an effective tumour antigen for DC-based cancer immunotherapy.

Synthesis and evaluation of a novel liposome containing BPA–peptide conjugate for BNCT

We aimed at securing sufficient concentrations of 10B in boron neutron capture therapy (BNCT) by developing a new drug delivery system. We have designed and developed a novel lipid analog and succeeded in using it to develop the new boron component liposome. It consisted of three different kinds of amino acid derivatives and two fatty acids, and could react directly with the peptide synthesized first on resin by Fmoc solid-phase synthesis. In this study, lipid analog conjugated with HIV-TAT peptide (domain of human immunodeficiency virus TAT protein) and boronophenylalanine (BPA) was synthesized and successfully incorporated into liposomes.

Insertional Gene Activation by Lentiviral and Gammaretroviral Vectors

Gammaretroviral and lentiviral vectors are promising tools for gene therapy, but they can be oncogenic. The development of safer vectors depends on a quantitative assay for insertional mutagenesis. Here we report a rapid, inexpensive, and reproducible assay which uses a murine cell line to measure the frequency of interleukin-3 (IL-3)-independent mutants. Lentiviral and gammaretroviral vectors cause insertional mutagenesis at similar frequencies; however, they use different mechanisms. Human immunodeficiency virus (HIV)-based vectors generate mutants by insertion only into the growth hormone receptor (Ghr) locus. The HIV enhancer/promoter is active in the absence of the HIV Tat protein in this locus, and an HIV/Ghr spliced transcript expresses GHR and cells respond to GH. Deletion of the enhancer/promoter in a self-inactivating HIV-based vector prevents this mechanism of insertional mutagenesis. In contrast, gammaretroviral vectors insert into other loci, including IL-3 and genes identified as common insertion sites in the Retroviral Tagged Cancer Gene Database (RTCGD).

Proteomic Analysis of Proteins Associated with Splicing Factor-2 Associated Protein p32 Revealed its Possible Involvement in Human Ribosome Biogenesis

Splicing factor-2 associated protein p32 (SF2p32) interacts with many cellular and viral proteins such as ASF/SF2, human immunodeficiency virus (HIV) Rev and Tat proteins etc. Among those, the interaction with HIV Rev is thought to be responsible for inhibition of the splicing of HIV transcripts, resulting in infection and production of progeny virions in human cells. Because of the ability of SF2p32 to interact with many proteins, it is believed to have many functions in the cell; however, its physiological role remains unclear. Our previous proteomic analysis identified SF2p32 as a component of the protein complexes associated with fibrillarin, which is the nucleolar antigen of the autoimmune disease scleroderma and is involved in early stage of ribosome biogenesis. In this study, we examined a possible involvement of SF2p32 in ribosome biogenesis by using proteomic methodologies.

Therapeutic benefits of intrathecal protein therapy in a mouse model of amyotrophic lateral sclerosis

When fused with the protein transduction domain (PTD) derived from the human immunodeficiency virus TAT protein, proteins can cross the blood-brain barrier and cell membrane and transfer into several tissues, including the brain, making protein therapy feasible for various neurological disorders. We have constructed a powerful antiapoptotic modified Bcl-X(L) protein (originally constructed from Bcl-X(L)) fused with PTD derived from TAT (TAT-modified Bcl-X(L)), and, to examine its clinical effectiveness in a mouse model of familial amyotrophic lateral sclerosis (ALS), transgenic mice expressing human Cu/Zn superoxide dismutase (SOD1) bearing a G93A mutation were treated by intrathecal infusion of TAT-modified Bcl-X(L). We demonstrate that intrathecally infused TAT-fused protein was effectively transferred into spinal cord neurons, including motor neurons, and that intrathecal infusion of TAT-modified Bcl-X(L) delayed disease onset, prolonged survival, and improved motor performance. Histological studies show an attenuation of motor neuron loss and a decrease in the number of cleaved caspase 9-, cleaved caspase 3-, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the lumbar cords of TAT-modified Bcl-X(L)-treated G93A mice. Our results indicate that intrathecal protein therapy using a TAT-fused protein is an effective clinical tool for the treatment of ALS.