| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Institute of Microbiology, University of Bologna, Bologna, and
Institute of Human Anatomy, University of Ferrara, Ferrara, Italy
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Data from different groups of investigators indicate that Tat protein can be actively secreted by either HIV-1-infected or tat-transfected cells (6, 7, 8, 9), and it displays pleiotropic activities on cell survival, growth, and function of bystander uninfected cells (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). In particular, it has been demonstrated that high (100 nM-10 µM) concentrations of extracellular Tat show a cytotoxic effect on the proliferation of isolated T lymphocytes driven by recall Ags (11, 12, 13, 14) as well as on Jurkat cells (15, 16, 17). On the other hand, lower (10 pM–1 nM) concentrations of Tat protein promote the survival/growth of different cell types, including primary PBMC and Jurkat cells (18).
Besides being released by HIV-1-infected cells, Tat protein can be taken up by different cell types both in vitro (19, 20, 21) and in vivo (22), reaching the nucleus quite rapidly. Therefore, it has been proposed that the biologic effects of high Tat concentrations on T cell survival/growth are mainly due to a direct action of Tat at the nuclear level on the promoters of cellular genes encoding for cytokines (23, 24, 25, 26, 27) or genes relevant to cell survival or transformation (14, 18, 28, 29). However, it has also been shown that Tat, at low concentrations, displays a cytokine-like activity, able to interact with a variety of surface receptors (30, 31, 32, 33, 34, 35) and to activate phosphatidylinositol 3-kinase (PI-3)3 (36) and protein kinase C (PKC) (37, 38).
The cAMP-responsive element binding protein (CREB) transcription factor has been identified as a substrate for several kinases (39, 40, 41). While the role of progressive phosphorylation by casein kinase I and II at other serine residues remains undefined, it is well established that phosphorylation of CREB at Ser133 is essential for gene transactivation mediated by CRE sites placed in the promoters of many cellular or viral genes (42, 43, 44, 45, 46). CREB plays a key role in the physiology of nervous and neuroendocrine systems (40, 45), whereas its potential relevance in the regulation of lymphoid cell survival/growth still remains to be clearly defined. In this respect, it has been recently demonstrated that CREB might be implicated also in T cell development (47) and proliferation (48).
To investigate the ability of Tat to activate intracellular signals in lymphoid cells, we evaluated here the effect of Tat on CREB phosphorylation levels, CRE-binding activity in nuclear extracts, and CRE-CAT activity in lymphoblastoid Jurkat T cell line. In parallel experiments, extracellular Tat was added to primary PBMC in the absence or presence of a variety of pharmacologic inhibitors of different signal transduction pathways.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Synthetic (Technogen, Caserta, Italy) HIV-1 Tat and recombinant HIV-1 p24 (Intracell, Cambridge, MA) proteins were dissolved in PBS containing 0.1% BSA and aliquoted at -70°C before use. Staurosporin, wortmannin, bisindolylmaleimide, chelerythrine, H-89, PD 98059, and forskolin, all purchased from Calbiochem (La Jolla, CA), were prepared in DMSO and stored at -20°C.
Cell lines, PBMC, and treatment of cells
Human Jurkat CD4+ lymphoblastoid T cell line was maintained in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% FCS (Life Technologies) at an optimal cell density of 0.3 to 1.5 x 106 cells/ml.
PBMC were obtained from normal blood donors, who gave their informed consent to this research according to the Helsinki Declaration of 1975. Briefly, heparinized (20 U/ml) blood samples were diluted 1:3 with RPMI plus 10% FCS, layered over Ficoll Histopaque (density = 1.077 g/ml; Pharmacia, Uppsala, Sweden) and centrifuged at 1500 rpm for 30 min. Light density mononuclear cells were collected, washed twice, counted, and seeded in culture in RPMI plus 0.1% BSA.
In most experiments, 48-h serum-starved (RPMI + 0.1% BSA) Jurkat cells or freshly isolated PBMC were seeded in culture in the absence or presence of increasing (0.1–1000 ng/ml) concentrations of Tat protein for 0 to 90 min. HIV-1 p24 was used as negative control.
In blocking experiments performed with the pharmacologic inhibitors staurosporin, wortmannin, bisindolylmaleimide, chelerythrine, and PD 98059, cells were pretreated for 1 h at 37°C with various doses of each inhibitor diluted in RPMI medium, while control cells were treated with DMSO diluted in RPMI.
Western blotting and indirect immunofluorescence staining revealed by flow cytometry or confocal microscopy
Samples derived from 2 x 106 viable Jurkat cells or PBMC, containing approximately 100 µg of proteins, were migrated in 10% acrylamide gels and blotted onto nitrocellulose filters. Blotted filters were blocked for 30 min in a 3% suspension of dried skimmed milk in PBS, then incubated overnight at 4°C with 1:1000 dilution of rat anti-CREB serum in PBS or serum directed against the phosphorylated Ser133 form of CREB (both from Upstate Biotechnology, Lake Placid, NY). Anti-Ser133 CREB serum was obtained from rabbits immunized with a phosphopeptide corresponding to amino acids 123 to 136 of CREB. Filters were washed and further incubated for 1 h at room temperature with 1:1500 dilution of peroxidase-conjugated anti-rabbit IgG (Sigma Chemical, Milan, Italy) in 1% BSA. Specific reactions were revealed with the enhanced chemiluminescence (ECL) Western blotting detection reagent (Amersham, Arlington Heights, IL).
For the indirect immunofluorescence analysis, Jurkat cells and PBMC were fixed in 2% paraformaldehyde/PBS for 20 min at room temperature, washed twice with PBS containing 10 mM glycin, and permeabilized in PBS/0.5% Triton X-100 for 5 min at 0°C. After two washings with PBS, cells were resuspended in PBS plus 10% normal goat serum for 10 min at room temperature before adding anti-Ser133 CREB serum (dilution 1:200) for 120 min at 37°C. After two washings with PBS, a polyclonal goat anti-rabbit IgG (dilution 1:100) conjugated to fluorescein (GAR-FITC) was added to cells and incubated for 45 min at 37°C. Finally, the cells were washed and analyzed. The negative controls consisted of normal rabbit serum (dilution, 1:200) followed by identical second-layer labeling as above. The expression of Ser133 CREB protein was investigated using either a FACScan flow cytometer (Becton Dickinson, San Jose, CA) or confocal microscopy.
For confocal microscopy, the cells were first spun on coverslips, then fixed and stained with the anti-Ser133 CREB serum as described above. The cells were analyzed by means of a MCR-1000 confocal microscopy (Bio-Rad Microscience, Hemel Hempstead, U.K.) equipped with a krypton/argon ion laser emitting at 488 nm. The signal was achieved through an Epidetector filter (passing band 522/35 nm), analyzed by CoMOS software and printed on a Ektachrome 64T Kodak film by a Focus Imagecorder Plus (Focus Graphics, Foster City, CA).
Electrophoretic mobility shift assay (EMSA)
A CRE site (5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3'),
containing double-stranded oligonucleotide (Promega, Madison, WI), was
5'-labeled with (32P)
ATP, purified, and used as a
probe in EMSAs. The underlined portion of the preceding sequence is the
CRE site. Briefly, nuclear protein extracts (4–8 µg protein)
prepared from Jurkat cells by the method of Dignam et al. (49) were
incubated with 3 µg of poly(dI-dC) and 20,000 cpm of 32P
end-labeled oligonucleotide probe for 20 min at room temperature in a
20-µl solution containing 15% glycerol, 20 mM HEPES-NaOH (pH 7.9),
60 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 1 mM DTT. For
supershift analysis, 5 µl of anti-CREB or
anti-Ser133 CREB sera or of normal rabbit serum were
added to reaction mixtures 15 min before the addition of labeled
CRE-containing oligonucleotide. Protein-DNA complexes and
protein-DNA-serum supershift complexes were resolved in 4%
polyacrylamide gels in 0.25x TBE buffer (22.5 mM Tris-borate and 0.5
mM EDTA, pH 8.3). Gels were dried and exposed overnight to radiographic
film with an intensifying screen at -70°C.
Plasmid and transfection experiments
The following plasmids were a generous gift of Dr. Enzo
Lalli (IGBMC, Strasbourg, France): 1) CRE-CAT (pSom-CAT) plasmid
(50), which represents the CRE motif of the rat somatostatin
promoter cloned in front to the chloramphenicol acetyl-transferase
(CAT) gene in pBLCAT2 vector; 2) pBLCAT2 backbone vector; 3) pGAL4
encoding for amino acids 1 to 147 of GAL4 yeast protein (DNA binding
domain, GAL41–147); 4) pGAL4-CREB
LZ, which lacks the
COOH-terminal 29 amino acids of GAL4-CREB, including the leucine repeat
dimerization motif (LZ); 5) pGAL4-CREBLZM1, representing the
pGAL4-CREBLZ with the Ser133 to Ala mutation (51); and
6) pG4-CAT (52), containing 4x GAL4 sites cloned in front of herpes
simplex virus thymidine kinase promoter.
Transient transfection experiments were performed using the DEAE-dextran method, as described previously (18). In single transfection experiments, 107 Jurkat cells were maintained in RPMI 1640 plus 1% FCS for 24 h and then transfected with 10 µg of CRE-CAT or pBLCAT2 in 500 µg DEAE-dextran for 90 min. Twelve hours after transfection, cells were treated with 10 ng/ml of Tat, 10 ng/ml of p24, or 10 µM forskolin or left untreated. In some experiments, Tat was pretreated with 1 µg of anti-Tat-neutralizing mAb (Intracell) for 1 h at 37°C before adding to the cells. Twenty-four hours after treatment with Tat and other agonists, Jurkat cells were lysed and assayed for CAT activity using volumes of extract corresponding to equal protein amounts. Protein determination was performed by Bradford assay (Bio-Rad, Richmond, CA).
In cotransfection experiments, 107 Jurkat cells were
transfected with 10 µg of pG4CAT plus pGAL4 or pGAL4-CREBLZ
or pGAL4-CREBLZM1 in 500 µg DEAE-dextran for 90 min. Twelve hours
after transfection, cells were treated with the same agonists described
above for 24 h and assayed for CAT activity.
Mitogen-activated protein kinase (MAPK) assay
In some experiments, the kinase catalytic activity of MAP was analyzed in whole homogenates obtained from serum-starved Jurkat cells or freshly isolated primary PBMC treated for different time points with 10 ng/ml of extracellular Tat or p24. For this purpose, the Biotrak kit p42/p44 MAPK enzyme assay system was used, according to the manufacturer’s instructions (Amersham). The data obtained were expressed in pmol of phosphate (Pi)/min.
Statistical analysis
Statistical analysis was performed using Student’s two-tailed t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Since previous data from our group and others clearly indicate
that extracellular HIV-1 Tat protein can act as a viral growth factor
(6–9), in the first group of current experiments we investigated
whether Tat was able to modulate the phosphorylation levels of the
transcription factor CREB. To do so, Jurkat cells were serum starved
(RPMI + 0.1% FCS) to lower the high level of endogenous
Ser133-phophorylated CREB in cells cultured in RPMI plus
10% FCS. Forty-eight hours after serum starvation, Jurkat cells were
stimulated with increasing concentrations of extracellular synthetic
Tat protein for 30 min. As positive control of CREB Ser133
phosphorylation, we used forskolin (10 µM), a well-known activator of
protein kinase A (PKA). Cell homogenates were next subjected to Western
blot analysis with both anti-Ser133 phosphorylated CREB
(Fig. 1
A) and
anti-CREB (Fig. 1B) sera.
|
A). Maximal
stimulation was achieved at 10 ng/ml, while 1000 ng/ml of Tat failed to
significantly stimulate CREB phosphorylation. A similar bell-shaped
response to increasing concentrations of extracellular Tat has been
previously reported from a study of Kaposi’s sarcoma cells by Ensoli
et al. (7). This phenomenon might reflect the saturation of
high-affinity surface receptors with occupancy of low-affinity
receptors. On the other hand, no significant modifications in the total
amount of CREB were noticed upon Tat treatment (Fig. 1B). The changes in intensity of the immunoreactive
bands were quantified by densitometry and expressed in arbitrary
units.
A time course of Tat-mediated CREB phosphorylation was next examined in
Jurkat cells (Fig. 2, A and
B) and primary PBMC (Fig. 2, C and
D). Extracellular Tat (10 ng/ml) induced a rapid
increase of Ser133 CREB phosphorylation, which reached the
maximal level after 30 to 60 min in both Jurkat and PBMC and showed a
decline toward basal levels after 90 min (Fig. 2, A and
C). Once again, no significant variations in total
CREB levels were noticed using an anti-CREB serum (Fig. 2, B and D). In this and other experiments,
instead of a single band, a doublet of immunoreactive proteins was
sometimes observed. The faster migrating band may represent a different
CREB isoform or a degradation product. As stripping and reprobing with
the anti-CREB serum did not allow for discrimination between these
two possibilities, due to the proximity of the two phosphorylated
bands, densitometric analysis was calculated considering an area that
comprised both phosphoproteins.
|
, A–C) or confocal
microscopy (Fig. 3, D–F).
|
Further studies were undertaken to examine whether extracellular
Tat had some effect on the nuclear CRE complexes in Jurkat cells. For
this purpose, a DNA-binding assay was performed in the absence or
presence of sera to CREB, Ser133 phosphorylated CREB,
or normal rabbit serum. As shown in Figure 4A, nuclear extracts from
Jurkat cells typically produced a single dominant nucleoprotein
complex. It was shown that this complex contained CREB by complete
immunoinhibition with anti-CREB serum (lanes 5
and 8 of Fig. 4A). By using a different,
supershifting serum that specifically recognizes only the
phosphorylated form of Ser133 CREB (lanes
4 and 7 of Fig. 4A), we probed the
presence of Ser133 CREB after Tat stimulation. On the other
hand, no supershift was observed when normal rabbit serum was used
(lane 3 of Fig. 4A). The presence
of two supershifted complexes in our assay was probably due to the
ability of CREB to heterodimerize with various trascription factors,
including ATF and c-Fos, that give rise to nuclear protein complexes of
different molecular weights (40). The specificity of the band was also
demonstrated by a competition analysis of CRE-binding activity by
excess cold CRE oligonucleotides (Fig. 4B).
|
). Moreover, pretreating Tat protein
with an anti-Tat-neutralizing mAb resulted in an almost complete
blocking of Tat-mediated CRE-CAT activation.
|
The ability of CREB to contribute to Tat signaling was further
investigated using a gene that encodes a chimeric protein in which CREB
is fused at its amino terminus to the DNA-binding and dimerization
domain of the yeast transcription factor, GAL41–147.
We employed a specific GAL4-CREB mutant (GAL4-CREBLZ) in which
the CREB leucine zipper region was deleted, while the GAL4 DNA-binding
and dimerization domains remained intact. This mutant was used to rule
out possible interactions of full length CREB with cellular proteins
containing leucine zipper regions. To determine the functional
relevance of CREB Ser133 phosphorylation, we also conducted
transfection experiments using GAL4-CREBLZM1 construct in which
Ser133 was mutated to Ala. It has been shown previously
that this specific mutation does not affect the stability of CREB
protein or its nuclear localization (51). All cotransfection
experiments were performed using a CAT reporter plasmid (pG4-CAT)
containing four GAL4 binding sites (Fig. 6).
|
LZ, CAT reporter gene expression showed
a threefold increase in induction levels of Jurkat cells upon treatment
with extracellular Tat (10 ng/ml) and a fivefold increase upon
treatment with forskolin. In the presence of GAL4-CREBLZM1 mutant,
Tat did not induce a significant activation of CAT expression.
Moreover, Tat failed to induce transcription of the reporter gene if
cells were transfected with only GAL41–147. Thus, Tat
induction of pG4-CAT reporter gene required the CREB moiety of the
GAL4-CREBLZ fusion protein. Tat stimulates CREB Ser133 phosphorylation through a MAPK-dependent pathway
In the next experiments, we sought to investigate whether Tat stimulated CREB Ser133 phosphorylation via an intracellular signal cascade. For this purpose, we used different concentrations of the broad-spectrum protein kinase inhibitor staurosporin (50–500 nM), or the more selective PKC inhibitors bisindolylmaleimide (100–1000 nM) and chelerythrine (10–100 µM), the PKA inhibitor H-89 (100–1000 nM), and the PI-3 kinase inhibitor wortmannin (100–1000 nM).
Western blot analysis of primary PBMC homogenates showed that only
staurosporin and wortmannin affected the levels of Tat-stimulated
Ser133 CREB phosphorylation (Fig. 7 A and B),
while no reproducible variations were noticed when selective PKC or PKA
inhibitors were used (data not shown). Of note, wortmannin induced some
suppression of Ser133 CREB phosphorylation only at
concentrations of 500 to 1000 nM, which are significantly higher than
those required to obtain a maximal inhibition of PI-3 kinase (100 nM)
(54), but are in the optimal range to inhibit the MAPK pathway.
Therefore, PBMC were treated next with 10 to 50 µM of PD 98059, a
specific inhibitor of MAPK kinase, the upstream enzyme involved in MAPK
activation (Fig. 7, C and D). At both
concentrations, PD 98059 was specifically able to inhibit the
Tat-mediated Ser133 phosphorylation of CREB.
|
, Tat induced a significant
(p < 0.05) increase of MAPK catalytic
activity, reaching a peak after 5 to 15 min from the beginning of the
treatment. Similar findings were observed using Jurkat cells instead of
PBMC (data not shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The specific DNA promoter/enhancer element located in the control regions of the genes serves as target sites for the binding of transcriptional transactivator proteins. The most thoroughly studied CRE-binding protein is a 43- to 46-kDa protein termed CREB, which is expressed constitutively. In fact, CREB activation results from posttranslational modifications such as phosphorylation of Ser133 in situ. This phosphorylation step is critical for transactivation of a CRE-dependent promoter.
Extensive studies have established the role of CREB in mediating transcriptional activation induced by cAMP via a direct phosphorylation of CREB by PKA on Ser133 (39-45). In particular, in the well-known model of PC12 rat pheochromocytoma cells, phosphorylation of CREB Ser133 occurs in response to pharmacologic agents that increase intracellular levels of cAMP, acting through PKA, but also in response to nerve growth factor, acting through the Ras/Raf/MAPK pathway (41). In addition to the PC12 cell model, it has been demonstrated also in hemopoietic cells that CREB appears downstream of a variety of intracellular signal transduction pathways that activate receptor tyrosine kinases (55). In fact, the kinase-inducible domain of the CREB gene contains consensus phosphorylation sites for a variety of protein kinases, including PKA, PKC, casein kinases I and II, and calcium-calmodulin-dependent protein kinases I and II (39, 40). Moreover, a specific CREB kinase has been recently identified downstream of the Ras/Raf/MAPK pathway (56).
In this study, we have demonstrated that HIV-1 Tat protein induces the rapid Ser133 phosphorylation of CREB in both the Jurkat cell line and primary PBMC. The binding to DNA CRE sites and the increased functional activity of phosphorylated CREB in Tat-stimulated Jurkat cells was then demonstrated by supershift EMSA assay and transient transfection experiments, respectively.
Although the potential physiologic significance of CREB in regulating lymphoid cell survival/growth remains to be defined, our findings may contribute to explaining the previously reported ability of extracellular Tat to modulate CD4+ T cell growth (9-17). In fact, it has been recently demonstrated that transgenic mice expressing a dominant-negative form of CREB show defective thymocyte proliferation and IL-2 production (47) and CREB/ATF regulate the cell cycle-controlled proliferating cell nuclear Ag, PCNA (48). Remarkably, a recent study reported that the U5 region of the HIV-1 long terminal repeat contains Tat-responsive element (TRE)-like cAMP-responsive elements binding both AP-1 and CREB/ATF proteins (57), which suggests that enhanced CREB expression is beneficial for HIV-1 replication.
As we have previously demonstrated that extracellular Tat rapidly stimulates PI-3 kinase (36) and PKC (37, 38), blocking experiments were performed using various pharmacologic inhibitors. The failure also of the PKC inhibitors bisindolylmaleimide (100–1000 nM) and chelerythrine (10–100 µM) to show any inhibition at the highest concentrations used indicated that PKC is presumably not involved in the Tat-mediated induction of CREB phosphorylation. Also, the PKA inhibitor H-89 did not show any inhibitory effect. On the other hand, a clear-cut suppressive effect of Tat-mediated CREB phosphorylation was noticed with staurosporin, a broad-spectrum protein kinase inhibitor and PD 98059, a specific MAPK inhibitor. Moreover, extracellular Tat stimulates the catalytic activity of MAPK showing a kinetics of activation (peak at 5–15 min) fully compatible with the Tat-mediated Ser133 CREB phosphorylation (peak at 30–60 min). Although these data suggest that extracellular HIV-1 Tat protein acts through an intracellular signal cascade after surface interactions with high affinity or integrin receptors (30, 31, 32, 33, 34), it is still possible that uptake of intracellular Tat may contribute to the induction of CREB phosphorylation. This issue clearly requires further investigation.
The Tat-mediated induction of CREB phosphorylation might also help to explain the ability of Tat to up-regulate c-fos gene expression in both lymphoid and monocytic cell lines (58). In fact, the minimal region still responsive to Tat contains both a serum-responsive element (SRE) and a CRE-like site. Therefore, the Tat-mediated enhancement of c-fos expression requires the activation of CREB via MAPK. While this study was under consideration for publication, the ability of extracellular Tat to stimulate MAPK in both lymphoid (59) and neuronal (60) cells has been reported by other groups of investigators.
In conclusion, our results represent the first evidence that extracellular HIV-1 Tat protein up-regulates the CREB phosphorylation levels in lymphoid T cells and may contribute to the explanation of some of the pleiotropic activities induced by Tat protein on these cells.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Giorgio Zauli, Institute of Human Anatomy, University of Ferrara, Via Fossato di Mortara 66, 44100 Ferrara, Italy.
3 Abbreviations used in this paper: PI-3, phosphatidylinositol-3 kinase; PKC, protein kinase C; PKA, protein kinase A; CRE, cAMP-responsive element; CREB, cAMP-responsive element binding protein; CAT, chloramphenicol acetyl-transferase; GAR, goat anti-rabbit; EMSA, electrophoretic mobility shift assay; MAPK, mitogen-activated protein kinase.
Received for publication April 15, 1997. Accepted for publication December 17, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
vß5 integrin to tha basic domain of the HIV tat protein and vitronectin. J. Cell. Biol. 121:461.
B binding and protein kinase C activity in primary human astrocytes. J. Virol. 70:1384.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  K. Gee, J. B. Angel, S. Mishra, M. A. Blahoianu, and A. Kumar IL-10 Regulation by HIV-Tat in Primary Human Monocytic Cells: Involvement of Calmodulin/Calmodulin-Dependent Protein Kinase-Activated p38 MAPK and Sp-1 and CREB-1 Transcription Factors J. Immunol., January 15, 2007; 178(2): 798 - 807. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Gee, J. B. Angel, W. Ma, S. Mishra, N. Gajanayaka, K. Parato, and A. Kumar Intracellular HIV-Tat Expression Induces IL-10 Synthesis by the CREB-1 Transcription Factor through Ser133 Phosphorylation and Its Regulation by the ERK1/2 MAPK in Human Monocytic Cells J. Biol. Chem., October 20, 2006; 281(42): 31647 - 31658. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Williams, D. Mondal, and K. C. Agrawal The HIV-1 Tat Protein Enhances Megakaryocytic Commitment of K562 Cells by Facilitating CREB Transcription Factor Coactivation by CBP Experimental Biology and Medicine, December 1, 2005; 230(11): 872 - 884. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Mondal, C. A. Williams, M. Ali, M. Eilers, and K. C. Agrawal The HIV-1 Tat Protein Selectively Enhances CXCR4 and Inhibits CCR5 Expression in Megakaryocytic K562 Cells Experimental Biology and Medicine, October 1, 2005; 230(9): 631 - 644. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Moreau, G. Belliard, C. D. Partidos, F. Pradezinsky, H. Le Buanec, S. Muller, and C. Desgranges Important B-cell epitopes for neutralization of human immunodeficiency virus type 1 Tat in serum samples of humans and different animal species immunized with Tat protein or peptides J. Gen. Virol., October 1, 2004; 85(10): 2893 - 2901. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Caldwell, B. S. Egan, and V. L. Shepherd HIV-1 Tat Represses Transcription from the Mannose Receptor Promoter J. Immunol., December 15, 2000; 165(12): 7035 - 7041. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Secchiero, D. Zella, S. Curreli, P. Mirandola, S. Capitani, R. C. Gallo, and G. Zauli Pivotal role of cyclic nucleoside phosphodiesterase 4 in Tat-mediated CD4+ T cell hyperactivation and HIV type 1 replication PNAS, December 8, 2000; (2000) 11512398. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. RUSNATI, G. TARABOLETTI, C. URBINATI, G. TULIPANO, R. GIULIANI, M. P. MOLINARI-TOSATTI, B. SENNINO, M. GIACCA, M. TYAGI, A. ALBINI, et al. Thrombospondin-1/HIV-1 Tat protein interaction: modulation of the biological activity of extracellular Tat FASEB J, October 1, 2000; 14(13): 1917 - 1930. [Abstract] [Full Text]
|
|
|
|
|
|
G. Zauli, P. Secchiero, L. Rodella, D. Gibellini, P. Mirandola, M. Mazzoni, D. Milani, D. R. Dowd, S. Capitani, and M. Vitale HIV-1 Tat-mediated Inhibition of the Tyrosine Hydroxylase Gene Expression in Dopaminergic Neuronal Cells J. Biol. Chem., February 11, 2000; 275(6): 4159 - 4165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Rusnati, G. Tulipano, D. Spillmann, E. Tanghetti, P. Oreste, G. Zoppetti, M. Giacca, and M. Presta Multiple Interactions of HIV-I Tat Protein with Size-defined Heparin Oligosaccharides J. Biol. Chem., October 1, 1999; 274(40): 28198 - 28205. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. C. Gallo Tat as one key to HIV-induced immune pathogenesis and Pat toxoid as an important component of a vaccine PNAS, July 20, 1999; 96(15): 8324 - 8326. [Full Text] [PDF]
|
|
|
|
|
|
E. Caselli, M. Betti, M. P. Grossi, P. G. Balboni, C. Rossi, C. Boarini, A. Cafaro, G. Barbanti-Brodano, B. Ensoli, and A. Caputo DNA Immunization with HIV-1 tat Mutated in the trans Activation Domain Induces Humoral and Cellular Immune Responses Against Wild-Type Tat J. Immunol., May 1, 1999; 162(9): 5631 - 5638. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Secchiero, D. Zella, S. Capitani, R. C. Gallo, and G. Zauli Extracellular HIV-1 Tat Protein Up-Regulates the Expression of Surface CXC-Chemokine Receptor 4 in Resting CD4+ T Cells J. Immunol., February 15, 1999; 162(4): 2427 - 2431. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Secchiero, D. Zella, S. Curreli, P. Mirandola, S. Capitani, R. C. Gallo, and G. Zauli Pivotal role of cyclic nucleoside phosphodiesterase 4 in Tat-mediated CD4+ T cell hyperactivation and HIV type 1 replication PNAS, December 19, 2000; 97(26): 14620 - 14625. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
