Inhibition of the expression and function of chemokine receptors on human CD4+ leukocytes by HIV-1 envelope protein gp120.
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
W Gong
Publications and source records attributed to W Gong.
Structure of a biological oxygen sensor: a new mechanism for heme-driven signal transduction.
The FixL proteins are biological oxygen sensors that restrict the expression of specific genes to hypoxic conditions. FixL's oxygen-detecting domain is a heme binding region that controls the activity of an attached histidine kinase. The FixL switch is regulated by binding of oxygen and other strong-field ligands. In the absence of bound ligand, the heme domain permits kinase activity. In the presence of bound ligand, this domain turns off kinase activity. Comparison of the structures of two forms of the Bradyrhizobium japonicum FixL heme domain, one in the "on" state without bound ligand and one in the "off" state with bound cyanide, reveals a mechanism of regulation by a heme that is distinct from the classical hemoglobin models. The close structural resemblance of the FixL heme domain to the photoactive yellow protein confirms the existence of a PAS structural motif but reveals the presence of an alternative regulatory gateway.
Amino Acid Sequence↗
Chemokines and their role in tumor growth and metastasis.
Chemokines are a superfamily of pro-inflammatory polypeptide cytokines that selectively attract and activate different cell types. Many patho-physiological conditions require the participation of chemokines, including inflammation, infection, tissue injury, allergy, cardiovascular diseases, as well as malignant tumors. Chemokines activate cells through their binding to shared or unique cell surface receptors which belong to the seven-transmembrane, G-protein-coupled Rhodopsin superfamily. The role of chemokines in malignant tumors is complex: while some chemokines may enhance innate or specific host immunity against tumor implantation, others may favor tumor growth and metastasis by promoting tumor cell proliferation, migration or neovascularization in tumor tissue. In this review, the authors summarize some of the recent advances in chemokine research and emphasis is made on the effect of chemokines in tumor growth and metastasis.
Animals↗
Crystal structures of acutolysin A, a three-disulfide hemorrhagic zinc metalloproteinase from the snake venom of Agkistrodon acutus.
Acutolysin A alias AaHI, a 22 kDa hemorrhagic toxin isolated from the snake venom of Agkistrodon acutus, is a member of the adamalysin subfamily of the metzincin family and is a snake venom zinc metalloproteinase possessing only one catalytic domain. Acutolysin A was found to have a high-activity and a low-activity under weakly alkaline and acidic conditions, respectively. With the adamalysin II structure as the initial trial-and-error model, the crystal structures were solved to the final crystallographic R-factors of 0. 168 and 0.171, against the diffraction data of crystals grown under pH 5.0 and pH 7.5 conditions to 1.9 A and 1.95 A resolution, respectively. One zinc ion, binding in the active-site, one structural calcium ion and some water molecules were localized in both of the structures. The catalytic zinc ion is coordinated in a tetrahedral manner with one catalytic water molecule anchoring to an intermediate glutamic acid residue (Glu143) and three imidazole Nepsilon2 atoms of His142, His146 and His152 in the highly conserved sequence H142E143XXH146XXGXXH152. There are two new disulfide bridges (Cys157-Cys181 and Cys159-Cys164) in acutolysin A in addition to the highly conserved disulfide bridge Cys117-Cys197. The calcium ion occurs on the molecular surface. The superposition showed that there was no significant conformational changes between the two structures except for a few slight changes of some flexible residue side-chains on the molecular surface, terminal residues and the active-site cleft. The average contact distance between the catalytic water molecule and oxygen atoms of the Glu143 carboxylate group in the weakly alkaline structure was also found to be closer than that in the weakly acidic structure. By comparing the available structural information of the members of the adamalysin subfamily, it seems that, when lowering the pH value, the polarization capability of the Glu143 carboxylate group to the catalytic water molecule become weaker, which might be the structural reason why the snake venom metalloproteinases are inactive or have a low activity under acidic conditions.
Acids↗
HIV-1 envelope gp120 inhibits the monocyte response to chemokines through CD4 signal-dependent chemokine receptor down-regulation.
Since HIV-1 infection results in severe immunosuppression, and the envelope protein gp120 has been reported to interact with some of the chemokine receptors on human T lymphocytes, we postulated that gp120 may also affect monocyte activation by a variety of chemokines. This study shows that human peripheral blood monocytes when preincubated with gp120 either purified from laboratory-adapted strains or as recombinant proteins exhibited markedly reduced binding, calcium mobilization, and chemotactic response to chemokines. The gp-120-pretreated monocytes also showed a decreased response to FMLP. This broad inhibition of monocyte activation by chemoattractants required interaction of gp120 with CD4, since the effect of gp120 was only observed in CD4+ monocytes and in HEK 293 cells only if cotransfected with both chemokine receptors and an intact CD4, but not a CD4 lacking its cytoplasmic domain. Anti-CD4 mAbs mimicked the effect of gp120, and both anti-CD4 Ab and gp120 caused internalization of CXCR4 in HEK 293 cells provided they also expressed CD4. Staurosporine blocked the inhibitory effect of gp120 on monocytes, suggesting that cellular signaling was required for gp120 to inhibit the response of CD4+ cells to chemoattractants. Our study demonstrates a broad suppressive effect of gp120 on monocyte activation by chemoattractants through the down-regulation of cell surface receptors. Thus, gp120 may be used by HIV-1 to disarm the monocyte response to inflammatory stimulation.
CD4 Antigens↗
HIV-1 envelope gp41 is a potent inhibitor of chemoattractant receptor expression and function in monocytes.
HIV-1 uses CD4 and chemokine receptors as cofactors for cellular entry. The viral envelope transmembrane protein gp41 is thought to participate in viral fusion with CD4(+) cells. We investigated whether gp41 interacts with chemokine receptors on human monocytes by testing its effect on the capacity of cells to respond to chemokine stimulation. Monocytes preincubated with gp41 of the MN strain showed markedly reduced binding, calcium mobilization, and chemotaxis in response to a variety of chemokines as well as to the bacterial peptide fMLP. This generalized inhibition of monocyte activation by chemoattractants required the presence of CD4, since the effect of gp41 was only observed in CD4(+) monocytes and in HEK293 cells cotransfected with chemokine receptors and an intact CD4, but not a CD4 lacking its cytoplasmic domain. Confocal microscopy showed that gp41 caused internalization of CXCR4 in HEK293 cells provided they were also cotransfected with intact CD4. In addition, pretreatment of monocytes with protein kinase C inhibitors partially reversed the inhibitory effect of gp41. Thus, gp41, which had not previously been implicated as interacting with HIV-1 fusion cofactors, downregulates chemoattractant receptors on monocytes by a CD4-dependent pathway.
CD4 Antigens↗
Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor.
The molecular mechanisms by which ovarian hormones stimulate growth of breast tumors are unclear. It has been reported previously that estrogens activate the signal-transducing Src/p21(ras)/Erk pathway in human breast cancer cells via an interaction of estrogen receptor (ER) with c-Src. We now show that progestins stimulate human breast cancer T47D cell proliferation and induce a similar rapid and transient activation of the pathway which, surprisingly, is blocked not only by anti-progestins but also by anti-estrogens. In Cos-7 cells transfected with the B isoform of progesterone receptor (PRB), progestin activation of the MAP kinase pathway depends on co-transfection of ER. A transcriptionally inactive PRB mutant also activates the signaling pathway, demonstrating that this activity is independent of transcriptional effects. PRB does not interact with c-Src but associates via the N-terminal 168 amino acids with ER. This association is required for the signaling pathway activation by progestins. We propose that ER transmits to the Src/p21(ras)/Erk pathway signals received from the agonist-activated PRB. These findings reveal a hitherto unrecognized cross-talk between ovarian hormones which could be crucial for their growth-promoting effects on cancer cells.
Animals↗
Purification and identification of chemokines potentially involved in kidney-specific metastasis by a murine lymphoma variant: induction of migration and NFkappaB activation.
The ESb-MP cell line is the subclone of a highly malignant variant of murine methylcholanthrene-induced T lymphoma, ESb. When injected in vivo, ESb-MP cells metastasize to the kidney with high frequency, whereas a non-adherent variant, ESb cells, rarely form metastatic foci in the kidney. Our previous results showed that ESb-MP, but not ESb, cells were able to migrate in response to murine kidney-conditioned media (KCM). In an effort to characterize the tumor cell chemoattractant(s) produced by kidney cells, we found that the murine kidney mesangial cell line MES-13 released more chemotactic activity for ESb-MP cells than present in KCM. A major heparin-binding chemotactic activity was purified to homogeneity by sequential fast-performance liquid chromatography and reversed phase high-performance liquid chromatography. Amino acid sequencing of the formic acid-digested active fractions revealed that the purified protein was identical to murine MCP-1(JE) and its activity was neutralized by an anti-MCP-1(JE) antibody. Another chemokine, RANTES, was also purified from MES-13 cell supernatant. The chemotactic activity contained in the MES-13 cell supernatant and in murine KCM was neutralized in part by a combination of anti-MCP-1(JE) and anti-RANTES antibodies. We further examined the differences in the ESb-MP and ESb cells. Binding studies using a variety of radio-iodinated chemokines showed that although both ESb-MP and ESb cells expressed substantial levels of high-affinity binding sites for CC chemokines, only ESb-MP cells migrated in response to CC chemokines and these cells constitutively expressed higher levels of beta2 integrin adhesion protein CD11b than their parental ESb cells. CC chemokines also activated NFkappaB in ESb-MP but not in ESb cells. Our results indicate that CC chemokines selectively chemoattract and activate ESb-MP cells. Thus, locally produced chemokines, MCP-1(JE) and RANTES in particular, may contribute to the preferential metastasis of ESb-MP cells to the kidneys.
Animals↗
Monocyte chemotactic protein-2 activates CCR5 and blocks CD4/CCR5-mediated HIV-1 entry/replication.
Human immunodeficiency virus, type I (HIV-1) cell-type tropism is dictated by chemokine receptor usage: T-cell line tropic viruses use CXCR4, whereas monocyte tropic viruses primarily use CCR5 as fusion coreceptors. CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation normal T cell expressed and secreted) inhibit CD4/CCR5-mediated HIV-1 cell fusion. MCP-2 is also a member of the CC chemokine subfamily and has the capacity to interact with at least two receptors including CCR-1 and CCR2B. In an effort to further characterize the binding properties of MCP-2 on leukocytes, we observed that MCP-2, but not MCP-1, effectively competed with MIP-1beta for binding to monocytes, suggesting that MCP-2 may interact with CCR5. As predicted, MCP-2 competitively inhibited MIP-1beta binding to HEK293 cells stably transfected with CCR5 (CCR5/293 cells). MCP-2 also bound to and induced chemotaxis of CCR5/293 cells with a potency comparable with that of MIP-1beta. Confocal microscopy indicates that MCP-2 caused remarkable and dose-dependent internalization of CCR5 in CCR5/293 cells. Furthermore, MCP-2 inhibited the entry/replication of HIV-1ADA in CCR5/293 cells coexpressing CD4. These results indicated that MCP-2 uses CCR5 as one of its functional receptors and is an additional potent natural inhibitor of HIV-1.
CD4 Antigens↗
GABAergic modulation of ventral pallidal dopamine release studied by in vivo microdialysis in the freely moving rat.
The mesopallidal dopamine system, which originates from the ventral tegmental area and projects to the ventral pallidum (VP), has been recently shown to play an important role in self-stimulation reward and cocaine reward. VP also receives a GABAergic projection from nucleus accumbens (NAS). The aim of the present study was to examine the involvement of this GABAergic projection in the modulation of VP dopamine release. Both the GABAA antagonist picrotoxin (2-200 microM) and the GABAB antagonist phaclofen (20-2,000 microM), perfused locally, dose-responsively increased VP extracellular dopamine 2-2.5-fold. Cocaine (10 microM) produced a 6.5-fold increase of VP dopamine. Neither picrotoxin (200 microM), phaclofen (2,000 microM), nor GABA (20-2,000 microM) altered the response of VP dopamine to locally applied cocaine. GBR 12909 (0.5 microM), a selective dopamine uptake blocker, induced a 3.5-fold increase of VP dopamine. The increase of VP dopamine in response to GBR 12909 was further augmented to 8.5-fold of baseline when picrotoxin (200 microM) was added to the perfusate. The data from the present study demonstrate that the GABAergic NAS-VP projection can modulate ventral pallidal dopamine release. However, the effect of GABA on the mesopallidal dopamine system's response to locally applied cocaine may be complicated by actions of cocaine other than dopamine uptake inhibition.
Animals↗
Analysis of the expression pattern of the latent transforming growth factor beta binding protein isoforms in normal and diseased human liver reveals a new splice variant missing the proteinase-sensitive hinge region.
Latent transforming growth factor beta binding protein (LTBP), a component of the extracellular matrix (ECM) of various tissues, is important for the secretion of TGF-beta and, furthermore, for the storage of TGF-beta in ECM. The proteolytic cleavage of LTBP is assumed to be the prerequisite for the activation of TGF-beta. We investigated the mRNA expression pattern of the three LTBP isoforms (LTBP-1, -2, -3) and the protein distribution of the components of the large latent TGF-beta complex, namely LTBP-1 and -2, latency-associated protein (LAP), and TGF-beta, in human liver using reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemical alkaline phosphatase anti-alkaline phosphatase (APAAP) staining. Parts of explanted livers diagnosed as hepatitis B, hepatitis C, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC) and normal liver tissue were examined. LTBP transcripts were detected in the same manner in all liver specimens. Interestingly, we found a new splice variant of LTBP-1 (LTBP-1D), in which the sequence coding for the proteinase-sensitive hinge region is deleted. The corresponding parts of the human LTBP-2 and LTBP-3 cDNA coding for the hinge region were sequenced and show neither similar proteinase cleavage sites nor deleted cDNA sequences. The proposed proteinase cleavage site of mouse LTBP-3 seems not to be conserved in the human LTBP-3 gene. By immunohistochemistry, LTBP-1, -2, and LAP were detectable in normal and diseased livers and showed a different staining pattern for both LTBP isoforms. By contrast, TGF-beta showed a spotted staining pattern in diseased livers only, predominantly in the area of parenchymal cells that are close to fibrotic tissue. This strongly suggests the release of active TGF-beta from preexisting latent complexes. The LTBP-1D splice variant, which is probably less sensitive against proteolytic degradation and therefore may protect TGF-beta from activation, may have importance for modulating the biological activity of TGF-beta in normal and diseased liver.
Amino Acid Sequence↗
Transformation-dependent susceptibility of rat hepatic stellate cells to apoptosis induced by soluble Fas ligand.
Cytokine-driven activation of hepatic stellate cells (HSC) in tissue injury and inflammation is a key pathogenetic event in liver fibrogenesis leading to an expanded pool of matrix producing myofibroblasts (MFB) which represent the transformed counterpart of HSC. We hypothesize that expansion of the pool of MFB might also be accomplished by modulation of apoptosis, which plays an opposite and complementary role to mitosis in the cellular homeostasis. We characterized the susceptibility of HSC in primary culture and of MFB in secondary culture to apoptosis induced by the soluble Fas ligand (sFasL) and related the effects to the expression levels of Fas (APO-1/CD95) and some major proapoptotic and contra-apoptotic protooncogenes. MFB showed a dose-dependent apoptotic reaction upon exposure to sFasL as evidenced by a strong increase of nucleosomal DNA fragments, loss of cellular DNA, positive TUNEL reaction, and annexin staining. The effect was found only if protein synthesis (cycloheximide) or RNA synthesis (actinomycin D) were arrested. HSC maintained for various times in primary culture were completely resistant to sFasL in combination with cycloheximide, but in late primary cultures (day 7 onward) an increasing susceptibility to sFasL-mediated apoptosis was developed. By semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and alkaline phosphatase-anti-alkaline phosphatase staining Fas receptor was identified both in HSC and MFB at comparable expression levels. The expression of the contra-apoptotic protooncogenes bcl-2 and bcl-xl was found to be much stronger in early HSC than in late HSC and MFB as shown by ribonuclease protection assay. The expression of bcl-2 was additionally confirmed by semiquantitative RT-PCR and immunoblotting. Proapoptotic bax was found in comparable quantities at the RNA level in HSC and MFB but at the protein level MFB showed increased bax expression. It is concluded that transformation of HSC to MFB is paralleled by an increasing sensitivity to sFasL-mediated apoptosis, which might be related to a strong decrease of bcl-2 and bcl-xl expression, leading to a preponderance of proapoptotic gene expression in MFB. Modulation of apoptotic susceptibility of transforming HSC could be an important complementary pathway in the pathogenesis of fibrosis.
Animals↗
Chemokines, receptors, and their role in cardiovascular pathology.
A superfamily of leukocyte chemotactic proteins, known as chemokines, has been identified during the past decade. Chemokines selectively attract and activate different leukocyte subpopulations and are key mediators of a variety of patho-physiological states, including hematopoiesis, inflammation, infection, allergy, atheroslerosis, reperfusion injury, as well as malignant tumors. Chemokines bind and activate a number of specific or promiscuous, G-protein-coupled seven-transmembrane receptors. Some of these receptors are utilized by human immuno-deficiency virus type 1 as essential fusion co-factors. Further understanding of the role of chemokines and their receptors in host defense will help develop means by which the beneficial versus detrimental effects of these molecules can be balanced.
Arteriosclerosis↗
Isoforms and splice variant of transforming growth factor beta-binding protein in rat hepatic stellate cells.
BACKGROUND & AIMS: Hepatic stellate cells (HSCs) are one important source for transforming growth factor beta (TGF-beta). They produce TGF-beta in a latent form associated with latency-associated peptide and latent TGF-beta-binding protein (LTBP). This study was designed to investigate, on RNA and protein levels, which isoforms of LTBP and TGF-beta are expressed in HSCs and myofibroblasts. METHODS: HSCs isolated from rat liver were analyzed for LTBP and TGF-beta at various times of culture during transdifferentiation into myofibroblasts using immunocytochemical staining, metabolic labeling and immunoprecipitation, reverse-transcription polymerase chain reaction (RT-PCR), and sequencing. RESULTS: Alkaline phosphatase-anti-alkaline phosphatase staining and fluorescence immunostainings indicated the expression of all three components of the large latent TGF-beta complex in HSCs and myofibroblasts. Transcripts of three TGF-beta and LTBP isoforms were detected by RT-PCR and confirmed by sequence analyses. A new LTBP-1 splice form was found lacking part of the hinge region with a potential proteinase cleavage site. Metabolic labeling followed by immunoprecipitation with LTBP antiserum confirmed the synthesis and secretion of various LTBP-related proteins. CONCLUSIONS: The existence of different LTBP isoforms and splice variants in HSCs and myofibroblasts suggests multiple functions of the LTBP family in rat liver, which might not be restricted to the maintenance of TGF-beta latency.
Amino Acid Sequence↗
Expression of different isoforms of TGF-beta and the latent TGF-beta binding protein (LTBP) by rat Kupffer cells.
BACKGROUND/AIMS: Kupffer cells (liver resident macrophages) make an important contribution to the perpetuation of liver diseases by synthesis and secretion of TGF-beta. In some cell types TGF-beta, is expressed as a large latent complex containing the latent TGF-beta binding protein (LTBP) in addition to the N-terminal TGF-beta precursor (latency associated peptide). This study aimed to identify LTBP expression in rat Kupffer cells. METHODS: Cells were isolated from rat liver by collagenase-pronase reperfusion, purified and cultured under standard conditions. TGF-beta and LTBP expression were characterized using alkaline phosphatase-anti-alkaline phosphatase immunostainings, reverse transcription-polymerase chain reaction and immunoprecipitation of metabolically labeled proteins. RESULTS: Immunostainings of Kupffer cells with anti-sera against LTBP-1 (ab 39) and LTBP-2 indicated the expression of both LTBP isoforms in addition to the expression of latency associated peptide and TGF-beta. Transcripts of three LTBP isoforms (LTBP-1,-2,-3) and TGF-beta isoforms (TGF-beta-1,-2,-3) were detectable by reverse transcription-polymerase chain reaction. The LTBP-1D splice variant missing a part of the proteinase sensitive hinge region which has recently been described in hepatic stellate cells is expressed in Kupffer cells, too. Metabolic labeling of Kupffer cells with [35S]-Met/Cys followed by immunoprecipitation of the conditioned media using antisera against LTBP-1 and LTBP-2 indicated the secretion of high molecular mass TGF-beta complexes containing LTBP proteins of 230 and 170 kDa (LTBP-1) or 230 kDa (LTBP-2). CONCLUSION: The results show that Kupffer cells partly synthesize and release TGF-beta as large latent complexes. This requires the extracellular activation of TGF-beta as a prerequisite for receptor binding and cellular signaling.
Animals↗
[Preliminary report of xenogenic bone graft for sixty-five cases].
From January 1984 to November 1997, the boiling xenogenic bone (porcine and bovine bone) was applied in 65 patients. The xenogenic bone was used to promote bone healing in 24 cases, intramedullary bone graft in 37 and osteomyelitis in 4. All of the patients were followed up for 2 to 35 months. The results showed that in five cases there was infection after operation, and all other the wounds had primary healing. The xenogenic bone seemed to induce rejection in vivo. In the sevious cases immunosuppressive treatment was often needed. The volume of the bone grafted and the extent of the periosteum being stripped seemed to be important whether xenogenic bone graft would be successful or. There were lots of problem needed investigation.
Adolescent↗
[A new approach to correction method for matrix effect in X-ray fluorescence spectroscopy analysis].
With the help of fundamental parameter program developed by the authors, the matrix effect of multicomponent complex sample and the composition of each element's X-ray fluorescence intensity are calculated. A new mathematical model for correcting multicomponent matrix effect is deduced theoretically. This model provides a new method to calculate influence coefficients alpha and beta. The correction model and the influence coefficients introduced by this paper have clear and definite physical meaning, which give satisfactory result when utilized in analyzing stainless steel sample. The paper also indicates that the effect of tertiary fluorescence can not be neglected in some cases. From the result of comparison experiments, we draw a conclusion that the tertiary fluorescence effect must be considered while analyzing Cr in a steel sample with high concentration of Ni.
English Abstract↗
[Determination of the valence of iron by the conventional XRF spectrometer].
FeKbeta emission spectra and parameters of mixtures are studied by conventional XRF spectrometer and PEAKFIT programs. The true spectra profile and it's parameters are obtained by profile resolving method. Integral area ratios are also calculated. The relations of these parameters and the valences of the iron are found. Fe3O4 sample and the iron ore which has different valences are quantified.
English Abstract↗