Bone enhancing effect of titanium-binding proteins isolated from bovine bone and implanted into rat calvaria with titanium scaffold.

Based on our previous finding that a chromatography with titanium beads selectively binds phosphoproteins, including caseins, phosvitin and dentin phosphoproteins, we investigated whether bone phosphoproteins also bind to titanium. Bovine bone matrix proteins were extracted with 2 M urea/PBS after demineralization. The 2 M urea extract was directly applied to the titanium chromatography column as reported. The chromatogram showed an initial large peak at breakthrough position (non-binding fraction) and a smaller second peak eluted later (titanium-binding fraction). Both peaks were analyzed by SDS polyacrylamide gel electrophoresis. Stains-all staining which preferentially identifies phospho-proteins revealed that the first peak contained no positively stained band, while the second peak showed 4 or 5 distinctive bands indicative of bone phosphoproteins. To investigate the biological functions of the titanium-binding bone proteins (TiBP), we implanted them into calvaria of rats, combined with titanium web (TW), a highly porous titanium scaffold of thin titanium-fibers. Bone TiBP induced significantly enhanced bone formation, and new bone appeared connected directly to titanium fibers, accompanied by active blood vessel formations. Control TW alone did not induce bone formation within the titanium framework. These results demonstrate that the bone titanium-binding proteins include phosphoproteins which enhance bone formation when implanted into bone with titanium.

Interaction between titanium and phosphoproteins revealed by chromatography column packed with titanium beads.

The biochemical mechanism behind the strong binding between titanium and living bone has not been fully elucidated, in spite of worldwide clinical application of this phenomenon. We hypothesized that one of the core mechanisms may reside in the interaction between certain proteins in the host tissues and the implanted titanium. To verify the interaction between titanium and proteins, we chose the technique of chromatography in that titanium spherical beads (45 µm) were packed into a column to obtain a bed volume of 16×50 mm, which was eluted with phosphate buffered saline (PBS) and a straight gradient system made by using PBS and 25 mM NaOH. Fetal calf serum, albumin, lysozyme, casein, phosvitin and dentin phosphoprotein (phosphophoryn) were applied to the column. Most part of albumin and lysozyme eluted with the breakthrough peak, indicating practically no affinity to titanium. Fetal bovine serum also eluted mostly as the breakthrough peak, but distinct retained peak was observed. On the other hand, α-casein, phosvitin and phosphophoryn exhibited a distinct retained peak separated from the breakthrough peak. We proposed that phosphate groups (phosphoserines) in the major phosphoproteins, α-casein, phosvitin and phosphophoryn may be involved in the binding of these proteins with titanium.

Acidic bone matrix proteins and their roles in calcification.

Mammalian bones are composed of calcium phosphate crystals in a protein matrix. The major form of the calcium phosphate is hydroxyapatite. The most abundant matrix protein in bone is type I collagen. Collagen contributes to the mechanical properties of bone and is necessary for calcification of the tissue. In addition to collagen, several acidic proteins are present as minor components. Osteocalcin is a gamma-carboxyglutamic acid-containing protein of bone, which has an affinity to hydroxyapatite and can prevent crystal growth. Bone sialoprotein (BSP) and osteopontin are acidic glycophosphoproteins of bone. These proteins have RGD cell-attachment sequences and consecutive sequences of acidic amino acids. The poly glutamic acid sequences of BSP act as possible nucleation sites for hydroxyapatite crystals. Dentin phosphoprotein is the major non-collagenous protein of dentin. This protein has (Asp-Ser-Ser) repeat sequences, in which most of the Ser residues are phosphorylated. Some of these acidic matrix proteins are immobilized on the collagen fibrils and induce nucleation of hydroxyapatite crystals. They can also modulate crystal shape by adsorption on a specific face of the crystals.

Chromatography of carbon nanotubes separated albumin from other serum proteins: a method for direct analysis of their interactions.

Chromatography technology was employed to clarify the mechanism of interaction between multi-wall carbon nanotubes (MWCNT) and proteins. A column (16x100 mm) was packed with purified MWCNT, and various proteins were eluted with phosphate buffered saline (PBS) with and without gradient systems. It was found that albumin in bovine serum was eluted immediately from the column without any adsorption to MWCNT. Conversely, the non-albumin proteins, including a protein of 85 kDa molecular mass and a group of proteins with molecular masses higher than 115 kDa, exhibited considerably high affinity towards MWCNT. A sample of pure bovine serum albumin was also eluted immediately from the column, while lysozyme did not elute as a peak with PBS, but eluted with 0.6 M NaCl. Fundamentally, carbon nanotubes are devoid of any electrical charge. Therefore, other forces including the hydrogen bonds, hydrophilic interactions, and van der Waals forces were most probably responsible for the differential elution behaviors. In conclusion, this chromatographic method provided a simple and direct analysis of the interactions between carbon nanotubes and the various proteins.

Host cell-specific effects of lentiviral accessory proteins on the eukaryotic cell cycle progression.

Lentiviral accessory proteins are thought to play important roles in regulating the viral replication through modulation of host cell functions. For example, Vpr of human immunodeficiency virus type 1 (HIV-1) induces the cell cycle G2 arrest in a host cell-specific manner. Similarly, HIV-2 Vpr, but not Vpx, has been shown to induce G2 arrest in primate cells. It has also been reported that Orf-A of feline immunodeficiency virus (FIV) induces G2 arrest in a simian cell line. However, activities of these non-HIV-1 accessory proteins in different cellular context are unclear. In this study, effects of HIV-2 Vpr, Vpx and FIV Orf-A on cell cycle progression were compared with those of HIV-1 Vpr in various mammalian cell lines and the fission yeast. These non-HIV-1 accessory proteins induced the cell cycle arrest in a host cell-specific manner, and their specificities were different from each other. Interestingly, HIV-2 Vpx-induced G2 arrest in bovine MDBK cells. It was also notable that HIV-2 Vpx and FIV Orf-A appeared to block the cell separation in the fission yeast. The host cell-specific activities of different lentiviral accessory proteins revealed in this study may provide a useful basis for elucidating the mechanism of their functions.

Effect of dentin phosphoprotein on phosphate-induced apoptosis of odontoblast-like cells.

Dentin phosphoprotein, the major noncollagenous protein in dentin, has effects on differentiation of odontoblast-like cells. This study was designed to investigate the effect of the protein on apoptosis of the cells. The odontoblast-like cells were prepared from the pulp cells of rat incisors. Apoptosis was detected by measuring caspase-3 activity by using DEVD-AMC as a fluorescent substrate. The cells formed calcification nodules in the presence of 2-glycerophosphate and expressed dentin sialophosphoprotein. Apoptosis was not observed in the cells through the differentiation stages. Then, apoptosis was induced by raising inorganic phosphate concentration in the medium. Elevation of phosphate concentration to 5 mM reduced the number of viable cells and increased caspase-3 activity, indicating the induction of apoptosis. Addition of bovine dentin phosphoprotein in the medium suppressed phosphate-induced apoptosis. Phosvitin and poly(Asp) had similar antiapoptotic effects.

Ecotropic murine leukemia virus envelope protein affects interaction of cationic amino acid transporter 1 with clathrin adaptor protein complexes, leading to receptor downregulation.

Mouse cationic amino acid transporter 1 (mCAT1) serves as the receptor for ecotropic murine leukemia virus (eMuLV). It has been shown that mCAT1 is expressed on the basolateral surface of polarized epithelial MDCK cells. However, little is known about the mechanisms involved in the intracellular trafficking of mCAT1. Using the green fluorescent protein-tagged mCAT1 expressed in MDCK cells, we report here that mCAT1 is physically associated with clathrin adaptor protein complex 1 (AP-1) implicated in protein trafficking from trans-Golgi network (TGN) to the basolateral surface. When the cells were infected with eMuLV, reduction of cell surface mCAT1, as well as a concomitant decrease in mCAT1-AP-1 association, was observed while association of mCAT1 with AP-3 involved in the TGN-to-lysosome trafficking was increased. Similar results were obtained when eMuLV envelope protein alone was expressed. The results may provide useful insights into the mechanism by which a simple retrovirus downregulates its receptor.

Cell cycle arrest induction by an adenoviral vector expressing HIV-1 Vpr in bovine and feline cells.

An accessory protein, Vpr, of human immunodeficiency virus type 1 (HIV-1) induces the cell cycle G(2)/M arrest in primate cells, but not in rodent cells, suggesting that a species-specific factor might be involved in the phenomenon. To study whether Vpr can cause G(2)/M arrest in non-primate cells, a novel adenoviral vector, Ad-VIG, co-expressing HIV-1 Vpr and green fluorescent protein (GFP) was constructed and infected on cell lines derived from various mammalian species. With its ability to express GFP, Ad-VIG enabled flow cytometric evaluation of transduction efficiency in the infected cells, and Western blot analysis showed successful expression of Vpr in the vector-transduced cells. Upon Ad-VIG infection, human HeLa, African green monkey Vero, feline CRFK, and bovine MDBK cells manifested cell cycle G(2)/M arrest. This is the first study showing that non-primate feline and bovine cells are susceptible to Vpr-induced cell cycle arrest.

IFN-gamma-inducible expression of thymus and activation-regulated chemokine/CCL17 and macrophage-derived chemokine/CCL22 in epidermal keratinocytes and their roles in atopic dermatitis.

Thymus and activation-regulated chemokine (TARC)/CCL17 and macrophage-derived chemokine (MDC)/CCL22 are a pair of CC chemokines known to selectively attract T(h)2 type memory T cells via CCR4. Here we examined circulating levels of TARC and MDC in patients with atopic dermatitis (AD) and control subjects by using plasma samples, which reflect blood contents of chemokines more accurately than serum samples. The plasma levels of TARC and MDC were significantly elevated in AD patients. These values also strongly correlated with disease severity and serum lactate dehydrogenase levels, and weakly correlated with serum total IgE levels and blood eosinophilia. Previous studies demonstrated TARC immunoreactivity in the epidermal layer of AD lesional skin and production of TARC by a human keratinocytic cell line HaCaT upon stimulation with IFN-gamma. Here we demonstrated MDC immunoreactivity in the epidermal layer of AD skin at levels stronger than that of TARC. Furthermore, primary epidermal keratinocytes expressed both TARC and MDC mRNA upon stimulation with IFN-gamma, but efficiently secreted only MDC. These results suggest a post-transcriptional regulation in TARC production. IFN-gamma also induced TARC and MDC mRNA in mouse skin. Collectively, both TARC and MDC play important roles in the local accumulation of T(h)2 cells in AD lesional skin. Production of T(h)2-attracting chemokines by epidermal keratinocytes upon treatment with IFN-gamma, which is also the potent inducer of T(h)1-attracting chemokines, may underline the pivotal role of IFN-gamma in the chronic phase of AD where both T(h)1 and T(h)2 responses are mixed.

Presence of high contents of thymus and activation-regulated chemokine in platelets and elevated plasma levels of thymus and activation-regulated chemokine and macrophage-derived chemokine in patients with atopic dermatitis.

BACKGROUND: T(H)2 cells and eosinophils selectively express CC chemokine receptor 4 and CCR3, respectively, and their chemokine ligands are likely to play important roles in the pathogenesis of atopic dermatitis (AD). OBJECTIVE: The purpose of this study was to demonstrate the presence of thymus and activation-regulated chemokine (TARC) in platelets and its release during clotting and to evaluate the circulating levels of TARC, macrophage-derived chemokine (MDC), and eotaxin in control subjects and patients with AD. METHODS: We compared plasma and serum contents of TARC, MDC, and eotaxin. We measured TARC contents in platelet lysates. We analyzed the correlation of plasma levels of TARC, MDC, and eotaxin with various clinicolaboratory parameters in patients with AD. RESULTS: Serum contents of TARC rapidly increased during clotting, whereas those of MDC and eotaxin increased only slightly. We demonstrated that platelets contained TARC, and its levels were dramatically elevated in patients with AD. Platelets also released TARC on stimulation with thrombin. We therefore evaluated circulating levels of these chemokines in control subjects and patients with AD by using plasma samples. Plasma TARC levels were significantly increased in patients with AD (P <.0001) and showed significant correlations with severity scoring of atopic dermatitis (SCORAD) index (r = 0.665, P <.00001), serum lactate dehydrogenese levels (r = 0.696, P =.00001), eosinophil counts (r = 0.381, P =.007), and platelet counts (r = 0.562, P <.0001). Similarly, plasma MDC levels were significantly increased in patients with AD (P <.0001) and showed significant correlations with SCORAD index (r = 0.727, P <.0001), serum lactate dehydrogenese levels (r = 0.861, P <.0001), eosinophil counts (r = 0.505, P =.005), and platelet counts (r = 0.370, P =.01). On treatment, plasma TARC and MDC levels were dramatically decreased in accordance with improved SCORAD scores (P =.0012 and P =.0007, respectively). On the other hand, plasma eotaxin levels did not show any significant increase or correlation with any of the clinical parameters in patients with AD. CONCLUSION: Platelets from patients with AD contain high levels of TARC. Thus platelets might play an important role in AD pathogenesis by releasing T(H)2-attracting TARC on activation. Furthermore, circulating levels of TARC and MDC, but not those of eotaxin, correlate well with the disease activity of AD.

Frequent expression of CCR4 in adult T-cell leukemia and human T-cell leukemia virus type 1-transformed T cells.

Chemokines and chemokine receptors play important roles in migration and tissue localization of various lymphocyte subsets. Here, we report the highly frequent expression of CCR4 in adult T-cell leukemia (ATL) and human T-cell leukemia virus type 1 (HTLV-1)-immortalized T cells. Flow cytometric analysis revealed that ATL and HTLV-1-immortalized T-cell lines consistently expressed CCR4. Inducible expression of HTLV-1 transcriptional activator tax in a human T-cell line Jurkat did not, however, up-regulate CCR4 mRNA. In vitro immortalization of peripheral blood T cells led to preferential outgrowth of CD4(+) T cells expressing CCR4. We further demonstrated highly frequent expression of CCR4 in fresh ATL cells by (1) reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of CCR4 expression in peripheral blood mononuclear cells (PBMCs) from patients with ATL and healthy controls; (2) flow cytometric analysis of CCR4-expressing cells in PBMCs from patients with ATL and healthy controls; (3) CCR4 staining of routine blood smears from patients with ATL; and (4) an efficient migration of fresh ATL cells to the CCR4 ligands, TARC/CCL17 and MDC/CCL22, in chemotaxis assays. Furthermore, we detected strong signals for CCR4, TARC, and MDC in ATL skin lesions by RT-PCR. Collectively, most ATL cases have apparently derived from CD4(+) T cells expressing CCR4. It is now known that circulating CCR4(+) T cells are mostly polarized to Th2 and also contain essentially all skin-seeking memory T cells. Thus, HTLV-1-infected CCR4(+) T cells may have growth advantages by deviating host immune responses to Th2. CCR4 expression may also account for frequent infiltration of ATL into tissues such as skin and lymph nodes.

Human B cells immortalized with Epstein-Barr virus upregulate CCR6 and CCR10 and downregulate CXCR4 and CXCR5.

Compared to peripheral blood resting B cells, Epstein-Barr virus (EBV)-immortalized B cells consistently express CCR6 and CCR10 at high levels and CXCR4 and CXCR5 at low levels. Accordingly, these cells vigorously responded to the ligands of CCR6 and CCR10 but not to those of CXCR4 and CXCR5. In a human EBV-negative B-cell line, BJAB, stable expression of EBNA2 upregulated CCR6, while stable expression of EBNA2 as well as LMP1 downregulated CXCR4. On the other hand, upregulation of CCR10 or downregulation of CXCR5 was not induced in BJAB by stable expression of EBNA2 or LMP1. Thus, these changes may be due to a plasmablast-like stage of B-cell differentiation fixed by EBV immortalization. EBV-infected B cells in infectious mononucleosis are known to avoid germinal centers and accumulate under the mucosal surfaces. EBV-associated opportunistic lymphomas also tend to occur in extranodal sites. These preferred sites of in vivo localization are consistent with the unique profile of chemokine receptor expression exhibited by EBV-immortalized B cells.