S100A12 is expressed exclusively by granulocytes and acts independently from MRP8 and MRP14.

Changes in cytosolic calcium concentrations regulate a wide variety of cellular processes, and calcium-binding proteins are the key molecules in signal transduction, differentiation, and cell cycle control. S100A12, a recently described member of the S100 protein family, has been shown to be coexpressed in granulocytes and monocytes together with two other S100 proteins, MRP8 (S100A8) and MRP14 (S100A9), and a functional relationship between these three S100 proteins has been suggested. Using Western blotting, calcium overlays, intracellular flow cytometry, and cytospin preparations, we demonstrate that S100A12 expression in leukocytes is specifically restricted to granulocytes and that S100A12 represents one of the major calcium-binding proteins in these cells. S100A12, MRP8, and MRP14 translocate simultaneously from the cytosol to cytoskeletal and membrane structures in a calcium-dependent manner. However, no evidence for direct protein-protein interactions of S100A12 with either MRP8 or MRP14 or the heterodimer was found by chemical cross-linking, density gradient centrifugation, mass spectrometric measurements, or yeast two hybrid detection. Thus, S100A12 acts individually during calcium-dependent signaling, independent of MRP8, MRP14, and the heterodimer MRP8/MRP14. This granulocyte-specific signal transduction pathway may offer attractive targets for therapeutic intervention with exaggerated granulocyte activity in pathological states.     

Adhesion of MRP8/14 to amastigotes in skin lesions of Leishmania major-infected mice

In murine experimental cutaneous leishmaniasis, parasite infection induces an accumulation of macrophages expressing migration inhibitory factor-related protein 8 (MRP8) and MRP14, two members of the S100 calcium-binding protein family. Although MRP8 and MRP14 are cytoplasmic proteins expressed by myeloid cells, recent studies have demonstrated that MRP8 and MRP14 have extracellular functions such as chemotactic activities. In this study, we examined whether extracellular MRP8 and MRP14 interact with Leishmania parasites during infection. By immunohistochemistry, positive staining by MRP8 and MRP14 was detected on amastigotes in skin lesions of Leishmania major-infected mice. Western blot analysis with amastigotes purified from the skin lesions demonstrated that both of these proteins adhered to amastigotes. The adhesion of MRP14 to amastigotes was reproduced in vitro and enhanced in the presence of Ca2+ and Zn2+. MRP14 adhered to not only amastigotes, but also promastigotes, suggesting receptor molecules for MRP14 are expressed commonly in both developmental stages.     

Characterization of the tissue-specific expression of the S100P gene which encodes an EF-hand Ca2+-binding protein*

S100 proteins are a calcium-binding protein family containing two EF-hand domains exclusively expressed in vertebrates and play roles in many cellular activities. Human S100P gene was first cloned as a 439 bp cDNA in placenta and it was found to be associated with human prostate cancer. Here we describe the cloning of the 1297 bp full-length cDNA, and the characterization of the tissue-specific expression of the human S100P gene. It is abundantly expressed in many tissues including placenta by Northern blot and RT-PCR analysis, unlike the expression pattern of other S100 family genes.     

Calprotectin - a pleiotropic molecule in acute and chronic inflammation

Calprotectin (MRP8/14, S100A8/S100A9, 27E10 antigen) is a heterodimer of two calcium-binding proteins present in the cytoplasm of neutrophils and expressed on the membrane of monocytes. Upon neutrophil activation or endothelial adhesion of monocytes, calprotectin is released and may be detected in serum or body fluids as potentially useful clinical inflammatory marker. The soluble form of calprotectin provides both bacteriostatic and cytokine-like effects in the local environment. When calprotectin metabolism is affected on a systemic level, the zinc-binding properties of protein may induce severe dysregulation of zinc homeostasis with severe clinical symptoms. The distribution of membrane form of calprotectin is restricted to monocytes and immature macrophages and the presence of calprotectin-positive infiltrating cells reflects the influx of mononuclear phagocytes to the site of inflammation. Calprotectin expression and release seems to be of particular importance in immune and immunopathological reactions.     

S100A8/S100A9 and their association with cartilage and bone

S100A8 and S100A9 are calcium-binding proteins expressed in myeloid cells and are markers of numerous inflammatory diseases in humans. S100A9 has been associated with dystrophic calcification in human atherosclerosis. Here we demonstrate S100A8 and S100A9 expression in murine and human bone and cartilage cells. Only S100A8 was seen in preosteogenic cells whereas osteoblasts had variable, but generally weak expression of both proteins. In keeping with their reported high-mRNA expression, S100A8 and S100A9 were prominent in osteoclasts. S100A8 was expressed in alkaline phosphatase-positive hypertrophic chondrocytes, but not in proliferating chondrocytes within the growth plate where the cartilaginous matrix was calcifying. S100A9 was only evident in the invading vascular osteogenic tissue penetrating the degenerating chondrocytic zone adjacent to the primary spongiosa, where S100A8 was also expressed. Whilst, S100A8 has been shown to be associated with osteoblast differentiation, both S100A8 and S100A9 may contribute to calcification of the cartilage matrix and its replacement with trabecular bone, and to regulation of redox in bone resorption.     

Changing the conformation state of cytochrome b558 initiates NADPH oxidase activation: MRP8/MRP14 regulation

Phagocyte NADPH oxidase generates O2. for defense mechanisms and cellular signaling. Myeloid-related proteins MRP8 and MRP14 of the S100 family are EF-hand calcium-binding proteins. MRP8 and MRP14 were co-isolated from neutrophils on an anti-p47phox matrix with oxidase cytosolic factors and identified by mass spectrometry. MRP8 and MRP14 are absent from Epstein-Barr virus-immortalized B lymphocytes, and, coincidentally, these cells display weak oxidase activity compared with neutrophils. MRP8/MRP14 that was purified from neutrophils enhanced oxidase turnover of B cells in vitro, suggesting that MRP8/MRP14 is involved in the activation process. This was confirmed ex vivo by co-transfection of Epstein-Barr virus-transformed B lymphocytes with genes encoding MRP8 and MRP14. In a semi-recombinant cell-free assay, recombinant MRP8/MRP14 increased the affinity of p67phox for cytochrome b558 synergistically with p47phox. Moreover, MRP8/MRP14 initiated oxidase activation on its own, through a calcium-dependent specific interaction with cytochrome b558 as shown by atomic force microscopy and a structure-function relationship investigation. The data suggest that the change of conformation in cytochrome b558, which initiates the electron transfer, can be mediated by effectors other than oxidase cytosolic factors p67phox and p47phox. Moreover, MRP8/MRP14 dimer behaves as a positive mediator of phagocyte NADPH oxidase regulation.     

Copurification of P6, MRP8, and MRP14 from Human Granulocytes and Separation of Individual Proteins

A method is described for purification of P6, MRP8, and MRP14, three calcium-binding proteins assigned to the S100 protein family. The purification procedure included preparation of human granulocytes, ammonium sulfate precipitation, and anion-exchange chromatography and resulted in the copurification of P6, MRP8, and MRP14. Individual proteins were separated by either preparative isoelectric focusing or preparative SDS–PAGE. The procedure was carried out in the course of 4 days and yielded several milligrams of essentially pure P6, MRP8, and MRP14 in either native or denatured form.     

Accumulation of macrophages expressing MRP8 and MRP14 in skin lesions during Leishmania major infection in BALB/c and RAG-2 knockout mice

Migration inhibitory factor-related protein 8 (MRP8) and MRP14 are expressed by myeloid cells and especially known as marker proteins of an immature and inflammatory subtype of macrophages. In this study, we immunohistochemically examined an accumulation of MRP8+ and MRP14+ macrophages in skin lesions during Leishmania major infection in susceptible BALB/c and RAG-2-/- mice. L. major infection caused the development of a nodular type of skin lesion at the infection site in mice and a massive accumulation of macrophages was observed in the lesions at four weeks after the infection. Immunohistochemical analyses showed MRP8+ and MRP14+ macrophages are predominant cell types in the skin lesions in both mouse strains. In contrast, F4/80+ cells, which correspond to mature macrophages, were rarely found in the skin lesions. These data suggest that the accumulation of inflammatory subtype of macrophages in BALB/c mice during L. major infection can be induced without acquired immune responses.     

DMSO and retinoic acid induce HL-60 differentiation by different but converging pathways

The expression of c-myc and two calcium-binding proteins, MRP8 and MRP14, has been analyzed in wild-type and differentiation-resistant HL-60 variants. In HL-R5 cells, resistant to the induction of differentiation by retinoic acid but not DMSO, the characteristic c-myc down-regulation which is associated with HL-60 differentiation, as well as increased levels of MRP8 and MRP14, is detectable only after DMSO treatment. By contrast HL-D4 cells, which were selected for resistance to the induction of differentiation by DMSO alone, are actually resistant to both DMSO and retinoic acid. However, treatment of HL-D4 cells with DMSO results in a transient c-myc down-regulation in the absence of either growth arrest or induction of differentiation. Neither agent can induce an increase in the level of either MRP8 or MRP14 in HL-D4. The resistance of HL-D4 cells to DMSO and retinoic acid, and the different effects of these agents on c-myc RNA levels, despite their common effect on the expression of MRP8 and MRP14, suggest that the two agents act through different pathways which coverage before the onset of myeloid differentiation in HL-60 cells.     

Identification of potential protein regulators bound to the tissue-specific positive and negative cis-acting elements of a green tissue-specific promoter in rice

Although some tissue-specific cis-acting elements have been identified, the molecular mechanisms of tissue-specific gene expression remain elusive. Here, we report the identification by a yeast one-hybrid screen of five proteins, Os10g31330/glycine-rich, Os01g10400/metallothionein-like, Os05g51180/nucleic acid-binding, Os05g37930/unknown and Os01g01689/phosphatidylinositol kinase that bound to either the positive or negative tissue-specific cis elements of a rice promoter from the green tissue-specific D54O gene. These proteins are localised in the nucleus and the genes encoding them are differentially expressed in different tissues, further suggesting their putative roles in regulating gene expression. These results suggest that the green tissue-specific expression of the D54O gene may be regulated by the interaction of multiple proteins with cis elements in the promoter region.     

Loss of S100A9 (MRP14) results in reduced interleukin-8-induced CD11b surface expression,a polarized microfilament system,and diminished responsiveness to chemoattractants in vitro

The S100A9 (MRP14) protein is abundantly expressed in myeloid cells and has been associated with various inflammatory diseases. The S100A9-deficient mice described here were viable, fertile, and generally of healthy appearance. The myelopoietic potential of the S100A9-null bone marrow was normal. S100A8, the heterodimerization partner of S100A9 was not detectable in peripheral blood cells, suggesting that even a deficiency in both S100A8 and S100A9 proteins was compatible with viable and mature neutrophils. Surprisingly, the invasion of S100A9-deficient leukocytes into the peritoneum and into the skin in vivo was indistinguishable from that in wild-type mice. However, stimulation of S100A9-deficient neutrophils with interleukin-8 in vitro failed to provoke an up-regulation of CD11b. Migration upon a chemotactic stimulus through an endothelial monolayer was markedly diminished in S100A9-deficient neutrophils. Attenuated chemokinesis of the S100A9-deficient neutrophils was observed by using a three-dimensional collagen matrix migration assay. The altered migratory behavior was associated with a microfilament system that was highly polarized in unstimulated S100A9-deficient neutrophils. Our data suggest that loss of the calcium-binding S100A9 protein reduces the responsiveness of the neutrophils upon chemoattractant stimuli at least in vitro. Alternative pathways for neutrophil emigration may be responsible for the lack of any effect in the two in vivo models we have investigated so far.     

The role of calcium in the conformational changes of the recombinant S100A8/S100A91

Calprotectin is a member of the EF-hand proteins, composed of two subunits, S100A8 (MRP8) and S100A9 (MRP14). These proteins are involved in important processes including cell signaling, regulation of inflammatory responses, cell cycle control, differentiation, regulation of ion channel activity and defense against microbial agents in a calcium dependent manner. In the present study, recombinant S100A8 and S100A9 were expressed in E. coli BL21 and then purified using Ni-NTA affinity chromatography. The structure of the S100A8/A9 complex in the presence and absence of calcium was assessed by circular dichroism and fluorescence spectroscopy. The intrinsic fluorescence emission spectra of the S100A8/A9 complex in the presence of calcium showed a reduction in fluorescence intensity, reflecting conformational changes within the protein with the exposure of aromatic residues to the protein surface. The far ultraviolet-circular dichroism spectra of the complex in the presence of calcium revealed minor changes in the regular secondary structure of the complex. Also, increased thermal stability of the S100A8/A9 complex in the presence of calcium was indicated.     

Tissue-specific and hormonally regulated expression of a rat alpha 2u globulin gene in transgenic mice.

To investigate the tissue-specific and hormonal regulation of the rat alpha 2u globulin gene family, we introduced one cloned member of the gene family into the mouse germ line and studied its expression in the resulting transgenic mice. Alpha 2u globulingene 207 was microinjected on a 7-kilobase DNA fragment, and four transgenic lines were analyzed. The transgene was expressed at very high levels, specifically in the liver and the preputial gland of adult male mice. The expression in male liver was first detected at puberty, and no expression was detected in female transgenic mice. This pattern of expression is similar to the expression of endogenous alpha 2u globulin genes in the rat but differs from the expression of the homologous mouse major urinary protein (MUP) gene family in that MUPs are synthesized in female liver and not in the male preputial gland. We conclude that these differences between rat alpha 2u globulin and mouse MUP gene expression are due to evolutionary differences in cis-acting regulatory elements. The expression of the alpha 2u globulin transgene in the liver was abolished by castration and fully restored after testosterone replacement. The expression could also be induced in the livers of female mice by treatment with either testosterone or dexamethasone, following ovariectomy and adrenalectomy. Therefore, the cis-acting elements responsible for regulation by these two hormones, as well as those responsible for tissue-specific expression, are closely linked to the alpha 2u globulin gene.     

A comparison of human S100A12 with MRP-14 (S100A9)

MRP-8 and -14 are two S100 proteins highly expressed as a complex by neutrophils, and to a lesser extent by monocytes and certain squamous epithelia. However, less is known about the close homologue S100A12. This S100 protein is expressed by neutrophils and here we show that it is also expressed by monocytes, but not lymphocytes. An absence of coimmunoprecipitation of MRP-14 and S100A12 indicates that S100A12 is not associated with the MRP proteins in vivo. When directly compared to MRP-14, S100A12 expression by squamous epithelia is more restricted. In esophagus and psoriatic skin, S100A12 is differentially regulated, like MRP-14, but the expression pattern of the two S100 proteins is quite different.     

Calprotectin (MRP8/14 protein complex) release during mycobacterial infection in vitro and in vivo

The calprotectin (MRP8/14) protein complex belongs to the S100 family of Ca2+ binding proteins and is expressed during myelomonocytic differentiation. MRP8/14 plasma levels were determined by ELISA in 35 patients with active pulmonary tuberculosis (TB) showing mild (n = 12), moderate (n = 11) or severe (n = 12) disease, 13 patients with active pulmonary sarcoidosis (SR) and 21 healthy controls. TB patients had significantly increased plasma levels of MRP8/14 in comparison with SR and controls, which significantly depended on the volume of lung tissue involved in the inflammatory process. In TB patients, there was no correlation between plasma levels of MRP8/14 and total white blood cell (WBC) count, and blood polymorphonuclear neutrophil (PMN) count. In SR patients, MRP8/14 plasma levels were twofold higher in comparison with controls, but were lower compared with mild TB, and correlated with PMN and WBC counts. Human monocytes infected and cultured for 7 days with Mycobacterium bovis bacillus Calmette-Guérin showed fivefold higher MRP8/14 levels in supernatants compared with unstimulated or purified protein derivative-stimulated cells. Human MRP8/14 significantly increased Mycobacterium tuberculosis H37Rv growth in liquid medium in a dose- and time-dependent manner. These findings suggest that MRP8/14 plays an important role in the immunopathogenesis of tuberculosis.     

毛竹萜类合成酶基因家族序列鉴定与表达分析

通过生物信息学方法,对毛竹(Phyllostachys edulis(Carrière)J.Houzeau)TPS基因家族的成员进行鉴定,并对其编码蛋白的理化性质、基因结构、进化关系、蛋白结构、启动子元件及表达模式进行了分析。结果表明,毛竹全基因组含有14个TPS候选基因,大小为693~2439 bp。编码蛋白等电点为5.08~8.17。系统发育分析结果显示,毛竹含有TPS-a、TPS-b、TPS-e/f、和TPS-g 4个亚家族,成员数目分别为6、5、2、1个。TPS蛋白质二级结构中,α-螺旋和无规则卷曲所占比重较大;毛竹TPS基因家族各成员蛋白三维结构比较相似。基因启动子分析共获得50个调控元件,可分为6大类,其中光响应相关元件数量最多,共包含17个顺式调控元件。基于转录组测序数据构建的基因表达谱热图分析结果表明,Pe TPS在叶、花和笋等7个组织中的表达差异明显,表现出组织特异性,其中Pe TPS9仅在早花期花序中表达,Pe TPS8仅在叶中表达。