Tissue inhibitor of metalloproteinase gene from pearl oyster Pinctada martensii participates in nacre formation

Tissue inhibitors of metalloproteinases (TIMPs) are nature inhibitors of matrix metalloproteinases and play a vital role in the regulation of extracellular matrix turnover, tissue remodeling and bone formation. In this study, the molecular characterization of TIMP and its potential function in nacre formation was described in pearl oyster Pinctada martensii. The cDNA of TIMP gene in P. martensii (Pm-TIMP) was 901 bp long, containing a 5′ untranslated region (UTR) of 51 bp, a 3′ UTR of 169 bp, and an open reading fragment (ORF) of 681 bp encoding 226 amino acids with an estimated molecular mass of 23.37 kDa and a theoretical isoelectric point of 5.42; The predicted amino acid sequence had a signal peptide, 13 cysteine residues, a N-terminal domain and a C-terminal domain, similar to that from other species. Amino acid multiple alignment showed Pm-TIMP had the highest (41%) identity to that from Crassostrea gigas. Tissue expression analysis indicated Pm-TIMP was highly expressed in nacre formation related-tissues, including mantle and pearl sac. After decreasing Pm-TIMP gene expression by RNA interference (RNAi) technology in the mantle pallium, the inner nacreous layer of the shells showed a disordered growth. These results indicated that the obtained Pm-TIMP in this study participated in nacre formation.     

Dermatopontin, a shell matrix protein gene from pearl oyster Pinctada martensii, participates in nacre formation

Dermatopontin (DPT) is identified as a major component of the shell matrix protein. However, its exact function in the shell formation remains obscure. In this study, we described the characteristic and function of DPT gene from Pinctada martensii. DPT cDNA was 797bp long, containing an open reading fragment (ORF) of 537bp encoding a polypeptide of 178 amino acids with an estimated molecular mass of 21.4kDa and theoretical isoelectric point of 5.97. The 5' untranslated region (UTR) was 11bp and the 3'UTR was 249 with 18bp poly (A) tail. In the peptide, there was a signal sequence, six potential phosphorylation sites, one glycosylation site and eight cysteine residues. Moreover, a sequence motif (D-R-X-W/F/Y-X-F/Y/I/L/M-X(1-2)-C) was contained and repeated itself three times in the entire sequence. DPT mRNA was constitutively expressed in all studied tissues with the most abundant mRNA in the mantle, which was nacre formation-related tissue. After decreasing DPT expression using RNA interference (RNAi) technology in the mantle, the nacreous layer showed a disordered growth; whereas the prismatic layer of the shells has no significant changes. These results suggested that DPT obtained in this study was a constitutive matrix protein and participated in nacre formation in P. martensii.     

Action range of BMP is defined by its N-terminal basic amino acid core

During early development, cells receive positional information from neighboring cells to form tissue patterns in initially uniform germ layers. Ligands of the transforming growth factor (TGF-beta) superfamily are known to participate in this pattern formation. In particular, activin has been shown to act as a long-range dorsalizing signal to establish a concentration gradient in Xenopus. In contrast, BMP-2 and BMP-4, other members of the family, appear to influence and induce ventral fates only where they are expressed. This raises a question as to how the action of BMPs is tightly restricted to the region within and around the cells that produce them. Here, we have demonstrated that a basic core of only three amino acids in the N-terminal region of BMP-4 is required for its restriction to the non-neural ectoderm as its expression domain. Our results also suggest that heparan sulfate proteoglycans bind to this basic core and thus play a role in trapping BMP-4. The present study is the first to identify the critical domain of BMP that is responsible for its interaction with the extracellular environment that restricts its diffusion in vivo.     

A new matrix protein family related to the nacreous layer formation of Pinctada fucata

We have isolated a new matrix protein family (N16) which is specific to the nacreous layer of the Japanese pearl oyster, Pinctada fucata, and have cloned and characterized the cDNAs coding for the components. Analysis of the deduced amino acid sequence revealed that N16 showed no definitive homology with other proteins. The in vitro studies of the crystallization clarified that N16 induced aragonite crystals when fixed on the substrate but inhibited crystal formation without it. The aragonite crystals showed platy morphology different from those formed inorganically, and long intervals of incubation resulted in crystalline layers highly similar to the nacreous layer.     

Molecular cloning and characterization of perlucin from the freshwater pearl mussel, Hyriopsis cumingii

Perlucin is an important functional protein that regulates shell and pearl formation. In this study, we cloned the perlucin gene from the freshwater pearl mussel Hyriopsis cumingii, designated as Hcperlucin. The full-length cDNA transcribed from the Hcperlucin gene was 1460 bp long, encoding a putative signal peptide of 20 amino acids and a mature protein of 141 amino acids. The mature Hcperlucin peptide contained six conserved cysteine residues and a carbohydrate recognition domain, similar to other members of the C-type lectin families. In addition, a “QPS” and an invariant “WND” motif near the C-terminal region were also found, which are extremely important for polysaccharide recognition and calcium binding of lectins. The mRNA of Hcperlucin was constitutively expressed in all tested H. cumingii tissues, with the highest expression levels observed in the mantle, adductor, gill and hemocytes. In situ hybridization was used to detect the presence of Hcperlucin mRNA in the mantle, and the result showed that the mRNA was specifically expressed in the epithelial cells of the dorsal mantle pallial, an area known to express genes involved in the biosynthesis of the nacreous layer of the shell. The significant Hcperlucin mRNA expression was detected on day 14 post shell damage and implantation, suggesting that the Hcperlucin might be an important gene in shell nacreous layer and pearl formation. The change of perlucin expression in pearl sac also confirmed that the mantle transplantation results in a new expression pattern of perlucin genes in pearl sac cells that are required for pearl biomineralization. These findings could help better understanding the function of perlucin in the shell and pearl formation.     

Identification of genes directly involved in shell formation and their functions in pearl oyster, Pinctada fucata

Mollusk shell formation is a fascinating aspect of biomineralization research. Shell matrix proteins play crucial roles in the control of calcium carbonate crystallization during shell formation in the pearl oyster, Pinctada fucata. Characterization of biomineralization-related genes during larval development could enhance our understanding of shell formation. Genes involved in shell biomineralization were isolated by constructing three suppression subtractive hybridization (SSH) libraries that represented genes expressed at key points during larval shell formation. A total of 2,923 ESTs from these libraries were sequenced and gave 990 unigenes. Unigenes coding for secreted proteins and proteins with tandem-arranged repeat units were screened in the three SSH libraries. A set of sequences coding for genes involved in shell formation was obtained. RT-PCR and in situ hybridization assays were carried out on five genes to investigate their spatial expression in several tissues, especially the mantle tissue. They all showed a different expression pattern from known biomineralization-related genes. Inhibition of the five genes by RNA interference resulted in different defects of the nacreous layer, indicating that they all were involved in aragonite crystallization. Intriguingly, one gene (UD_Cluster94.seq.Singlet1) was restricted to the 'aragonitic line'. The current data has yielded for the first time, to our knowledge, a suite of biomineralization-related genes active during the developmental stages of P. fucata, five of which were responsible for nacreous layer formation. This provides a useful starting point for isolating new genes involved in shell formation. The effects of genes on the formation of the 'aragonitic line', and other areas of the nacreous layer, suggests a different control mechanism for aragonite crystallization initiation from that of mature aragonite growth.     

Perlinhibin, a cysteine-, histidine-, and arginine-rich miniprotein from abalone (Haliotis laevigata) nacre, inhibits in vitro calcium carbonate crystallization

We have isolated a 4.785 Da protein from the nacreous layer of the sea snail Haliotis laevigata (greenlip abalone) shell after demineralization with acetic acid. The sequence of 41 amino acids was determined by Edman degradation supported by mass spectrometry. The most abundant amino acids were cysteine (19.5%), histidine (17%), and arginine (14.6%). The positively charged amino acids were almost counterbalanced by negatively charged ones resulting in a calculated isoelectric point of 7.86. Atomic-force microscopy studies of the interaction of the protein with calcite surfaces in supersaturated calcium carbonate solution or calcium chloride solution showed that the protein bound specifically to calcite steps, inhibiting further crystal growth at these sites in carbonate solution and preventing crystal dissolution when carbonate was substituted with chloride. Therefore this protein was named perlinhibin. X-ray diffraction investigation of the crystal after atomic-force microscopy growth experiments showed that the formation of aragonite was induced on the calcite substrate around holes caused by perlinhibin crystal-growth inhibition. The strong interaction of the protein with calcium carbonate was also shown by vapor diffusion crystallization. In the presence of the protein, the crystal surfaces were covered with holes due to protein binding and local inhibition of crystal growth. In addition to perlinhibin, we isolated and sequenced a perlinhibin-related protein, indicating that perlinhibin may be a member of a family of closely related proteins.     

Specific inhibition of procollagen C-endopeptidase activity by synthetic peptide with conservative sequence found in chordin

Procollagen C-endopeptidase (BMP-1) and N-endopeptidase (ADAMTS-2) are key enzymes for correct and efficient conversion of fibrillar procollagens to their self assembling monomers. Thus, they have an essential role in building and controlling the quality of extracellular matrices (ECMs). Here, we tested inhibition of activity of the largest variant of BMP-1, a recombinant mammalian tolloid (mTld), in vitro by three synthetic peptides with conservative amino-acid sequences found in chordin using procollagen type I as a substrate. We also verified the specific action of best inhibitory 16 amino-acid peptide in the procollagen type I cleavage assay with the use of ADAMTS-2 (procollagen N-endopeptidase). Subsequently, we determined the critical residues and minimal sequence of six amino acids in the original 16 amino-acid peptide required to maintain the inhibitory potential. Studies on the interactions of 6 and 16 amino acid long peptides with the enzyme revealed their binding to non-catalytic, regulatory domains of mTld; the inhibitory activity was not due to the competition of peptides with the substrate for the enzyme active center, because mTld did not cleave the peptides. However, in the presence of mTld both peptides underwent cyclization by disulfide bond formation. Concluding, we have shown that procollagen C-endopeptidase may be specifically blocked via its non-catalytic domains by synthetic peptide consisting of 6 amino acids in the sequence found in highly conservative region of chordin. Thus, we hypothesize that the 6 amino-acid peptide could be a good candidate for anti-fibrotic drug development.     

草鱼生肌因子5基因的克隆及其组织表达谱分析

目的:获得草鱼生肌因子5(Myf-5)基因序列,分析其在草鱼不同组织和不同发育阶段中的表达规律。方法:根据鲤鱼Myf-5基因序列设计引物,用草鱼肌肉组织总RNA,经RT-PCR扩增其Myf-5基因序列;利用半定量RT-PCR分析草鱼Myf-5基因在草鱼不同组织和不同发育阶段的mRNA表达特性。结果:获得了草鱼Myf-5基因开放读框序列723 bp,GenBank登录号为GU290227;该基因编码由240个氨基酸残基组成的蛋白,具有MyoD家族基因的典型性碱性螺旋-环-螺旋(bHLH)结构,其氨基酸序列与斑马鱼、鲤鱼、虹鳟、大西洋鲑等的同源性较高(74%~97%),与哺乳动物和禽类如人、小鼠、大鼠、猪、牛和鸡的同源性较低(56%~60%);在草鱼红肌、白肌、肝胰脏、肾脏、脑和肠中均检测到Myf-5基因的表达,红肌、白肌和脑组织中Myf-5基因mRNA的表达量显著高于其他组织(P<0.05);草鱼Myf-5基因的表达随着其生长发育呈下降趋势,在较大规格试验鱼(500 g)中的表达显著低于其他2种规格(50~60 g、120~130 g)的试验鱼(P<0.05)。结论:获得了草鱼Myf-5基因序列,其在红肌、白肌和脑组织中的表达量显著高于其他组织,并随生长发育呈下降趋势,为研究Myf-5在草鱼肌肉发育过程中的作用提供了基础资料。     

hBMP－2cDNA在COS细胞和小鼠肌肉中的表达

研究了骨形态发生蛋白ＢＭＰ－２ｃＤＮＡ在ＣＯＳ细胞和小鼠肌肉中的表达的情况,从ｐＳＰＳ６５ＢＭＰ－２质粒中回收ＢＭＰ－２ｃＤＮＡ,删除５＇端的非翻译序列,插入ｐＳＶＬ载体中,构建了含有ＢＭＰ－２全长编码序列的重组表达质粒ｐＳＶＬＢＭＰ－２。将表达质粒导入ＣＯＳ－７细胞中,细胞ＲＮＡ点杂交结果表明,转染ＢＭＰ－２基因的细胞内ＢＭＰ－２的ｍＲＮＡ水平明显升高;细胞培养上清的ＥＬＩＳＡ显示,转染ＢＭＰ－２ｃＤＮＡ后,细胞分泌产生的ＢＭＰ－２显著增加。小鼠实验发现,在肌肉内用注射法导入ＢＭＰ－２重组质粒后,局部组织内ＢＭＰ－２的ｍＲＮＡ转录水平也明显提高。     

Stimulation of chondrogenesis in limb bud mesoderm cells by recombinant human bone morphogenetic protein 2B (BMP-2B) and modulation by transforming growth factor beta 1 and beta 2

Bone morphogenetic protein 2B (BMP-2B) also called BMP-4 is one of a family of cartilage and bone-inductive proteins derived from bone matrix and belongs to the transforming growth factor beta (TGF-beta) superfamily. These bone-inductive proteins isolated from adult bone may be involved in bone repair. However, they may also play a role in cartilage and bone formation during embryonic development. To test whether BMP-2B influences cartilage formation by embryonic cells, recombinant human BMP-2B was applied to cultured limb bud mesoderm plated at three different densities. BMP-2B stimulated cartilage formation as assessed by Alcian blue staining and incorporation of radioactive sulfate into sulfated proteoglycans. Cells cultured at all three densities in the presence of 10 ng/ml BMP-2B formed a nearly continuous sheet of cartilage with abundant extracellular matrix and type II collagen. In addition, when cells were cultured in 0.5% serum in the presence of 10 ng/ml of BMP-2B for 5 days there was an increase in alkaline phosphatase as detected by histochemical and biochemical methods. Transforming growth factor beta isoforms (TGF-beta 1 and TGF-beta 2) inhibited sulfate incorporation into proteoglycans in a dose-dependent manner. This inhibition by TGF beta was overcome by recombinant BMP-2B. This study demonstrates that recombinant BMP-2B stimulates cartilage formation by chick limb bud mesoderm in vitro and is further modulated by TGF-beta isoforms.     

Molecular cloning of a novel immunoglobulin superfamily gene preferentially expressed by brain and testis

We have cloned and characterized a novel gene from both human and mouse that encodes a new member of the immunoglobulin superfamily. The gene is preferentially expressed in both brain and testis, and hence, termed BT-IgSF (brain- and testis-specific immunoglobulin superfamily). The predicted protein consists of V-type and C2-type immunoglobulin domains as well as a hydrophobic signal sequence, a single transmembrane region, and a cytoplasmic domain. Human BT-IgSF protein (431 amino acids) is 88% identical to the mouse protein (428 amino acids) and both show significant homology to coxsackie and adenovirus receptor (CAR) and endothelial cell-selective adhesion molecule (ESAM). We examined the expression of BT-IgSF with various cultured cells and found that the gene was expressed in both neurons and glial cells in vitro. Furthermore, the expression was preferentially detected in pyramidal cell layers of the dentate gyrus and hippocampus and in commissure fibers of the corpus callosum, in brain tissue sections examined. These findings suggest that BT-IgSF plays a role in the development or function of the central nervous system.     

Retinoic acid induces gene expression of fibroblast growth factor-9 during induction of neuronal differentiation of mouse embryonal carcinoma P19 cells

We have found that the gene expression of the ninth member of the fibroblast growth factor (FGF) family, FGF9 was induced during retinoic acid(RA)-induced neuronal differentiation of murine embryonal carcinoma P19 cells. We have reported here the nucleotide sequence of the mouse FGF9 cDNA. The murine cDNA showed 92.4% nucleotide sequence homology to the human FGF9 cDNA and 98.2% homology to that of rats. This mouse FGF9 cDNA encoded a polypeptide consisting of 208 amino acids with amino acid sequence identical to that of rats. Only one amino acid was replaced compared to the human homolog. The highly conserved sequence homology of FGF9 suggests its functional importance. FGF9 was originally isolated from a culture medium of a human glioma cell line as a growth-promoting factor for glial cells [5]. Upon induction of neuronal differentiation by forming cell aggregates with 10⁻⁶ M RA, the gene expression of FGF9 was increased biphasically during the first 96 hours when cells were aggregating and from 168 hours to 192 hours followed by plating onto a tissue culture dish as glia-like cells proliferated. Neither undifferentiated P19 cells nor the cells aggregated without RA remaining undifferentiated expressed FGF9. This indicates that RA regulates the gene expression of FGF9 that may play an important role in neuronal differentiation in both early and late developmental process.     

Bone morphogenetic proteins act synergistically with haematopoietic cytokines in the differentiation of haematopoietic progenitors

We examined the effects of bone morphogenetic protein-2 (BMP-2), -3, -4, -5, -6, and -7 on the proliferation and differentiation of bone marrow CD34+ haematopoietic progenitors in semi-solid medium. The BMPs had no effect on haematopoietic colony development when added to medium containing erythropoietin (Epo) or Interleukin-3 plus Epo. Synergistic effects with the haematopoietic cytokines stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were observed. In conjunction with GM-CSF and Epo, BMP-4 increased the number of both erythroid and granulocyte/monocyte colonies formed in semi-solid medium (P<0.01). No other BMP stimulated erythroid colony development under these conditions, while BMP-3, BMP-7 (P<0.01), BMP-5, and BMP-6 (P<0.05) stimulated granulocyte/monocyte colony formation. BMP-7 acted synergistically with stem cell factor to increase granulocyte/monocyte colony formation but not erythroid colony formation. The other BMPs did not affect either erythroid or granulocyte/monocyte colony development under these conditions. These results suggest that individual BMPs form part of the complement of cytokines regulating the development of haematopoietic progenitors, and in particular, point to a role for BMP-4 in the control of definitive, as well as embryonic erythropoiesis.     

Cloning and sequence analysis of thymidine kinase from the oral bacterium Streptococcus gordonii

Abstract Thymidine kinase is an important enzyme in the pyrimidine nucleotide salvage pathway and catalyzes the formation of thymidylate from thymidine using ATP as a phosphate donor. The gene encoding thymidine kinase of the oral bacterium Streptococcus gordonii was cloned and the nucleptide sequence determined. The inferred amino acid sequence of thymidine kinase (191 amino acids) exhibited 43% identity with type H thymidine kinase from Escherichia coli . The S. gordonii thymidine kinase expressed in Escherichia coli KY895 ( tdk⁻ ) was inhibited by thymidline triphosphate, a feature typical of type II thymidine kinases. Immediately 3' to the tdk gene, and possibly co-transcribed with it, was the gene encoding release factor 1 ( prfA ).