Epithelin/granulin growth factors: extracellular cofactors for HIV-1 and HIV-2 Tat proteins

Epithelin/granulin growth factor is synthesized as a 593 amino acid precursor protein that contains 7.5 imperfectly conserved repeats of approximately 57 amino acids. Processed epithelin/granulin peptides have been isolated from vertebrate/invertebrate species and are growth factors implicated in epithelial and haemic cell function. Here they are identified as Human Immunodeficiency Virus (HIV) Tat binding proteins using the yeast two-hybrid assay. Intracellularly in yeast, mutation of selected cysteines in an epithelin/granulin dimeric repeat caused loss of binding to Tat exon 1. In vitro binding of HIV-1 and HIV-2 Tat to epithelin/granulin dimeric and monomeric repeats was also observed by GST-glutathione bead "pulldown" assays. Because Tat is actively secreted from HIV-infected cells and has been shown to serve as a mitogenic factor for angiogenesis and for Kaposi-like cells, our observations suggest that epithelin/granulin growth factors may function as biologically important extracellular Tat co-factors.     

Granulins, a novel class of peptide from leukocytes

We report the isolation and characterization of a novel class of leukocyte peptides with possible cytokine-like activities which we call granulins. They are cystine-rich with molecular weights of approximately 6 Kda, except for granulin D, which appears to be a dimer. We present the sequence of one member of this family, a 56 residue peptide, granulin A, and amino-terminal sequences for three other granulins from human peripheral leukocytes. A fifth related peptide was isolated and partially sequenced from rat bone marrow, suggesting that at least some of the granulin in peripheral leukocytes is preformed in the marrow. Rat granulin, and human granulin A, are closely related, showing that the granulin structures are highly conserved between species.     

Granulin-like peptide in the mid-gut gland of the bivalve mollusk, Patinopecten yessoensis

A cysteine-rich polypeptide, termed CRP1, with a molecular mass of 5829 Da was found to occur in the mid-gut gland of the scallop Patinopecten yessoensis. CRP1 was purified by reverse phase and cation-exchange chromatographies. The amino acid sequence of CRP1 was deduced from its N-terminal amino acid sequence, amino acid composition and the sequence of a partial cDNA, indicating that CRP1 is a 57-amino-acid polypeptide containing 12 cysteine residues with a calculated molecular mass of 5841 Da (5829 Da when oxidized to form six disulfide bridges). A homology search of databases revealed that the deduced amino acid sequence of CRP1 displays significant similarity to those of granulin/epithelins, a family of growth-modulating factors; all cysteine residues in CRP1 are located at the same positions as those conserved characteristically in other known granulin/epithelins. Purified CRP1 inhibited the proliferation of mouse embryo cells. The results suggest that CRP1 functions as a growth-modulating factor in the scallop, and that granulin/epithelin family polypeptides and their precursors play physiologically important roles in invertebrates.     

极性蛋白Crumbs胞内域功能保守性研究

跨膜蛋白Crumbs(Crb)是细胞顶部的决定因子,对上皮细胞顶-底极性的建立和维持起着关键的作用。其胞内域虽然仅有37个氨基酸,但对Crb的功能必不可少。在果蝇(Drosophila melanogaster)中,如果胞内域发生突变,将造成胚胎发育异常、上皮细胞顶底极性丧失等严重后果。Crb胞内域从果蝇到小鼠(Mus musculus)和人类(Homo sapiens)具有很高的同源性,但线虫(Caenorhabditis elegans)两个Crb蛋白的胞内域与果蝇和哺乳动物却较为不同。为验证线虫Crb蛋白胞内域是否功能保守,本文利用基因组工程法(Genomic engineering),将果蝇基因组中Crb基因编码胞内域的部分替换为一致性和相似性较远的线虫Crb2基因的相应区段。与其他Crb胞内域突变果蝇不同,替换突变体胚胎发育正常,Crb及其他极性蛋白的表达和定位正常,胚胎上皮细胞顶底极性能够正确的建立和维持。这些结果证实虽然线虫和果蝇Crb蛋白胞内域之间存在大量序列变异,但重要的氨基酸位点和功能模块则完全保守。     

The Evolution of the Secreted Regulatory Protein Progranulin

Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i): the origins of metazoan progranulins (ii): the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii): the evolution of granulin module architectures of vertebrate progranulins (iv): the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl-terminus. Polypeptide sequence conservation of mammalian granulin modules identified potential structure-activity relationships that may be informative in designing progranulin based therapeutics.     

Wingless signaling initiates mitosis of primordial germ cells during development in Drosophila

The germline cells of Drosophila are derived from pole cells, which form at the posterior pole of the blastoderm and become primordial germ cells (PGCs). To elucidate the signal transduction pathways for the development of embryonic PGCs, we examined the effects of various growth factors on the proliferation of PGCs. Up- and down-regulation of Wingless (Wg) in both of soma and PGCs caused an increase and a decrease in the number of PGCs, respectively. The Wg/β-catenin signaling pathway began to occur in PGCs at the same time as the PGCs began to divide during the embryonic stage in both sexes. In addition, PGCs were found to produce wg mRNA as they begin to divide. Thus, Wg functions as an autocrine factor to initiate mitosis in embryonic PGCs. Decapentaplegic affected the growth of PGCs from the end of the embryonic stage. The results indicate that these growth factors regulate the division of embryonic PGCs in a stage-specific manner.     

Standardization of a colorimetric method to quantify growth and metabolic activity of Wolbachia-infected mosquito cells

The Aedes albopictus Aa23 cell line, which is persistently infected with Wolbachia pipientis strain wAlbB, tends to grow as aggregated clusters of cells that are difficult to disperse for conventional quantification based on cell number. We used A. albopictus C7-10 cells to validate conversion of methylthiazole tetrazolium (MTT) to a colored formazan product with respect to incubation time, cell number over a 40-fold range, and metabolic activity as cells enter stationary phase. Using this assay, we showed that the doubling time of Aa23 cells increases from about 45 h early after plating to more than 70 h as the cells reach stationary levels. Growth of Aa23 cells proceeds at similar rates in the presence or absence of tetracycline concentrations that decrease the abundance of Wolbachia. Insofar as the MTT assay reflects mitochondrial function, our results indicate that, in Aa23 cells, abundance of intracellular Wolbachia has no measurable effect on mitochondrial activity in the presence of tetracycline.     

Quick identification of acetic acid bacteria based on nucleotide sequences of the 16S-23S rDNA internal transcribed spacer region and of the PQQ-dependent alcohol dehydrogenase gene

Acetic acid bacteria (AAB) are well known for oxidizing different ethanol-containing substrates into various types of vinegar. They are also used for production of some biotechnologically important products, such as sorbose and gluconic acids. However, their presence is not always appreciated since certain species also spoil wine, juice, beer and fruits. To be able to follow AAB in all these processes, the species involved must be identified accurately and quickly. Because of inaccuracy and very time-consuming phenotypic analysis of AAB, the application of molecular methods is necessary. Since the pairwise comparison among the 16S rRNA gene sequences of AAB shows very high similarity (up to 99.9%) other DNA-targets should be used. Our previous studies showed that the restriction analysis of 16S–23S rDNA internal transcribed spacer region is a suitable approach for quick affiliation of an acetic acid bacterium to a distinct group of restriction types and also for quick identification of a potentially novel species of acetic acid bacterium (Trcek & Teuber 2002; Trcek 2002). However, with the exception of two conserved genes, encoding tRNA^Ile and tRNA^Ala, the sequences of 16S–23S rDNA are highly divergent among AAB species. For this reason we analyzed in this study a gene encoding PQQ-dependent ADH as a possible DNA-target. First we confirmed the expression of subunit I of PQQ-dependent ADH (AdhA) also in Asaia, the only genus of AAB which exhibits little or no ADH-activity. Further we analyzed the partial sequences of adhA among some representative species of the genera Acetobacter, Gluconobacter and Gluconacetobacter. The conserved and variable regions in these sequences made possible the construction of A. aceti-specific oligonucleotide the specificity of which was confirmed in PCR-reaction using 45 well-defined strains of AAB as DNA-templates. The primer was also successfully used in direct identification of A. aceti from home made cider vinegar as well as for revealing the misclassification of strain IFO 3283 into the species A. aceti.     

Stimulation of PC cell-derived growth factor (epithelin/granulin precursor) expression by estradiol in human breast cancer cells

PC cell-derived growth factor (PCDGF) is an 88 kDa glycosylated protein isolated from a highly tumorigenic mouse teratoma derived cell line which is similar to the epithelin/granulin precursor. Using Northern blot and western blot analyses, we detect the expression of PCDGF mRNA and protein in MCF-7 human breast cancer cells. We show that 17-beta-estradiol stimulates PCDGF mRNA and protein expression in a time and dose-dependent manner. The stimulation of PCDGF expression by 17-beta-estradiol was observed as early as 4 hours and reached a maximum at 12 hours. Maximal stimulation of PCDGF mRNA and protein expression by 17-beta-estradiol was observed at a concentration of 10(-8) M. The stimulation of PCDGF expression by 17-beta-estradiol was completely inhibited by treatment with actinomycin D and with the antiestrogen 4-hydroxytamoxifen. The stimulation of PCDGF expression was also demonstrated in another human estrogen-responsive cell line T47D. The results presented here provide evidence of a novel estradiol responsive gene product in human breast cancer cell lines and give information about the hormonal control of epithelin/granulin (PCDGF) expression in these cells.     

Structural and immunological characterization of the amino-terminal domain of mammalian neural cell adhesion molecules

The neural cell adhesion molecules (N-CAMs) are a group of structurally and immunologically related glycoproteins found in vertebrate neural tissues. Adult brain N-CAMs have apparent molecular weights of 180,000 and 140,000 with an additional form at 120,000 in murine brain. In embryonic brain, N-CAMs are represented by a highly sialylated form with an apparent molecular weight greater than 180,000. We have used monoclonal antibodies that cross-react with N-CAMs of various mammalian species to purify N-CAMs from adult murine and bovine brains and from embryonic murine brains. We determined the amino acid sequences of the amino-terminal domains of these molecules: Leu-Gln-Val-Asp-Ile-Val-Pro-Ser-Gln-Gly-Glu-Ile-Ser-Val-Gly-Glu-Ser. This sequence is highly conserved among all three forms of adult murine brain N-CAM as well as embryonic murine brain N-CAM and adult bovine brain N-CAM. Based on this sequence, we synthesized an undecapeptide and used it to raise a site-directed polyclonal antiserum. This antiserum reacted with the intact N-CAM in liquid phase radioimmunoassays, immunoblotting experiments, and immunofluorescent labeling of cells. The antiserum also reacted with N-CAMs in extracts of brain tissues from different species, confirming the highly conserved nature of the amino-terminal domain of mammalian N-CAMs. Immunofluorescence experiments indicated that this domain resides on the outer surfaces of cells that express N-CAMs, in both primary neuronal cell culture and in cell lines.