Complementary DNA for human glioblastoma-derived T cell suppressor factor, a novel member of the transforming growth factor-beta gene family.

Human glioblastoma cells secrete a peptide, termed glioblastoma-derived T cell suppressor factor (G-TsF), which has suppressive effects on interleukin-2-dependent T cell growth. As shown here, complementary DNA for G-TsF reveals that G-TsF shares 71% amino acid homology with transforming growth factor-beta (TGF-beta). In analogy to TGF-beta it is apparently synthesized as the carboxy-terminal end of a precursor polypeptide which undergoes proteolytic cleavage to yield the 112 amino-acid-long mature form of G-TsF. Comparison of the amino-terminal sequence of G-TsF with that of porcine TGF-beta 2 and bovine cartilage-inducing factor B shows complete homology, which indicates that we have cloned the human analogue of these factors. It is tempting to consider a role for G-TsF in tumor growth where it may enhance tumor cell proliferation in an autocrine way and/or reduce immunosurveillance of tumor development.     

Hepatocyte growth factor and transforming growth factor-beta stimulate both cell growth and migration of human gastric adenocarcinoma cells.

Hepatocyte growth factor (HGF) induced scattering and cell migration of human gastric adenocarcinoma MKN-74. HGF also significantly promoted the growth of MKN-74 cells in a dose-dependent manner, although HGF is reported to be antiproliferative for the growth of tumor cell lines. This result indicates that HGF stimulates cell proliferation of not only normal epithelial cells but also certain carcinoma cells. Furthermore, transforming growth factor-beta (TGF-beta), which is recognized to inhibit the growth of most epithelial cells, additively enhanced both the cell proliferation and migration induced by HGF. These additive effects of HGF and TGF-beta may be responsible for the tumor invasiveness and uncontrolled growth of certain types of carcinoma.     

cDNA cloning of porcine transforming growth factor-beta 1 mRNAs. Evidence for alternate splicing and polyadenylation

Most eukaryotic cells encode principally a 2.5-kilobase (kb) transforming growth factor (TGF)-beta 1 mRNA. However, we have found two major TGF-beta 1 RNA species, 3.5 and 2.5 kb long, in porcine tissues. The 3.5-kb species has a longer 3'-untranslated sequence generated by the selection of an alternate polyadenylation site. There is a 117-nucleotide sequence within this unique 3' region, which is similar to the PRE-1 repetitive sequence of unknown function, reported earlier in the porcine genome. We have also cloned and characterized an alternately spliced mRNA species specific for the TGF-beta 1 gene, in which exons IV and V of the corresponding human TGF-beta 1 gene are deleted. The nucleotide sequence of this cDNA clone predicts a putative precursor protein of 256 amino acids; the N-terminal 211 amino acids of this putative protein are identical to the TGF-beta 1 precursor protein (exons I, II, and III of the human TGF-beta 1 gene), but the C-terminal 45 amino acids are distinct, due to a frameshift in the translation of exons VI and VII. In addition we provide data for the existence of other mRNA species generated in a tissue-specific manner either by alternate splicing or by heterogeneous 5' leader sequences.     

Murine transforming growth factor-beta 2 cDNA sequence and expression in adult tissues and embryos

Murine transforming growth factor-beta 2 (TGF-beta 2) cDNAs were isolated from cDNA libraries derived from a differentiated murine embryonic carcinoma cell line, PCC3. The composite cDNA sequence is 4267 nucleotides long, including a 1217 nucleotides 5'-untranslated sequence, and encodes a murine TGF-beta 2 precursor of 414 amino acids with 96% identity to its human counterpart. Several consensus polyadenylation sequences are present in the 1807 nucleotides 3'-untranslated sequence. Five TGF-beta 2 mRNA species are observed in the developing mouse fetus and they show different patterns of expression during development. TGF-beta 2 mRNA expression was also examined in adult mouse tissues, in which four of the five RNA species were observed. TGF-beta 2 mRNAs were present in all adult mouse tissues examined, except liver, and was most abundant in placenta, the male submaxillary gland and lung. The patterns of expression suggest a physiological role for TGF-beta 2 both in embryonic development and in the maintenance of adult tissues.     

Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor

Amino acid sequences of four peptide fragments of human hepatocyte growth factor purified from the plasma of patients with fulminant hepatic failure were determined. Based on the amino acid sequence of one of the fragments, two oligodeoxyribonucleotide mixtures were synthesized and used to screen a human placenta cDNA library. On the screening, two overlapping cDNA clones for human hepatocyte growth factor were isolated and the nucleotide sequence of the cDNA was determined. The entire primary structure of the protein was deduced from the sequence. The protein consists of 728 amino acid residues, including a possible signal peptide at the N-terminus. The sequence revealed that the heavy and light chains which comprise the protein are encoded by the same mRNA and are produced from a common translation product by proteolytic processing.     

Identification of a novel mammalian annexin. cDNA cloning, sequence analysis, and ubiquitous expression of the annexin XI gene.

Annexins (or lipocortins) are a family of at least 10 structurally related calcium- and phospholipid-binding proteins. Each protein consists of a conserved core domain having four (or eight) repeats of a segment approximately 70 amino acids in length and a nonconserved, usually short, amino-terminal domain. To date, amino acid sequences for eight distinct mammalian annexins have been predicted from cDNAs. This report describes an additional member of this family, bovine annexin XI, identified by cDNA cloning and sequence analysis. The 503-amino acid deduced protein consists of a core domain of four annexin repeats and a long amino-terminal domain rich in glycine, proline, and tyrosine. This novel annexin gene is expressed in a wide variety of tissues and isolated cells in culture.     

Latent form of transforming growth factor-beta 1 acts as a potent growth inhibitor on a human erythroleukemia cell line

Recombinant latent form of transforming growth factor-beta 1 (L-TGF-beta 1) is activated by various chemical treatments, including acidification and heating. However, cellular mechanisms that release transforming growth factor-beta (TGF-beta) in an active form have not been fully elucidated. Investigated herein are the effects of L-TGF-beta 1 on various leukemic cell lines. Heat-activated L-TGF-beta 1 inhibited colony formation of U937, KG-1 and HL-60, whereas untreated L-TGF-beta 1 had only a marginal effect on these cells. In contrast, colony formation of human erythroleukemia cell line (HEL) was markedly inhibited by both heat-activated and untreated L-TGF-beta 1. In vitro incubation of L-TGF-beta 1 with HEL cells did not release the active form in the culture supernatants. These results suggest that HEL cells are capable of activating L-TGF-beta 1, but only in a cell-associated manner. Since HEL cells produce L-TGF-beta 1, it may act as an autocrine negative growth factor on these cells.     

Human transforming growth factor type beta 2: production by a prostatic adenocarcinoma cell line, purification, and initial characterization

Human type beta 2 transforming growth factor (hTGF-beta 2) was purified from tamoxifen-supplemented, serum-free medium conditioned by the human prostatic adenocarcinoma cell line PC-3. The purification of hTGF-beta 2 was monitored in a growth inhibition assay and was achieved by batch purification on methylsilyl-controlled pore glass, followed by gel permeation chromatography and reversed-phase high-performance liquid chromatography. The overall recovery of hTGF-beta 2 was 75% of the initial activity and yielded 22 micrograms of hTGF-beta 2/L of conditioned medium. The concentration of hTGF-beta 2 required for half-maximal inhibition of Mv 1 Lu mink lung epithelial cells (CCl-64) was approximately 5 pM when assayed in the presence of 10% fetal bovine serum. The purified hTGF-beta 2 has a molecular weight of 24,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consists of two disulfide-linked, apparently identical polypeptide chains, with a molecular weight of 13,000. The amino-terminal sequence of hTGF-beta 2 was determined. Alignment of the amino acid sequences of hTGF-beta 2 and hTGF-beta reveals statistically significant sequence homology. On the basis of the extensive amino acid sequence homology, we propose the term TGF-beta 2 for this newly isolated polypeptide. The reported results suggest that TGF-beta (TGF-beta 1) and TGF-beta 2 may have evolved from a common progenitor.     

Transforming growth factor-beta1, transforming growth factor-beta2, and transforming growth factor-beta3 enhance ovarian cancer metastatic potential by inducing a Smad3-dependent epithelial-to-mesenchymal transition

Transforming growth factor-beta (TGF-beta) is thought to play a role in the pathobiological progression of ovarian cancer because this peptide hormone is overexpressed in cancer tissue, plasma, and peritoneal fluid. In the current study, we investigated the role of the TGF-beta/Smad3 pathway in ovarian cancer metastasis by regulation of an epithelial-to-mesenchymal transition. When cancer cells were cultured on plastic, TGF-beta1, TGF-beta2, and TGF-beta3 induced pro-matrix metalloproteinase (MMP) secretion, loss of cell-cell junctions, down-regulation of E-cadherin, up-regulation of N-cadherin, and acquisition of a fibroblastoid phenotype, consistent with an epithelial-to-mesenchymal transition. Furthermore, Smad3 small interfering RNA transfection inhibited TGF-beta-mediated changes to a fibroblastic morphology, but not MMP secretion. When cancer cells were cultured on a three-dimensional collagen matrix, TGF-beta1, TGF-beta2, and TGF-beta3 stimulated both pro-MMP and active MMP secretion and invasion. Smad3 small interfering RNA transfection of cells cultured on a collagen matrix abrogated TGF-beta-stimulated invasion and MMP secretion. Analysis of Smad3 nuclear expression in microarrays of serous benign tumors, borderline tumors, and cystadenocarcinoma revealed that Smad3 expression could be used to distinguish benign and borderline tumors from carcinoma (P = 0.006). Higher Smad3 expression also correlated with poor survival (P = 0.031). Furthermore, a direct relationship exists between Smad3 nuclear expression and expression of the mesenchymal marker N-cadherin in cancer patients (P = 0.0057). Collectively, these results implicate an important role for the TGF-beta/Smad3 pathway in mediating ovarian oncogenesis by enhancing metastatic potential.     

Effects of N,N-dimethylformamide and extracellular matrix on transforming growth factor-beta binding to a human colon carcinoma cell line

Alterations in the binding of transforming growth factor-beta (TGF-beta) to the MOSER human colon carcinoma cell line caused by N,N-dimethylformamide (DMF) or extracellular matrix (ECM) were examined. DMF induced a more differentiated phenotype in the MOSER cells and resulted in a twofold increase in TGF-beta binding to the cells. This was due to an increase in receptor number with no significant alteration in the KD. The extent of increased TGF-beta binding was dependent on the dose and time of exposure to DMF. Upon removal of DMF, the receptor level returned to that of untreated cells within 6 hr. The binding of TGF-beta 1 and TGF-beta 2 to the cells was increased equally. Despite this increase in TGF-beta binding in the presence of DMF, the sensitivity of the MOSER cells to the growth inhibitory effects of TGF-beta was unaltered. Growth of the MOSER cells on ECM derived from a well-differentiated colon cell line increased the TGF-beta receptor number twofold without altering the KD. No change was observed if the MOSER cells were grown on ECM derived from a poorly differentiated cell line. While no alteration in sensitivity to TGF-beta was observed on cells grown in the presence of DMF, MOSER cells grown on the ECM derived from well-differentiated colon carcinoma cell lines were twofold more sensitive to the growth inhibitory effects of TGF-beta. These results indicated that growth conditions which resulted in a more differentiated phenotype resulted in an increase in the cellular receptors for TGF-beta.     

棉铃虫酚氧化酶原基因的克隆、序列分析和组织表达

利用RT-PCR和RACE方法,获得了棉铃虫Helicoverpa armigera酚氧化酶原(prophenoloxidase,PPO)基因一个亚型cDNA的完整序列。该序列全长2 405 bp,含有一个2 097 bp的开放阅读框,编码一个由698个氨基酸残基组成的蛋白质。推导的氨基酸序列与其他鳞翅目昆虫PPO2基因相应氨基酸序列有较高的同源性(76％～80％),同时该序列具有铜离子结合位点等PPO基因所具有的典型特征。组织特异性表达分析表明,该基因在棉铃虫血细胞、体壁和中肠中均有表达。     

Molecular cloning and characterization of the human and porcine transforming growth factor-beta type III receptors.

Full-length cDNAs for the transforming growth factor-beta (TGF-beta) type III receptors were isolated from porcine uterus and human placenta cDNA libraries. The human TGF-beta type III receptor coding region encodes a protein of 849 amino acids with a single transmembrane domain and a short stretch of the intracellular domain. Potential glycosaminoglycan attachment sites were found in the extracellular domain. The overall amino acid sequence identities with those of the porcine and rat TGF-beta type III receptors were 83% and 81%, respectively. A high degree of sequence conservation was observed in the transmembrane and intracellular domains, which also have sequence similarity with human endoglin. In addition, two portions with 29 and 52 amino acids in the extracellular domain were found to be substantially similar with human endoglin.     

苜蓿夜蛾保幼激素酯酶cDNA的克隆、序列分析及表达研究

【目的】研究苜蓿夜蛾Heliothis viriplaca保幼激素酯酶(Juvenile hormone esterase,JHE)的功能和作用机理。【方法】本试验提取苜蓿夜蛾的总RNA,并利用RT-PCR和RACE技术,扩增得到苜蓿夜蛾保幼激素酯酶基因的全长c DNA序列,命名为Hvjhe(Gen Bank登录号:JQ901384)。【结果】该基因含有3 106 bp,包括一个1 746 bp的开放阅读框,编码一个含581个氨基酸的多肽,分子量约为63.9 ku,多肽的等电点为5.11,且氨基酸序列具含有5个保幼激素酯酶特有的氨基酸保守模块。推导的氨基酸序列与烟芽夜蛾Heliothis virescens和棉铃虫Helicoverpa armigera中获得的保幼激素酯酶相似性较高,分别达到83%和82%。荧光定量PCR结果表明,该基因在苜蓿夜蛾不同发育时期和不同组织中都有m RNA水平的特异性表达,且在预蛹期表达量相对较高,取食期、蛹期期和成虫期表达量相对较低;在中肠和脂肪体内的表达量相对于其它组织较高。该基因在大肠杆菌Escherich coli表达系统中进行了诱导表达,经SDS-PAGE和Western blot检测结果表明,表达出与预测的蛋白分子量相符的融合蛋白。【结论】研究结果表明本试验获得了苜蓿夜蛾中一个新的保幼激素酯酶基因的c DNA序列。     

Molecular cloning and sequence analysis of a novel chalcone synthase cDNA from Ginkgo biloba.

A chalcone synthase (CHS) gene was cloned from Ginkgo biloba for the first time and it was also the first cloned gene involved in flavonoids metabolic pathway in G. biloba. The full-length cDNA of G. biloba CHS (designated as Gbchs) was 1608bp with poly(A) tailing and it contained a 1173bp open reading frame (ORF) encoding a 391 amino acid protein. Gbchs was found to have extensive homology with those of other plant chs genes via multiple alignments. The active sites of the CoA binding, coumaroyl pocket and cyclization pocket in CHS protein of Medicago sativa were also found in GbCHS. Molecular modeling of GbCHS indicated that the three-dimensional structure of GbCHS strongly resembled that of M. sativa (MsCHS2), implying GbCHS may have similar functions with MsCHS2. Phylogenetic tree analysis revealed that GbCHS had closer relationship with CHSs from gymnosperm plants than from other plants. Gbchs is a useful tool to study the regulation of flavonoids metabolism in G. biloba.     

Nucleotide sequence of transforming human c-sis cDNA clones with homology to platelet-derived growth factor.

Three c-sis cDNA clones were obtained from polyadenylated RNA of a human T-cell lymphotropic virus (HTLV) type I transformed cell line. Two clones, designated pSM-1 and pSM-2, have cDNA inserts of 2498 and 2509 base pairs (bp), respectively, excluding the sizes of the guanylate tails, and the polyadenylate tracts. These clones are shorter than the estimated size of the c-sis mRNA of 4200 bp. Both of these clones can transform NIH 3T3 cells. The third clone, designated pSM-3 has a cDNA insert of 1421 bp and lacks transforming activity. The sequence of clone pSM-1 reveals a single long open reading frame (nucleotides 118-840) encoding chain A of platelet-derived growth factor, and two segments with homology to v-sis (nucleotides 182-871 and 1021-1325). Sequence homology is noted in the 3' untranslated region to the corresponding regions of the beta 1 interferon (IFN), human and murine beta-nerve growth factor (NGF), human interleukin 2 (IL2) genes, and tubulin pseudogenes. However, no typical AATAAA polyadenylation signal is present. An alternating (dCdA)n X (dGdT)n sequence is present in the 3' flanking cellular sequences similar to those in the corresponding position of the human proenkephalin gene, in the first intron of the gamma-IFN gene, and the second intron of the beta-NGF gene.     

Differentially regulated production of platelet-derived growth factor and of transforming growth factor beta by a human teratocarcinoma cell line

The human teratocarcinoma stem cell line Tera-2 clone 13 is induced by retinoic acid to differentiate in vitro into endodermal or neuroectodermal cell types. In the absence of externally added growth factors, Tera-2 clone 13 cells proliferated at the same rate as in the presence of serum growth factors. Analysis of serum-free medium conditioned by Tera-2 clone 13 cells showed the presence of a polypeptide immunologically and biochemically related to platelet-derived growth factor (PDGF). In addition transforming growth factor beta (TGF-beta), but no TGF-alpha production could be detected. Tera-2 clone 13 cells specifically expressed high levels of the A-chain mRNA, but not the B-chain mRNA of PDGF. During retinoic acid induced differentiation the level of A-chain mRNA became markedly reduced. In contrast the TGF-beta mRNA levels increased significantly upon differentiation. The implications of these findings are discussed in terms of regulation of growth and differentiation in early embryos as well as in (human) teratocarcinomas.     

Human hepatic triglyceride lipase: cDNA cloning, amino acid sequence and expression in a cultured cell line

By immunoscreening of a human cDNA expression library and hybridization of colonies, four partially overlapping cDNA clones of human hepatic triglyceride lipase (HTGL) mRNA were isolated. The clones included the complete coding sequence, the 3'- and at least part of the 5'-untranslated region. The length of the composite HTGL cDNA segment (1.7 kb) was consistent with the size of the mRNA identified in an established human hepatoma cell line. DNA-sequence analysis of cDNAs of partially unspliced mRNAs, and of cloned genomic DNA indicated that the HTGL coding sequence comprises at least six exons. As predicted from the cDNA, the unprocessed HTGL protein has a molecular weight of 56, three potential glycosylation sites, and a signal peptide of 23 amino acids. Sequence comparison with cDNA of other lipases, including rat hepatic lipase, revealed 30%-75% protein-sequence homology. The data establish that HTGL is a secretory protein produced in the hepatocyte, and that its synthesis can be continued in permanent cell lines of hepatoma origin. Our studies also showed that HTGL is another member of a lipase gene family which has interfacial binding sites and possibly other functional domains in common.