Structural and immunological similarities between simian sarcoma virus gene product(s) and human platelet-derived growth factor

The predicted amino acid sequence of the simian sarcoma virus (SSV) transforming gene product, p28sis, closely corresponds to that of human platelet-derived growth factor (PDGF). We demonstrate that p28sis rapidly undergoes a series of discrete processing steps including dimer formation and proteolytic digestion to yield molecules structurally and immunologically resembling biologically active PDGF.     

Antibodies against platelet-derived growth factor inhibit acute transformation by simian sarcoma virus

A clue to the molecular mechanism of neoplastic transformation was provided by the finding of a near identity in amino-acid sequence between the platelet-derived growth factor (PDGF) B-chain and a region in the transforming protein, p28sis, of simian sarcoma virus (SSV), an agent that causes sarcomas and gliomas in experimental animals. This finding infers a direct link between the molecular biology of normal mitogenesis and oncogenesis since it suggests that the transforming activity of SSV is caused by a growth factor. Although PDGF agonist activity has been isolated from conditioned medium of SSV-transformed cells, it is not clear whether infection of responsive cells by SSV leads solely to autocrine stimulation of growth by a secreted PDGF-like factor or whether other, possibly intracellular, activities of p28sis or its processed products contribute to the transformation. To distinguish between these possibilities, we have studied the effect of anti-PDGF antibodies on acute SSV-transformation, and report here that these antibodies inhibit both proliferation and SSV-induced morphological changes in human diploid fibroblasts.     

Platelet-derived growth factor is structurally related to the putative transforming protein p28sis of simian sarcoma virus

A partial amino acid sequence of human platelet-derived growth factor, the major mitogen in serum for cells of mesenchymal origin, has been determined. A region of 104 contiguous amino acids shows virtual identity with the predicted sequence of p28sis, the putative transforming protein of simian sarcoma virus (SSV). This similarity suggests a mechanism for transformation by SSV and other agents, involving expression of growth factors.     

Mutants of avian myelocytomatosis virus with smaller gag gene-related proteins have an altered transforming ability

Avian myelocytomatosis virus strain MC29 is a replication-defective avian oncovirus which in newborn chickens causes myelocytomatosis and liver and kidney tumours. In vitro infection of bone marrow cells gives rise to colonies of transformed macrophage-like cells, and cloned viruses is also capable of transforming fibroblasts. The genome of MC29 contains cellular sequences which are closely related to those in other defective leukaemia viruses with similar transforming spectra. Consequently, these cellular sequences have been postulated to represent a new oncogene which has been designated mac, for macrophage transformation. MC29-transformed cells contain a gag gene-related protein of a 110,000 molecular weight (MW) (p110), which by tryptic peptide analysis has been shown to be a fusion product comprised of a gag gene-derived sequences and sequences which are presumed to be coded by the adjacent mac gene. These findings suggest that this protein may be implicated in transformation by MC29. We now describe three mutants of MC29 and synthesize smaller gag gene-related proteins. These mutants have an altered ability to transform bone marrow cells but not fibroblasts. This demonstrates for the first time a direct involvement of the p110 protein of MC29 in transformation.     

Sequence analysis of peptides with biological activities using electrospray-Fourier trans form ion cyclotron resonance mass spectrometry

The mass spectra of five peptides with biological activities are reported. All mass spectra were recorded using a 4.7-T Fourier transform ion cyclotron resonance mass spectrometer equipped with an external electrospray source. The accurate molecular weights for the five peptides prepared by solid phase synthesis were measured as 1765.9013, 1063.5420, 1092.5254, 820.3804 and 1078.5193, respectively. All the data were obtained with the external calibration. Differences between observed and theoretical monoisotopic molecular weights were in the (0.2—1.0)×10^-6 range. The complete primary sequence for the five polypeptides were determined using the method of in-source electrospray ionization/collision induced dissociation (ESI/CID). All the intact y series ions and b series ions were obtained from various peptides respectively, thus determining the sequences of the five polypeptides. We found that the measured accurate molecular mass of sample 4 was not in agreement with that expected from the planned synthetic peptide. The sequences of sample 4 were determined through analysis. The corresponding accurate masses of b series ions and y series ions were gained, which proved that it was correct to re-determine the sequences.     

Structural alteration of viral homologue of receptor proto-oncogene fms at carboxyl terminus

A role for proto-oncogenes in the regulation and modulation of cell proliferation has been suggested by the findings that the B-chain of platelet-derived growth factor (PDGF) is encoded by the proto-oncogene sis and that the erb-B oncogene product is a truncated form of the epidermal growth factor (EGF) receptor. Furthermore, the product of the proto-oncogene fms (c-fms) may be related or identical to the receptor for macrophage colony-stimulating factor (CSF-1). v-fms is the transforming gene of the McDonough strain of feline sarcoma virus (SM-FeSV) and belongs to the family of src-related oncogenes which have tyrosine-specific kinase activity. Furthermore, nucleotide sequence analysis of the v-fms gene product revealed topological properties of a cell-surface receptor protein. To elucidate the features involved in the conversion of a normal cell-surface receptor gene into an oncogenic one, we have now determined the complete nucleotide sequence of a human c-fms complementary DNA. The 972-amino-acid c-fms protein has an extracellular domain, a membrane-spanning region, and a cytoplasmic tyrosine protein kinase domain. Comparison of the feline v-fms and human c-fms sequences reveals that the proteins share extensive homology but have different carboxyl termini.     

Identification of a 32K plasma membrane protein that binds to the myristylated amino-terminal sequence of p60v-src

The transforming protein of Rous sarcoma virus, p60v-src, is a myristylated membrane-bound phosphoprotein. Interaction of p60v-src with the plasma membrane is essential for transforming activity, and is mediated by association with a membrane-bound Src receptor protein. Evidence for the existence of an Src receptor is based on the ability of a myristylated peptide containing the N-terminal Src sequence to inhibit binding of p60v-src to plasma membranes in vitro: binding of p60v-src to a plasma membrane receptor is therefore mediated by N-terminal Src sequences. Here we report that a myristyl-Src peptide, but not the corresponding non-myristylated peptide, can be specifically crosslinked to a plasma membrane protein of relative molecular mass 32,000 (Mr32K). The 32K protein represents an Src-binding protein in the plasma membrane that is likely to be a component of the myristyl-Src receptor, and which could be involved in cellular transformation.     

A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family

A new acute transforming feline retrovirus, the Hardy-Zuckerman 4 feline sarcoma virus (HZ4-FeSV), has been isolated from a feline fibrosarcoma. The viral genome of HZ4-FeSV contains a new oncogene designated v-kit, has the structure 5' delta gag-kit-delta pol-delta env 3' and specifies a gag-kit polyprotein of relative molecular mass 80,000. The predicted kit amino-acid sequence displays partial homology with tyrosine-specific protein kinase oncogenes. HZ4-FeSV appears to have been generated by transduction of feline c-kit sequences with feline leukaemia virus.     

异柠檬酸裂解酶肽类抑制剂的优化筛选

利用噬菌体肽库筛选与计算机模拟分子对接技术, 优化异柠檬酸裂解酶肽类抑制剂的筛选. 先通过噬菌体肽库筛选出与异柠檬酸裂解酶（ICL）具有高亲和力的结合肽, 再利用Discovery Studio 2.1模拟多肽与ICL蛋白晶体（1F8I）的分子对接, 最后用Fmoc固相合成法合成多肽, 并对其生物活性进行检测. 实验结果表明, 通过噬菌体肽库筛选得到了29条七肽序列, 其中12条可与ICL蛋白晶体成功对接. 体外生物活性检测结果显示, 得到的12条七肽均对ICL的活性有明显抑制作用（抑制率均超过50%）.     

A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences

A biologically active complementary DNA clone of a transforming gene present in a human colon carcinoma contains gene sequences of both tropomyosin and a previously unknown protein tyrosine kinase. The predicted protein (641 amino acids) encoded by this oncogene seems to have been formed by a somatic rearrangement that replaced the extracellular domain of a putative transmembrane receptor by the first 221 amino acids of a non-muscle tropomyosin molecule.     

Cloning and sequence analysis of cDNAs encoding two distinct somatostatin precursors found in the endocrine pancreas of anglerfish

Complementary DNAs for two distinct anglerfish somatostatin peptides (termed I and II) have been cloned in bacterial plasmids and sequenced. The nucleotide sequence for somatostatin I encodes a large percursor peptide (molecular weight 13,300) in which the somatostatin hormones is at the carboxyl terminus. The predicted 14-amino acid sequence for anglerfish somatostatin I is the same as mammalian somatostatin. Somatostatin II is also synthesized as part of a larger precursor (molecular weight 14,100) with the presumptive somatostatin hormone also at the carboxyl terminus. The 14-amino acid sequence of somatostatin II differs from somatostatin I at two internal residues (Tyr in place of Phe 7 and Gly in place of Thr 10). The two different somatostatins may have distinct biological activities. Homologies in the amino acid sequences of the two peptides outside the somatostatin moiety suggest other regions of the molecules have biological functions.     

Non-responsiveness to a foot-and-mouth disease virus peptide overcome by addition of foreign helper T-cell determinants

Study of the immune response to synthetic antigens has shown that uncoupled peptides can realize their potential as vaccines only if they contain domains that react with helper T-cell receptors and Ia antigens in addition to antibody binding sites. Here we consider whether genetically restricted non-responsiveness to an uncoupled peptide could be overcome by synthesizing a peptide with an additional helper T-cell epitope from a different protein. We demonstrate that H-2d mice, which are non-responders to the 141-160 VP1 peptide of foot-and-mouth disease virus (FMDV), can be converted into responders by immunization with peptides containing the FMDV sequence with defined 'foreign' helper T-cell determinants from ovalbumin or sperm whale myoglobin. Furthermore, the virus-neutralizing activity of the antibody raised against peptide was dependent on the determinant used. Thus, FMDV peptides with the added sequences 323-339 from ovalbumin and 132-148 from sperm-whale myoglobin elicited a high degree of neutralizing activity in B10.D2 mice. The sera from mice which received the peptide with the added sequence 105-121 from sperm whale myoglobin did not neutralize the virus, although they had high levels of anti-141-160 FMDV peptide activity. Our data indicate that the T-cell help given by the 'foreign' epitopes is B-cell clone specific. These results are likely to have important implications for the design of peptide vaccines.     

Nucleotide sequence and formation of the transforming gene of a mouse sarcoma virus

The complete nucleotide sequence of the transforming gene of a mouse sarcoma virus has been determined. It codes for a protein of 374 amino acids. The nucleotide sequence of the junctions between a murine leukaemia virus and cellular sequences leading to the formation of the viral transforming gene have also been elucidated. The viral transforming sequence and its cellular homologue share an uninterrupted stretch of 1,159 nucleotides, with few base substitutions. The predicted amino acid sequence of the mouse sarcoma virus transforming gene was found to share considerable homology with the proposed amino acid sequence of the avian sarcoma virus oncogene (src) product.     

Use of a cDNA clone to identify a supposed precursor protein containing valosin

Valosin, a novel 25-amino-acid peptide isolated recently from pig intestine, has several effects on the digestive system of dogs. We report here that the valosin-specific complementary DNA clone from pigs codes for a polypeptide unlike most precursors of biologically active peptides. The predicted protein lacks a characteristic amino-terminal hydrophobic signal sequence and contains no processing signals of the type acted upon by endopeptidases to generate other active peptides from precursors. Antibodies to synthetic valosin have been used to show that nearly all valosin immunoreactivity is in the cytoplasm and that the protein detected (valosin-containing protein, VCP), although smaller than the predicted product of the cDNA sequence, is much larger than valosin. Valosin-specific messenger RNA is found in extracts from many pig tissues, which contrasts with the restricted occurrence expected of a biologically active peptide. We conclude that valosin is an artefact of the purification procedure and does not occur in vivo.     

In vivo competition between self peptides and foreign antigens in T-cell activation

Cytotoxic and helper T lymphocytes recognize foreign antigen in the form of short peptides associated with class I and class II major histocompatibility complex (MHC) molecules, respectively. A recent study of the three-dimensional structure of a class I MHC molecule revealed a cleft formed by the amino-terminal half of the protein, which could serve as the binding site for these peptides. Because an individual possesses only a limited set of different MHC molecules, each molecule of this set must have the ability to bind a large number of different peptides in order to ensure full immunocompetence. Thus, it can be anticipated that peptides with unrelated sequences compete for binding to the same MHC molecule, and, indeed, this has been shown to occur in vitro. We therefore decided to see whether such competition could also regulate the cell responses in vivo. We have found that a synthetic peptide corresponding to residues 46-62 of mouse lysozyme, although not immunogenic itself, effectively inhibits the priming for T-cell responses when injected into mice together with foreign protein or peptide antigens. The inhibition observed strictly correlates with the capacity of the competitor to bind to the particular MHC molecule presenting the foreign antigen, and its extent depends on the molar ratio between antigen and competitor.     

HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides

Current candidate vaccines fail to protect primates against challenge with human immunodeficiency virus (HIV) in the presence of antibody responses; this underlines the importance of studying cell-mediated immunity to HIV and identifying specific epitopes that stimulate cytotoxic T lymphocytes (CTL). Using a recombinant vaccinia virus to express the gag protein of HIV-1 we found HLA class-I-restricted gag-specific CTL in thirteen out of fifteen healthy HIV seropositive patients. We then used short synthetic peptides in the lysis assay to screen for gag CTL epitopes. In one patient we have identified a peptide in p24 that is recognized by CTL in association with HLA-B27. This peptide, and further peptide sequences defined by these methods, could be incorporated in vaccines designed to induce cell-mediated immunity against HIV.