Molecular cloning of a new human insulin-like growth factor binding protein.

We have cloned a new insulin-like growth factor's binding protein (IGFBP) from a human osteosarcoma cDNA library. Two conserved regions in the COOH-terminal third of the five known human IGFBPs were used to design primers and to perform polymerase chain reaction (PCR) with osteosarcoma cDNA as a template. One of the eight PCR products encoded a unique IGFBP sequence. The DNA sequence was used to synthesize probes to screen an osteosarcoma cDNA library and isolate full length cDNA clones. The amino acid sequence was deduced from one of them. It contains two possible signal peptidase cleavage sites yielding a mature molecule of 257 or 252 amino acids, and 18 cysteines in identical positions to the other IGFBPs. The most pronounced homology exists with human IGFBP-3 (50% in the NH2- and 45% in the COOH-terminal region).     

Efficacy of combination gene therapy with multiple growth factor cDNAs to enhance skin flap survival in a rat model

The objective of this study was to investigate the efficacy of combination gene therapy with multiple angiogenic growth factor cDNAs to enhance survival of ischemic skin flaps in a rat model. Sixty Sprague-Dawley rats were divided into six groups. Varying combinations of VEGF165, PDGF-B, and bFGF-plasmids were injected to prefabricate the flaps. Random skin flaps were raised on the dorsal aspect of rats following prefabrication with growth factor cDNAs. Flap viability was determined by measurement of percentage area of survival. The efficacy of gene therapy was evaluated by flap survival and neovascularization of representative histologic sections stained immunohistologically. The VEGF165 plus bFGF cDNAs enhanced the viability of the flap and neovascularization most effectively; the flap survival area was 64.3 +/- 8.7% after transfer of these two growth factor genes. Addition of PDGF-B cDNA is deleterious to the effects of combined VEGF165 and bFGF, leading to a significant decrease in flap viability (44.9 +/- 2.7%). Viability of the flaps with combined VEGF165 and bFGF cDNA transfer was significantly greater than that of the flaps with VEGF165 transfer alone (57.6 +/- 5.2%) or sham plasmid control (52.3 +/- 5.0%). Combined transfer of VEGF165 and bFGF cDNA is the most effective combination of multiple growth factor genes to improve flap viability in this model. Simultaneous transfer of three growth factor genes (VEGF165, PDGF-B, and bFGF) is deleterious to flap survival, at least for the ratio of lipofectin:transgene employed.     

A cDNA presumptively coding for the alpha subunit of the receptor with high affinity for immunoglobulin E

Rat mast cells and a neoplastic analogue such as rat basophilic leukemia (RBL) cells have receptors that have exceptionally high affinity for immunoglobulin E (IgE). When aggregated, these receptors induce cellular degranulation. The alpha chain of the receptor contains the binding site for IgE; the function(s) of the noncovalently associated beta and gamma chains is (are) still undefined. Using a cDNA library constructed from the mRNA of RBL cells, we have isolated a cDNA clone whose sequence predicts a putative 23-residue signal peptide, followed by a sequence that accurately predicts the amino acid composition, the peptide molecular weight, and six peptide sequences (encompassing 59 residues or 26% of the total number) determined for the alpha chain by direct analysis. These findings provide strong evidence that the cDNA codes for the alpha chain, even though expression has not been unambiguously achieved. The sequence suggests that the alpha chain contains a 180-residue extracellular portion with two homologous domains of approximately 35 residues, a 20-residue transmembrane segment containing an aspartic acid, and a 27-residue cytoplasmic portion containing 9 basic amino acids. The sequence shows no homology with the low-affinity receptor for IgE from lymphocytes but over 30% homology with an Fc gamma receptor.     

Structure of the murine c-sis proto-oncogene (Sis, PDGFB) encoding the B chain of platelet-derived growth factor

The murine proto-oncogene c-sis (Sis, PDGFB), encoding the B chain of platelet-derived growth factor, has been cloned. Its structure, with seven exons spanning approximately 20 kb, closely resembles that of the human and feline homologs. The predicted amino acid sequence of murine PDGF-B has residues 89% identical to those of human PDGF-B. A noncoding region at the start of exon 7, which is deleted by alternative splicing during the generation of the viral v-sis oncogene, is highly conserved in human, mouse, and cat and may represent an important regulatory element.     

Coexpression of tumor-associated alpha 2-macroglobulin and growth factors in human melanoma cell lines.

alpha 2-Macroglobulin (alpha 2M) is known as an inhibitor of various proteinases and to bind several of the growth factors. We previously demonstrated that clonal variation exists in the production of alpha 2M in a human melanoma and that this variation may be associated with growth stimulation. We have now analyzed six human melanoma cell lines for the simultaneous expression of TGF-alpha, TGF-beta, PDGF-A chain, PDGF-B chain, and tumor-associated alpha 2M. In Northern blot analysis TGF-alpha was detected in four of the cell lines, TGF-beta in all, PDGF-A chain in three, and PDGF-B chain in none of the cell lines. alpha 2M, detected by immunoblotting, varied significantly between the different melanoma cell lines and only one cell line was found to be negative. Evaluation of growth-promoting activity in conditioned media suggested that alpha 2-macroglobulin, secreted by these tumor cell lines, is a significant modulator of melanoma cell growth.     

Cloning and sequence of the cDNA corresponding to the variable region of immunoglobulin heavy chain MPC11.

Poly(A)-containing mRNA from mouse myeloma MPC11 was transcribed into cDNA which was cloned in the PstI site of the plasmid pBR322. The transformants were screened by hybridization with a cDNA fragment, derived from plasmid p gamma(11)7, corresponding to the 5' portion of the constant region of MPC11 heavy chain. Several positive transformants were found to contain various lengths of the variable region of the heavy chain. We describe the structure and sequence of one of these clones, pV(11)2, which contains cDNA corresponding to the entire variable region of MPC11 heavy chain and extends to codon 248 in the constant region. The protein sequence deduced from the DNA sequence indicates that the variable region of MPC11 heavy chain contains 121 amino acids and belongs to subgroup II of mouse heavy chains. Comparison of this sequence with other heavy chain sequences suggests a J (joining) segment of 16 residues which overlaps five residues of the third hypervariable region. The cDNA sequence shows that there is no discontinuity between the end of the variable region and the beginning of the constant region.     

Bovine high molecular weight kininogen. The amino acid sequence, positions of carbohydrate chains and disulfide bridges in the heavy chain portion

The existence of two types of circulating bovine plasma high molecular weight kininogen (HMWK) was predicted from analyses of complementary DNAs coding for this protein (Kitamura, N., Takagaki, Y., Furuto, S., Tanaka, T., Nawa, H., and Nakanishi, S. (1983) Nature 305, 545-549). The present protein-based study provided evidence in support of the proposed amino acid sequence derived from analysis of the cDNA clone, and the results confirm the existence of two types of circulating HMWK. Type I HMWK contains a heavy chain composed of 361 residues, while the heavy chain of type II HMWK contains 359 residues. The amino acid sequences of type I and type II HMWK determined in this study were identical to that inferred from the cDNA sequence with the exception of microheterogeneity observed in the cDNA at position 87 (Glu/Gln) and 168 (Lys/Arg). The heavy chain of type I HMWK contains 4 asparagine-linked carbohydrate chains at Asn-69, -150 (or -151), -179, and -186, while the heavy chain of type II HMWK contains these and an additional carbohydrate chain at Asn-264. In addition, a carbohydrate chain was found to be O-glycosidically linked to Thr-118 in both chains. Among nine disulfide linkages found in HMWK, eight intrachain disulfide pairs were established in the heavy chain. One interchain disulfide bridge occurs between the heavy chain and the light chain. This disulfide pairing, as well as repeating amino acid sequences observed in the heavy chain, provides strong evidence for the existence of three homologous domains in the heavy chain of bovine HMWK.     

Molecular cloning of a bovine immunoglobulin lambda chain cDNA

A cDNA library of the bovine mammary gland constructed in pBR322 was screened by mRNA hybrid-selected translation and by differential hybridization. Several immunoglobulin (Ig) lambda light-chain clones were identified and sequenced. Nucleotide sequence comparison of bovine and human Ig lambda chains showed a high degree of homology for constant regions and for J regions. The amino acid (aa) sequence encoded by the constant region of the bovine Ig lambda chain cDNA contains 107 aa with differences at 24 aa positions from the human Ig lambda chain. Three complementarity-determining regions (CDR1,2,3) characteristic of the variable region of bovine Ig lambda chain cDNA can be distinguished. The bovine and human sequences display good homology in the framework region 3 (FR3) but only patches of homology throughout the FR2 region. The 5' end of the bovine Ig lambda chain cDNA fragment of clone 1-14E contains five stop codons: two in CDR1, one in FR1 and two in the hydrophobic prepeptide region. These data suggest that the Ig lambda mRNA of clone 1-14E is transcribed from the V lambda pseudogene.     

缺氧性肺动脉高压大鼠肺组织中血小板源性生长因子和c－myc基因表达增强

本研究分析了大鼠肺组织中血小板源性生长因子Ａ链、Ｂ链（ＰＤＧＦ－Ａ,ＰＤＧＦ－Ｂ）和ｃ－ｍｙｃ原癌基因ｍＲＮＡ在正常和缺氧时的含量变化。正常肺组织可表达１．７ｋｂ的ＰＤＧＦ－ＡｍＲＮＡ和３．５ｋｂ的ＰＤＧＦ－ＢｍＲＮＡ,还有少量２．２ｋｂｃ－ｍｙｃｍＲＮＡ。在缺氧过程中,ＰＤＧＦ－Ｂ链ｍＲＮＡ和ｃ－ｍｙｃｍＲＮＡ迅速增加,至缺氧１４ｄ时,分别为正常的３倍和５倍。而ＰＤＧＦ－ＡｍＲＮＡ在缺氧７ｄ时增高,而后又略有降低。结果表明：缺氧的肺组织局部生成的ＰＤＧＦ激活了ｃ－ｍｙｃ原癌基因,这对于缺氧性肺动脉高压的形成具有重要作用。     

Preparation of biologically active platelet-derived growth factor type BB from a fusion protein expressed in Escherichia coli

Preparations of the mitogen platelet-derived growth factor (PDGF) from human platelets contain two related polypeptides termed A chain and B chain. PDGF-B is highly homologous to a portion of p28v-sis, the transforming protein of simian sarcoma virus. We have studied the mitogenic potential of a PDGF-BB-like homodimer by expressing the sequence coding for the mature part of PDGF-B in Escherichia coli. Expression was achieved as cro-beta-gal-PDGF-B fusion protein which was exclusively found in the "inclusion bodies". A monomeric PDGF-B fragment shortened by 12 amino acid residues from the NH2 terminus was excised from the fusion protein by CNBr cleavage. After protection of thiols by S-sulfonation, this fragment was purified by gel permeation chromatography and reversed-phase high-performance liquid chromatography. This monomeric protein was dimerized in the presence of a mixture of reduced and oxidized glutathione to yield biologically active rPDGF-BB with an overall yield of approximately 0.7 mg of rPDGF-BB/L of culture. Escherichia coli rPDGF-BB stimulated [3H]thymidine incorporation into AKR2B fibroblast at concentrations of about 1 ng/mL.     

Deletions in the N-terminal coding region of the v-sis gene: determination of the minimal transforming region.

The gene product of the v-sis gene is closely related to the B chain of platelet-derived growth factor (PDGF). However, v-sis also encodes additional amino acids at its N and C termini, which are not represented in the sequence data of PDGF. We have constructed a series of N-terminal deletion mutants in the v-sis gene to define the minimum region required for transformation. These mutants were assayed for biological activity by using retroviral expression vectors which donate a signal sequence, required for translocation across the rough endoplasmic reticulum, to the mutant gene product. The minimal transforming region of the v-sis gene product defined by this analysis has 15 residues missing at the N terminus when compared with the PDGF-B chain. There are only two residues separating the closest transforming and nontransforming gene products. Mutant gene products lacking both the basic dipeptide processing site and the N-linked glycosylation site were found to be biologically active, indicating the dispensability of those processing steps. These results delimit the minimal transforming region of the v-sis gene product to residues 127 through 214, a total of 21 residues smaller than the PDGF-B chain.