Stimulation of plasminogen activator and inhibitor in the lymphatic endothelium.

Chromogenic assays, immunoblotting, and Northern blot hybridization methods were employed to assess the effects of various agonists on the production of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) by the lymphatic endothelium (LEC). Fibrin autography showed that plasminogen-dependent fibrinolytic activity occurred at M(r) of 110 kDa, which represents a complex of tPA with PAI-1, and 65- and 55-kDa bands corresponding to tPA and uPA, respectively. The fractionation of lymph collected from ovine lymphatic vessels also produced a prominent lytic band of approximately 110 kDa, suggesting the formation of PA/PAI complexes in lymph. The stimulation of various agonists produced large-scale increases in tPA mRNA, as shown by Northern blot hybridization analyses. The effects of ECGF, histamine, and LPS on the presence of tPA and on enhancing the levels of mRNA reached maximum activity at 4 h and declined to levels below that of controls by 8 h. However, phorbol-treated cells exhibited reduced levels of tPA mRNA at 4 h, but was significantly increased by 8 h. A large-scale increase in PAI-1 mRNA steady-state levels was also stimulated by the agonists used in these studies. Both the 3.4- and 2.4-kb species of PAI-1 mRNA were increased. These observations demonstrated that tPA and PAI-1 are produced and secreted by LEC monolayer cultures and are also present in lymph.

BRCA1 mutations in African Americans.

The breast cancer predisposing gene, BRCA1, was analyzed for germline mutations in 45 African American families at high-risk for hereditary breast cancer. Patients were considered high-risk if they had a family history of the disease, early onset breast cancer, bilateral breast cancer, or breast and ovarian cancer. The entire BRCA1 coding and flanking intron regions have been examined by single stranded conformation polymorphism analysis followed by sequencing of variant bands. Eleven different BRCA1 germline mutations/variations were identified in 7 patients from the 45 high-risk families. Two pathogenic, protein-truncating mutations were detected in exon 11. A ten base pair tandem duplication, 943ins10, was present in a woman with breast and ovarian cancer whose first-degree relatives had prostate cancer. A four base pair deletion, 3450del4, was detected in a breast cancer patient with five cases of breast cancer in the family; two of the proband's sisters with breast cancer also carried the same mutation. Four amino acid substitutions (Lys1183Arg, Leu1564Pro, Gln1785His, and Glu1794Asp) and four nucleotide substitutions in intron 22 (IVS22+78 C/A, IVS22+67 T/C, IVS22+8 T/A and IVS22+7 T/C) were observed in patients and not in control subjects. One early onset breast cancer patient carried five distinct BRCA1 variations, two amino acid substitutions and three substitutions in intron 22. An amino acid substitution in exon 11, Ser1140Gly, was identified in 3 different unrelated patients and in 6 of 92 control samples. The latter probably represents a benign polymorphism.

Structure and expression of osteonectin mRNA in human tissue.

A cDNA encoding osteonectin was isolated from a human bone cell cDNA library and used to examine osteonectin protein structure, mRNA structure and expression in human tissue. The deduced protein sequence shows complete identity with a recently isolated placental form and extensive homology to mouse and bovine counterparts. The protein is rich in cysteine residues, which are conserved between species except for cys 194 which is only present in the bovine. In the human, osteonectin mRNA is of two sizes, 2.3 and 3.0 kb, the former being dominant in all tissues studied. Human mRNA was detected in the Ewing sarcoma and in non-bone cell and tissue sources. The potential folded structure of osteonectin mRNA was estimated, based on computer predictions, and indicates the presence of a bulge at the 5' end of the message which includes the start of translation. Southern analysis of human genomic DNA using radiolabeled osteonectin cDNA as probe demonstrates a simple banding pattern confirming earlier studies that the osteonectin gene is present in one copy per haploid human genome.

Molecular cloning and sequence analysis of the cDNA for small proteoglycan II of bovine bone.

The cDNA for the full-length core protein of the small chondroitin sulphate proteoglycan II of bovine bone was cloned and sequenced. A 1.3 kb clone (lambda Pg28) was identified by plaque hybridization with a previously isolated 1.0 kb proteoglycan cDNA clone (lambda Pg20), positively identified previously by polyclonal and monoclonal antibody reactivity and by hybrid-selected translation in vitro [Day, Ramis, Fisher, Gehron Robey, Termine & Young (1986) Nucleic Acids Res. 14, 9861-9876]. The cDNA sequences of both clones were identical in areas of overlap. The 360-amino-acid-residue protein contains a 30-residue propeptide of which the first 15 residues are highly hydrophobic. The mature protein consists of 330 amino acid residues corresponding to an Mr of 36,383. The core protein contains three potential glycosaminoglycan-attachment sites (Ser-Gly), only one of which is within a ten-amino-acid-residue homologous sequence seen at the known attachment sites of related small proteoglycans. Comparisons of the published 24-residue N-terminal protein sequence of bovine skin proteoglycan II core protein with the corresponding region in the deduced sequence of the bovine core protein reveals complete homology. Comparison of the cDNA-derived sequences of bovine bone and human embryonic fibroblast proteoglycans shows a hypervariable region near the N-terminus. Nucleotide homology between bone and fibroblast core proteins was 87% and amino acid homology was 90%.

Characterization of bone PG II cDNA and its relationship to PG II mRNA from other connective tissues.

Two cDNA clones encoding the small proteoglycan II (PG II) of bone were isolated from a lambda gt11 expression library. These clones expressed recombinant protein which was cross-reactive with polyclonal and monoclonal antisera to PG II molecules from several connective tissues. The longest clone, lambda Pg 20 was studied in detail. The clone was shown to encode PG II by hybrid selected translation and immunoprecipitation. Northern analysis showed two species of the PG II message of approximately 1.4 and 1.8 kb. Substantial amounts of PG II message were found in bone, tendon, articular cartilage, skin, smooth muscle and cornea. Trace amounts of message were also detected in liver and brain. Radiolabeled bovine PG II cDNA hybridized to RNA from several other species including the human, rat and chicken. The level of PG II mRNA in chick embryonic fibroblasts was sensitive to transformation by Rous sarcoma virus.

Osteonectin mRNA: distribution in normal and transformed cells.

Overlapping cDNA clones encoding bovine osteonectin were isolated from a lambda gt11 expression library constructed from bovine bone cell mRNA. The longest clone, lambda On 17 (insert size 2.0 kb) was studied in detail. The clone was shown to encode osteonectin by hybrid select translation experiments and by DNA sequence analysis. Northern analysis of bone cell RNA showed the length of the osteonectin mRNA to be 2.0 kb. Osteonectin message was found in bone but not in soft tissue (liver and brain) preparations consistent with the distribution of the protein in these tissues. On the other hand, osteonectin message was observed in tendon, a tissue in which little or no osteonectin protein is found in vivo. Hybridization of osteonectin cDNA was detected in cells from a number of species including human, rat, mouse and chick. The level of osteonectin mRNA was drastically decreased in chick embryo fibroblasts transformed by Rous sarcoma virus.