ABSTRACT: Fibrinogen synthesis in the intact rat was perturbed by treatment with cycloheximide. Specific radioactivities of fibrinogen in plasma and liver both decreased at 2 h after treatment and increased over 2-fold by 18 h. Labelled-antibody--polyribosome binding experiments showed that more polyribosomes were engaged in fibrinogen synthesis at 18 h after treatment. Radioactivity of plasma fibrinogen chains from untreated control rats showed a constant ratio of A alpha--B beta/gamma = 1.03. At 2 h after cycloheximide treatment the A alpha- and B beta-chains showed the greatest decrease in labelling (A alpha--B beta/gamma = 0.66) and at 18 h all chains were much more labelled (the A alpha--B beta/gamma ratio chainged to 1.39). The observed imbalance in fibrinogen-chain synthesis suggests that cycloheximide has a selective effect on gene expression.
Project description:The message for a second fibrinogen alpha chain has been cloned from a lamprey liver cDNA library. The sequence is unique in that the amino-terminal half is homologous to all other known alpha chains, including another from lamprey, but its carboxyl-terminal half is homologous to the carboxyl-terminal portions of beta and gamma chains, segments that compose the distal globular regions of fibrinogen. The structural pattern of this newly discovered alpha chain suggests that it could be a direct descendant of the archetypal chain that existed prior to the gene duplications that led to unique beta and gamma chains and before the dislocating events that gave rise to contemporary alpha chains.
Project description:The biosynthesis, assembly and secretion of fibrinogen were investigated in cultured rat hepatocytes which were incubated with [35S]methionine. When initial rates of the synthesis of three fibrinogen subunits were compared, the A alpha-subunit was found to be synthesized significantly slower than the B beta- and gamma-subunits. Pulse-chase experiments revealed that the secreted fibrinogen contained different proportions of the newly synthesized subunits, depending upon the chase times. Radioactivity in the A alpha subunit, which initially had the highest level of the three, was rapidly decreased in parallel with the chase time. The gamma-subunit had an increasing amount of the radioactivity in the secreted molecule during the chase periods, whereas that in the B beta-subunit was gradually decreased at the later stages of chase. Analysis of intracellular components of fibrinogen confirmed that the nascent A alpha-subunit was most rapidly exhausted, and the gamma-subunit occupied the largest proportion among the non-assembled subunits at later stages of chase. Taken together, these results suggest that the synthesis of A alpha-subunit, which has the lowest rate, could be the rate-limiting step in the production and secretion of fibrinogen in cultured rat hepatocytes, in contrast with what has been proposed for human and rabbit fibrinogen, namely that the synthesis of B beta-subunit is the rate-limiting step. The results also indicate that there is a large intracellular pool of gamma-subunit.
Project description:Snake venom contains large amounts of active proteins and peptides. In this study, a novel snake protein, metalloproteinase SP, was successfully isolated from the venom of Agkistrodon acutus by multi-gel chromatography. The isolated protein exhibits anti-platelet aggregation activity. Animal experiments showed that it exhibited defibration, anticoagulation, and antithrombotic effects and contributes to improved blood rheology and antiplatelet aggregation. In vivo experiments demonstrated that it prolonged clotting time, partial thromboplastin time, prothrombin time, thrombin time, fibrinogen time and reduced fibrinogen content of mice. Also, metalloproteinase SP inhibited carrageenan-induced tail thrombosis, ADP-induced acute pulmonary embolism, and ADP, Arachidonic acid (AA), or collagen-induced platelet aggregation. In vitro experiments showed that the protein cleaved the α, β, and γ chains of fibrinogen. Metabolomic analysis upon metalloproteinase SP treatment revealed that 14 metabolites, which are mainly involved in phenylalanine, tyrosine, and tryptophan biosynthesis, responded to metalloproteinase SP treatment. In summary, the isolated snake venom protein inhibits formation of acute pulmonary embolism probably through regulating and restoring perturbed energy, lipid, and amino acid metabolism.
Project description:Chondroitin sulphate synthesis on proteoglycans was decreased in rat chondrosarcoma cell cultures in the presence of cycloheximide (0.1-1.0 muM) or p-nitrophenyl beta-D-xyloside (50 microM). In the presence of cycloheximide the proteoglycan monomer was of larger size, the chondroitin sulphate chains were increased in length, but a similar number of chains was attached to each proteoglycan and the size of the core protein was unaltered. In the presence of p-nitrophenyl beta-D-xyloside (50 microM), chondroitin sulphate synthesis was increased (by 60-80%), but the incorporation into proteoglycans was decreased (by 70%). The chondroitin sulphate chains were of shorter length than in control cultured and the number of chains attached to each proteoglycan was decreased. In cultures with cycloheximide or actinomycin D the synthesis of chondroitin sulphate was less inhibited on beta-xyloside than on endogenous proteoglycan. When the rate of chondroitin sulphate synthesis was decreased by lowering the temperature of cultures, the chains synthesized at 22 and 4 degrees C were much longer than at 37 degrees C, but in the presence of p-nitrophenyl beta-D-xyloside the chains were of the same length at all three temperatures. A model of chain elongation is thus proposed in which the rate of chain synthesis is determined by the concentration of xylosyl acceptor and the length of the chains is determined by the ratio of elongation activity to xylosyl-acceptor concentration.
Project description:Sequence comparisons of the three homologous polypeptide chains that compose vertebrate fibrinogens imply that the molecule evolved before the divergence of vertebrates and invertebrates, but, to our knowledge, no protein resembling vertebrate fibrinogen has even been reported from an invertebrate. We used primers based on sequences conserved between lamprey and human fibrinogens and applied the polymerase chain reaction (PCR) to cDNA preparations from various invertebrates. A fibrinogen-like sequence was identified in cDNA prepared from the soft tissues of a sea cucumber, parastichopus parvimensis. The PCR-prepared material was then used to clone two closely related mRNA sequences from a sea cucumber soft tissue cDNA library. The putative fibrinogen-related proteins, FReP-A and FReP-B, correspond to the carboxyl-terminal two-thirds of vertebrate fibrinogen beta and gamma chains. Computer comparisons of various fibrinogen-related sequences indicate that the sea cucumber proteins diverged before the beta-gamma gene duplication.
Project description:Amino acids labelled with 18(O) on both carboxy oxygen atoms have the potential for use as non-recyclable tracers to measure protein turnover. During protein synthesis one of the labelled oxygen atoms is removed, and thus release of the mono-labelled amino acid could be used to determine proteolysis. Primary cultures of embryonic-chick skeletal-muscle cells were used to test the use of 18(O2)-labelled Leu to measure proteolysis. For 9-day cultures, prelabelled on days 2-8 with medium containing one-half the Leu as [18O2]Leu and one-half as [2H3]Leu, release of [18(O)]Leu was less than 50% that of [2H]Leu over 24 h, suggesting a loss of the 18O label by a mechanism other than protein synthesis. Medium containing [18(O2)]Leu, [2H3]Leu, [18O2]Phe and [13C]Phe was then incubated with 9-day cultures to compare the rate of loss of the 18(O)-label from Leu and Phe with the rate of uptake of the non-carboxy-oxygen-labelled amino acids. Results for Leu demonstrated an 81% loss of the 18(O) label compared with a 33% decrease in [2H]Leu over 12 h. Loss of the 18(O) label was four times as great for Leu as for Phe. Loss of the 18(O) label was not decreased by addition of cycloheximide or by addition of a 3-fold excess of Ile, Val and Tyr; thus the loss of label was not due to protein synthesis alone or to misbinding to incorrect tRNAs. Infusion of the isotopes into pigs showed that the 18(O) label of Leu was not lost during transamination to alpha-ketoisocaproate (alpha-oxoisohexanoate). The most probable explanation is that the 18(O) label is lost as a result of the enzymic deacylation of tRNA, that this process is substantially faster for Leu than for Phe, and that this represents a potentially costly futile cycle for Leu.
Project description:We have examined the combined effects of transforming growth factor-beta (TGF-beta), serum and gamma-interferon (gamma-IFN) on collagen synthesis by fibroblasts and compared the response of fibroblast subpopulations to TGF-beta. Human diploid fibroblasts were treated with TGF-beta alone and with serum of gamma-IFN. Cells were labelled with radioactive amino acids, and collagen production was measured as collagenase-digestible radioactivity. Collagen mRNA was determined by a solution-hybridization assay using procollagen-alpha 1[I] cDNA clone HF 677. The results showed that either serum or TGF-beta increased incorporation, collagen production and mRNA by fibroblasts approx. 2-fold; however, collagen synthesis relative to total protein synthesis and collagen mRNA relative to total polyadenylated [poly(A)+] RNA were not affected. Only serum activated cell growth. Collagen production increased approx. 4-fold in cells exposed to both TGF-beta and serum, and this increase was equal to that expected for an additive effect by both components. Treatment with gamma-IFN decreased collagen production and collagen mRNA to 44 and 40% respectively, whereas total incorporation and poly(A)+ RNA were affected only marginally. Cells exposed simultaneously to both gamma-IFN and TGF-beta produced less collagen and contained less mRNA than did those treated with TGF-beta alone. The gamma-IFN decreased collagen synthesis in control and TGF-beta-treated cultures to a similar extent, and TGF-beta increased collagen synthesis 2-fold in cells pre-treated with gamma-IFN. Fibroblast strains obtained in medium containing plasma-derived serum synthesized approximately half as much collagen as did cells derived from the same explant in the presence of fresh human serum, and TGF-beta stimulated collagen production and mRNA in both cell strains. We conclude that TGF-beta, serum and gamma-IFN regulate collagen synthesis by independent mechanisms, and that the combined action of these components plays a significant role in regulating collagen synthesis during wound healing and tissue repair.
Project description:The present study was designed to examine the interaction of the purified platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa or integrin alpha IIb beta 3) and the individual subunits of the complex with immobilized fibrinogen. Although 125I-GP IIb-IIIa binding to fibrinogen immobilized on Sepharose was specific, this interaction exhibited properties distinct from those of reversible fibrinogen binding to platelets: 125I-GP IIb-IIIa binding appeared irreversible, but non-covalent, Ca(2+)-independent, and was inhibited only weakly, or not at all, by the anti-(GP IIb-IIIa) monoclonal antibodies 10E5 and 7E3 and synthetic peptides from known platelet-binding domains of fibrinogen. Reversibly dissociated GP IIb or GP IIIa subunits inhibited 125I-GP IIb-IIIa binding to immobilized fibrinogen and bound directly to the fibrinogen. However, these subunits did not bind to peptides derived from known platelet-binding domains within the fibrinogen alpha- and gamma-chains, although the GP IIb-IIIa complex did. These results show that the complexed form of full-length GP IIb and GP IIIa is required for binding to these synthetic peptides, but not necessarily for binding to immobilized fibrinogen. Thus GP IIb-IIIa can bind to immobilized fibrinogen by a distinct mechanism that appears to involve novel binding sites on each subunit of the GP IIb-IIIa complex and on fibrinogen.
Project description:Drosophila laminin alphabetagamma trimer assembly in Kc167 cells was perturbed by chain-specific RNA interference (RNAi). The intracellular pool of alpha and gamma chains remained unchanged under beta-chain RNAi by lipofection of double-stranded RNA encoding a beta-chain partial sequence. This was also the case for the intracellular pool of alpha and beta chains under gamma-chain-specific RNAi. Nonetheless, the intracellular pool of beta and gamma chains increased markedly under alpha-chain-specific RNAi. Non-reducing SDS/PAGE revealed that some of the increased beta and gamma chains migrated as disulphide-linked betagamma dimers but that the rest migrated as monomers. Since the monomeric beta and gamma bands detected under alpha-chain RNAi were denser than the beta band under gamma-chain RNAi and the gamma band under beta-chain RNAi, respectively, beta and gamma also appeared to accumulate by forming betagamma dimers without the disulphide linkage. We suggest that interconversion of these betagamma dimers is crucial for the replaceable and selective assembly of the alpha chain for alphabetagamma trimer formation.
Project description:BACKGROUND:Congenital fibrinogen disorders are caused by variants occurring within the fibrinogen gene cluster. We describe ten subjects with disease-causative variants, adding information on such disorders. MATERIALS AND METHODS:Ten subjects were referred to our Centre because of likely hypo/dysfibrinogenaemia. We evaluated the function and quantity of fibrinogen, using Clauss and immunoreactive assays, and performed genetic investigations by direct sequencing of alpha, beta and gamma chain-encoding genes. Mutations were analysed using SIFT and Polyphen-2 algorithms. RESULTS:We identified one afibrinogenaemic patient (alpha p.Arg178* homozygote) with bleeding/thrombotic events, three heterozygous patients with hypo/dysfibrinogenaemia (gamma p.Thr47ILeu combined with beta IVS7+1G>T; beta p.Cys95Ser; beta p.Arg196Cys) referred for bleeding or thrombotic episodes and six heterozygous subjects with hypofibrinogenaemia (alpha p.Glu41Lys; gamma p.Gly191Val; beta p.Gly288Ser; gamma p.His333Arg; gamma p.Asp342Glu and p.343-344 duplication; gamma p.Asp356Val), of whom four were symptomatic. Five novel missense changes and one novel duplication variant were found, all in hypofibrinogenaemic subjects: p.Glu41Lys (SIFT score 0, Polyphen-2 score 0.986) was identified in a woman with bleeding after major orthopaedic surgery; p.Gly191Val (SIFT score 0.02, Polyphen-2 score 1) in an asymptomatic woman; p.His333Arg (SIFT score 0, Polyphen-2 score 1) in a woman with a post-partum haemorrhage; and p.Asp342Glu (SIFT score 0.23, Polyphen-2 score 0.931); and an Asn-343 and Asp-344 duplication in a child who developed a haematoma following a fall. DISCUSSION:All but one of the novel mutations were in symptomatic subjects and are predicted to be deleterious. Our findings shed more light on genotype-phenotype relationships in congenital fibrinogen disorders.