Molecular characterization and expression analysis of fibrinogen related protein (FREP) genes of Manila clam (Ruditapes philippinarum) after lipopolysaccharides challenge.

Ruditapes philippinarum has high economic value and is distributed all over the world. Fibrinogen associated protein (FREP) is a type of pattern recognition receptor, participates in the innate immune response to eliminate pathogens after the invasion of pathogenic microorganisms. In this study, three FREP genes (FREP-1, FREP-2, and FREP-3) were identified and characterized from R. philippinarum. The protein sequence of FREPs were highly conserved with those homologous in vertebrates, and FBG domain possessed significantly high structural conservation in polypeptide binding site and Ca2+ binding site. The tissues expression analysis of FREPs in three shell color strains and two population of R. philippinarum were examined, with the highest expression level in gill and hepatopancreas. Besides, FREP genes were demonstrated to be induced by lipopolysaccharides injection, the significantly changes were observed after LPS injected. Our results suggest the involvement of FREPs in response to LPS injection, and it might exert a significant function on the innate immune defense of the Manila clam.

Clinical significance of plasma fibrinogen and D-dimer in predicting the chemotherapy efficacy and prognosis for small cell lung cancer patients

Purpose. Elevated plasma fibrinogen and D-dimer levels indicate activation of hemostasis and fibrinolysis, and this activation is required for tumor angiogenesis, metastasis, and invasion. Previous studies demonstrated that the plasma fibrinogen and D-dimer levels correlate with patient’s prognosis in several solid tumors. The aim of this study is to examine the relationship between plasma fibrinogen and D-dimer levels before and during chemotherapy and treatment response and survival in patients with small cell lung cancer (SCLC). Methods. Plasma fibrinogen and D-dimer levels before and during chemotherapy were prospectively measured in 74 SCLC patients who received first-line therapy. The results were analyzed for correlation between fibrinogen and D-dimer levels and treatment response, as well as progressive-free survival (PFS) and overall survival (OS). Results. The levels of fibrinogen and D-dimer in SCLC patients before (C0) and after two cycles (C2) of chemotherapy were significantly higher than those in controls. Fibrinogen and D-dimer levels decreased during chemotherapy, and changes in fibrinogen and D-dimer levels between at C0 and at C2 were associated with treatment response. No matter which disease stage, patients with fibrinogen or D-dimer positivities at C0 and C2 time points had worse PFS and OS than those with fibrinogen or D-dimer negativities. Multivariate analyses revealed that fibrinogen and D-dimer positivities after two chemotherapy cycles were independently unfavorable factors for PFS and OS. Conclusion. Fibrinogen and D-dimer levels after two cycles of chemotherapy are predictors for response on chemotherapy and prognosis in SCLC patients (AU)

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A molecular phylogeny of Asian barbets: speciation and extinction in the tropics.

We reconstruct the phylogeny of all recognized species of the tropical forest associated Asian barbets based on mitochondrial and nuclear sequence data and test for the monophyly of species and genera. Tropical regions are well known for their extraordinarily high levels of biodiversity, but we still have a poor understanding of how this richness was generated and maintained through evolutionary time. Multiple theoretical frameworks have been developed to explain this diversity, including the Pleistocene pump hypothesis and the museum hypothesis. We use our phylogeny of the Asian barbets to test these hypotheses. Our data do not find an increase in speciation in the Pleistocene as predicted by the Pleistocene pump hypothesis. We do find evidence of extinctions, which apparently contradicts the museum hypothesis. However, the extinctions are only in a part of the phylogeny that is distributed mainly across Sundaland (the Malay peninsula and the islands off southeast Asia). The theory of island biogeography predicts a higher rate of extinction on islands than on mainland areas. The data from the part of the phylogeny primarily distributed on the mainland best fit a pure birth model of speciation, and thus supports the museum hypothesis.

Plasma fibrinogen.

Fibrinogen is the major plasma protein coagulation factor. Low plasma fibrinogen concentrations are therefore associated with an increased risk of bleeding due to impaired primary and secondary haemostasis. Fibrinogen is a classical positive acute-phase reactant protein and is an independent predictor of coronary heart disease events. This review considers available methods for measurement of fibrinogen and makes recommendations as to their appropriate use. Total clottable fibrinogen assays are the definitive and reference method for plasma fibrinogen measurement. However, they are time-consuming and are rarely required in clinical practice. Clotting rate assays remain the routine method of choice for investigation, monitoring and treatment of bleeding disorders associated with low plasma fibrinogen concentrations. They are appropriately sited in haematology or haemostasis laboratories, with facilities for further relevant investigations, and expert advice from consultant haematologists on appropriate management. They have also been used in the majority of studies investigating increasing fibrinogen concentrations as a cardiovascular risk factor. Prothrombin-time derived assays are widely used, because they are less expensive and come at no extra cost with prothrombin time assays. However, their results vary widely with analysers and reagents, show discrepancies with clotting rate assays for both low and normal plasma fibrinogen samples, and at present they are not recommended by routine clinical use. Immunoassays (radial immunodiffusion, enzyme-linked immunosorbent assay or nephelometric) are useful in (a) differentiating hypofibrinogenaemia from dysfibrinogenaemia, and (b) assessing cardiovascular risk and acute-phase reactions. Unlike clottable fibrinogen assays, immunoassays can be performed in dipotassium edetate anticoagulated samples.

Fibrinogen and high molecular weight fibrinogen during and after normal pregnancy.

INTRODUCTION: Pregnancy has recently been described as a generalized intravascular inflammatory response to the conceptus. Total fibrinogen concentrations increase during pregnancy. The percentage high molecular weight fibrinogen (HMW-Fg) of the concentration total fibrinogen is known to increase during acute-phase conditions like inflammation. Therefore, we investigated whether the percentage high molecular weight fibrinogen increases during normal pregnancy. MATERIALS AND METHODS: Eighteen healthy nulliparous women with uncomplicated pregnancies with normal course and outcome participated in this study. Five blood samples were drawn from every woman in the gestational age periods 9 to 16, 17 to 24, 25 to 33 and 34 to 42 weeks and at 12 to 20 weeks after delivery. Total fibrinogen concentrations were determined according to Clauss and the percentage high molecular weight fibrinogen was assessed by SDS-electrophoresis and densitometry after isolation of fibrinogen by precipitation. One-way analysis of variance (ANOVA) was used to evaluate differences between gestational age periods and correlation coefficients were calculated by Pearson's method. RESULTS: Total fibrinogen concentrations increased with advancing gestational age and decreased after delivery. The percentage high molecular weight fibrinogen of the total fibrinogen remained unaltered during and after pregnancy. CONCLUSIONS: During normal pregnancy, there is an increase of total fibrinogen concentrations with advancing gestational age, without a rise in percentage high molecular weight fibrinogen. After delivery, the total fibrinogen returns to baseline concentrations.

Fib420, the novel fibrinogen subclass: newborn levels are higher than adult.

Fib420 is a recently identified subclass of normal human fibrinogen in which two extended alpha chain isoforms (alphaE) replace the common alpha chains, yielding a molecule (ca. 420 kD) which is larger than the more abundant 340-kD form. Evidence for preservation of this subclass throughout vertebrate evolution suggests it performs some as yet unidentified vital function. A survey was undertaken to establish the range of plasma Fib420 levels in normal, healthy adults and in placental cord (fetal) blood. For measuring Fib420, a quantitative Western blot assay was developed using monoclonal antibody against the exon-VI encoded C-terminus of the molecule's unique alphaE chain. This alphaE chain signal was normalized to that of the beta chain, common to both fibrinogen forms. Analysis of plasma samples from the adult and newborn cohorts (n = 25 each; total fibrinogen ca. 2.6 mg/mL in both) revealed a statistically significant difference, with a mean level of 100 +/- 28 microg/mL in the neonate compared to 34 +/- 7 microg/mL in the adult. On average, 1 out of every 100 fibrinogen molecules in adult plasma belongs to the Fib420 subclass. Unlike in the newborn, adult Fib420 levels remained the same over a wide range of total plasma fibrinogen. The striking difference observed between these two cohorts suggests a changing developmental expression of the Fib420 subclass and a homeostatic control operating in later stages of life.

Genetic regulation of fibrinogen.

Prospective epidemiological studies have shown that elevated levels of fibrinogen are associated with thrombosis and ischaemic heart disease. Several sequence changes in the promoter region of the beta-fibrinogen gene have been detected that are associated with slightly raised plasma fibrinogen levels in healthy, non-smoking carriers, but which have much larger genotype-associated effects in smokers. In in vitro assays, these sequence changes affect the binding of liver nuclear proteins and may alter the rate of transcription of the gene and thus the rate of fibrinogen production. One sequence change is close to the consensus sequence for the binding of a nuclear factor responsive to interleukin-6, one of the cytokines responsible for the acute-phase changes seen upon infection or injury. This provides a molecular explanation for the different effects on fibrinogen levels seen in smokers, who are experiencing a 'chronic' and low-grade response to injury. Thus, for elevated plasma fibrinogen, which is associated with a risk of thrombosis, a genetic variation has been detected that determines, in part, its plasma level; but the variability in an individual's response to environmental changes may also be determined in part by their genotype at this locus. In the future, such individual-specific genetic information may be of prognostic and therapeutic use.

Biochemistry and measurement of fibrinogen.

Fibrinogen is a large heterogeneous family of closely related molecules consisting of three pairs of non-identical polypeptide chains: two A alpha-, two B beta- and two gamma-chains, held together by disulphide bridges. The heterogeneity of fibrinogen is due to heterogeneities in all three chains. Four main types of assay are used to determine fibrinogen:clotting rate (Clauss), clottable protein, precipitation and immunological assays. Heterogeneities may differ from person to person and may affect the apparent fibrinogen concentrations in different assays. A further complicating factor was, until recently, the lack of an international fibrinogen standard. The ratio of Clauss:enzyme immunoassay (EIA) for high + low molecular weight fibrinogen decreases during therapy for acute myocardial infarction and increases again after thrombolytic therapy to above normal values. Furthermore, high molecular weight fibrinogen tends to clot more easily than low molecular weight fibrinogen. This suggests that high molecular weight fibrinogen might be associated with increased thrombotic risk. Fibrinogen assessed by a functional assay (Clauss) alone is strongly associated with ischaemic heart disease. Although not proven, it is conceivable that a fibrinogen with a Clauss:EIA ratio of > 1 has an even stronger association in epidemiological studies.

Coordinate transcriptional regulation of the three fibrinogen subunit genes by glucocorticoids in cultured primary liver cells from Xenopus laevis.

Xenopus laevis primary hepatocytes in culture are induced by glucocorticoid hormones to synthesize and secrete fibrinogen. The increase in production of the protein is preceded by a 10-to 30-fold elevation of the mRNAs coding for the three fibrinogen subunits, A alpha, B beta, and gamma. To analyze the mechanisms underlying this coordinate control of independent genes in a common regulatory network, we show here that the steroid hormone induced simultaneous activation of transcription of the three fibrinogen subunit genes. Using an optimized transcription run-on assay for nuclei from Xenopus primary liver cells, we demonstrate that glucocorticoids rapidly stimulated transcription of the A alpha fibrinogen subunit gene by 15- to 20-fold, the B beta gene by 5- to 10-fold, and the gamma gene by 5- to 15-fold. The three genes exhibited a highly concerted response to the hormone, in which maximal stimulation occurred by 30 min and was maintained for at least 16 h. Blocking new protein synthesis before hormone treatment reduced total transcription by 45% and partially inhibited specific hormonal induction of all three fibrinogen subunit genes. The effect of glucocorticoids on fibrinogen transcription, therefore, was dependent in part on ongoing protein synthesis, suggesting that hormonal stimulation uses already synthesized stable factors, but also requires labile or newly synthesized factors for the full effect.

Factor I reference typing report.

Complement factor I (IF) reference typing has been examined using polyacrylamide gel isoelectric focusing followed by electroblotting with enzyme immunoassay. The submitted samples could be classified into three common and two new variants, and these were controlled by two common and two rare alleles. Two common alleles were IF*B (the most common allele) and IF*A, and two new rare alleles were designated IF*A1 and IF*B1, respectively.

Interactions of human blood platelets with three circulating plasma fibrinogens of different molecular weights.

The ability of three naturally occurring human fibrinogen species, HMW, LMW and LMW', to support ADP-induced platelet aggregation was investigated and compared to their ability to bind to gel-filtered platelets. Whereas HMW had intact subunit chains, LMW and LMW' had defined lesions in the C-terminal part of one (LMW) or both (LMW') of the A alpha-chains. The ADP-induced aggregation of gel-filtered platelets in the presence of LMW was about 75 per cent of that obtained with HMW (0.2 mumol/l of fibrinogen and 10 mumol/l of ADP). A mixture of equal amounts of LMW and LMW' showed around 50% decrease in aggregation. Compared to these difference in aggregation co-factor function, direct binding to gel-filtered platelets was less affected by the A alpha-chain degradation. However, a difference between LMW, LMW' and HMW binding was significant when the fibrinogens labelled with two different isotopes of iodine were present simultaneously. These results demonstrate that naturally occurring plasma fibrinogens differ in their interactions with platelets. As the HMW/LMW ratio changes during the acute phase, this may be of physiological significance.

A hereditary dysfibrinogenemia: fibrinogen Awaji.

Abnormal function of fibrinogen was observed in a 25-year-old woman with no symptoms attributable to dysfibrinogenemia. Disturbed polymerization of fibrin monomer was identified, but the release of fibrinopeptide from the purified fibrinogen and the cross-linking by factor XIII were normal. Other abnormal findings included a high value of fibrinogen degradation products, rapid mobility on immunoelectrophoresis and abnormal spot of gamma-chain on two-dimensional polyacrylamide gel electrophoresis. Similar abnormalities were also observed among the patient's family members.

[Studies on fibrinogen heterogeneity in disseminated intravascular coagulation].

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis utilizing a large pore gel showed that human fibrinogen (Fbg) in plasma resolved into 3 components which were tentatively designated as high molecular weight Fbg (HMW), low molecular weight Fbg1 (LMW1) and low molecular weight Fbg2 (LMW2) in order of their electrophoretic mobilities. Their estimated molecular weight were 360,000, 325,000 and 290,000 respectively. In normal plasma the ratio of HMW: LMW1: LMW2 was 71: 27: 2. Subunit chain analysis of Fbg species suggests that one of the A alpha chains in the predominant type of LMW1 is an A alpha remnant designated as A alpha', the molecular weight of which is about half that of the A alpha chain of HMW. The subunit chain formulas for the major forms of HMW and LMW1 are considered as (A alpha/1, 2)2 (B beta)2 (gamma)2 and (A alpha/1, 2) (A alpha') (B beta)2 (gamma)2 respectively. Although it is widely accepted that limited proteolysis of A alpha chain by plasmin might be the cause of Fbg heterogeneity, the conversion of HMW to LMW1 was not observed in the earliest stage of fibrinogenolysis induced by urokinase. However incubation of plasma induced the conversion of HMW to LMW1, even when t-aminomethyl-cyclohexane carboxylic acid or heparin was added to the plasma. These findings suggest that an unknown mechanism independent of plasmin or thrombin is responsible for the Fbg heterogeneity in blood. Clinically it is noticeable that the relative amount of LMW1 decreased markedly in disseminated intravascular coagulation (DIC). Animal models of DIC also exhibited essentially the same Fbg heterogeneity pattern as observed in DIC patients, when the levels of Fbg were recovering after the consumption caused by intravascular coagulation. In DIC it is considered that the consumption and compensatory production of Fbg results in the relative increase of HMW which is the freshly synthesized Fbg.

Congenital dysfibrinogenemia: fibrinogen Detroit.

A 17 yr old female with a congenital bleeding disorder was found to suffer from dysfibrinogenemia. Whole blood and plasma coagulation times were delayed and thrombelastograms were grossly abnormal. Clottability of plasma fibrinogen by addition of thrombin was not demonstrated during the 30 min test period. Fibrinogen was revealed by turbidometric and immunologic techniques. Other coagulation factors were present in normal amounts and prothrombin activation was normal. Patient's plasma inhibited thrombin clotting times of normal plasma and purified normal fibrinogen. Fibrinolysis was not detected. The plasma fibrinogen migrated normally on paper and cellulose acetate electrophoresis, but on immunoelectrophoresis it displayed a faster mobility than normal fibrinogen. On immunodiffusion the antigenic determinants were similar to those of normal fibrinogen. The patient's fibrinogen-antifibrinogen precipitins required longer to appear and the resultant precipitin was broader and hazier than those elicited with normal fibrinogen. These findings suggest the presence of two discrete populations of fibrinogen molecules. Investigation of the family of the patient suggested that the defect has an autosomal dominant pattern of heredity. Immunologic comparisons of our patient's plasma and of her relatives with plasma of patients with "Fibrinogen Baltimore" and "Fibrinogen Cleveland" revealed certain differences in immunoelectrophoretic mobility as well as in immunodiffusion. In keeping with the nomenclatures of abnormal fibrinogens in the literature, we propose the term "Fibrinogen Detroit" for this fibrinogen.Physicochemical properties of "Fibrinogen Detroit" were investigated also and compared with those of normal fibrinogen. Purified normal fibrinogen (clottability 96.7%) and "Fibrinogen Detroit" revealed homogeneity when studied by ultracentrifugation and immunoelectrophoresis. Native and cleaved "Fibrinogen Detroit" had the same sedimentation constants and molecular weights as the normal. In fresh samples. 3 moles of free SH groups/mole of fibrinogen were titrated in both. Determination of the amino acid composition revealed a decreased content of lysine, glucosamine, and galactosamine in abnormal fibrinogen. Total carbohydrates, protein-bound hexoses, sialic acid, and hexosamine were decreased in the abnormal fibrinogen. In an investigation with Doctors Blombäck a specific molecular defect was revealed in the N-terminal disulfide knot of the alpha (A) chain in which the arginine at the 19th position was replaced by serine. It is believed that the substitution of a strongly basic amino acid with a neutral hydroxy acid may result in considerable conformational changes in the N-terminal disulfide knot of fibrinogen which might affect the "active site" for polymerization. The lower carbohydrate content observed in "Fibrinogen Detroit" may have been the result of a change in primary and tertiary structure of the protein.