Carbohydrate heterogeneity of fibronectins. Synovial fluid fibronectin resembles the form secreted by cultured synoviocytes but differs from the plasma form.

Large quantities of fibronectin (Fn) are present in inflammatory synovial fluid. Inflammatory synovial fluid Fn, while indistinguishable from plasma Fn on the basis of reactivity to polyclonal antibodies, displays alterations in molecular size and charge. Since biochemical differences between plasma and synovial fluid fibronectins might be in part due to differences in glycosylation we have compared the carbohydrate composition of plasma Fn, synovial fluid Fn, and Fn from synoviocyte conditioned medium by biochemical assay, glycopeptide analysis, and binding to a series of lectins. Synovial fluid Fn has a greater carbohydrate content but contains less sialic acid when compared with plasma Fn. Glycopeptides formed from synovial fluid Fn are smaller than plasma Fn glycopeptides. These data suggest the presence of an additional N-linked oligosaccharide chain on synovial fluid Fn. In addition, synovial fluid Fn contains N-acetyl galactosamine indicating the presence of O-linked oligosaccharides. Synovial fluid Fn and Fn isolated from rheumatoid synoviocyte-conditioned medium display strong reactivity with the lectins wheat germ agglutinin (WGA) and peanut agglutinin (PNA), whereas normal and rheumatoid plasma Fn react weakly. The PNA reactivity of synovial fluid Fn is mediated by terminal beta-galactose residues on the gelatin-binding domain, whereas the enhanced WGA reactivity of synovial Fn is mediated by a sialic acid containing oligosaccharide located on a 27-kD C-terminal fragment. These data demonstrate domain-specific biochemical differences between plasma and synovial fluid fibronectins. These differences suggest a local origin for synovial fluid Fn and may contribute to functional differences between these forms of the protein.     

Fibronectin determination in pregnancy

The levels of fibronectin were determined by immunoturbidimetric assay in two populations: (a) plasma of healthy nonpregnant and pregnant women, and in amniotic fluid of healthy pregnant females; (b) plasma of umbilical cord blood of healthy newborns and of newborns with sepsis. Fibronectin concentrations of amniotic fluid showed a significant decrease during pregnancy, but the changes of plasma fibronectin levels were not significant in this period. In newborn sepsis, the levels of plasma fibronectin were significantly decreased. We did not find a significant difference between the fibronectin concentration of umbilical cord blood of premature infants compared to mature infants.     

Changes in alpha 1-acid glycoprotein serum concentrations and glycoforms in the developing human fetus.

alpha 1-Acid glycoprotein concentrations and reactivity to concanavalin A were measured in maternal and fetal serum and amniotic fluid obtained from 24 women undergoing diagnostic cordocentesis at 20 to 33 wk gestation and in 30 additional fetal sera (19 to 34 weeks gestation). Maternal alpha 1-acid glycoprotein serum levels were five to ten times higher than fetal and amniotic levels. Fetal alpha 1-acid glycoprotein levels were found to increase with advancing gestational age. Using crossed immunoaffino electrophoresis with concanavalin A, alpha 1-acid glycoprotein patterns were identical in maternal serum and amniotic fluid but totally different in fetal serum. The fetal concanavalin A pattern changed progressively during fetal life towards that of the newborn. These data confirm earlier assumptions of fetal synthesis of alpha 1-acid glycoprotein and provide normal reference values for alpha 1-acid glycoprotein in fetal serum. In addition, the specific fetal concanavalin A pattern indicates that the alpha 1-acid glycoprotein glycosylation process during fetal life differs from that in post-natal life.     

Affinity of fibronectin to collagens of different genetic types and to fibrinogen

Fibronectin, a fibroblast surface protein, was purified from human and chicken plasma and extracts of cultured chicken fibroblasts with affinity chromatography on gelatin coupled to Sepharose particles. A fibronectin-like protein was also isolated from the plasma of Torpedo fish. The collagen binding properties of fibronectin were studied with several genetically distinct collagens. Heat denatured types I, II, and III collagens were equal in their binding capacity and more active than the native collagens or A and B chains. Native type III collagen was more active than the other native collagens. Human and chicken fibronectins showed approximately the same pattern of specificity. Identical specificities were shown by the plasma and fibroblast forms of chicken fibronectin. Two cyanogen bromide peptides of the collagen alpha1 (II) chain, CB8 and CB12, derived from different parts of the chain, were active in fibronectin binding. A polymer of the tripeptide pro-gly-pro, and polyproline were inactive. Fibronectin also binds to fibrinogen and fibrin. Comparison of this binding to collagen binding showed that fibrinogen inhibited binding of fibronectin to collagen, but was less active than native collagen. Two other fibrous proteins, tropoelastin and keratin, did not bind fibronectin. The binding of fibronectin to fibrinogen was inhibited by collagen and incorporation of fibronectin into blood clot in the cold was inhibited by gelatin. These results suggest that the binding of fibronectin to collagen and fibrinogen depends on the same binding site in the fibronectin molecule. It is proposed that cell surface fibronectin mediates attachment of cells to the collagenous extracellular matrix and to a temporary fibrin matrix in a wound.     

A serine protease activity of human serum albumin towards 4-methylumbelliferyl-guanidinobenzoate (MUGB) and diisopropyl fluorophosphate (DEP): implications for the use of MUGB reactivity in amniotic fluid in prenatal diagnosis of cystic fibrosis

4-methylumbelliferylguanidinobenzoate (MUGB) reactivity in plasma from patients with cystic fibrosis and in amniotic fluid from pregnancies leading to children with cystic fibrosis, has been reported to be significantly decreased. We have so far been unable to confirm these findings and have therefore reexamined this reactivity using diisopropylfluorophosphate (DEF), another active site titrant of serine proteases. We have shown that MUGB and DFP are reacting with the same molecules in plasma and amniotic fluid. Using crossed immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis of 3H-DFP labelled plasma and amniotic fluid we have obtained strong evidence that the main contribution of MUGB and DFP reactive protein in plasma and amniotic fluid is identical to serum albumin. The use of MUGB reactivity in amniotic fluid in pregnancies at risk for cystic fibrosis must therefore be reconsidered.     

Levels of free and conjugated catecholamines in amniotic fluid and in umbilical and maternal blood in patients undergoing cesarean section

A new radioenzymatic assay was used to evaluate free and conjugate catecholamines in six pregnant women who underwent cesarean section at term, in their newborns and in the amniotic fluid. Free adrenaline in maternal plasma was higher while noradrenaline and dopamine were lower at the moment of surgery than 24 hours before the operation. In umbilical plasma adrenaline and noradrenaline are higher than in maternal plasma while only traces of free dopamine and salsolinol are present. In the amniotic fluid high levels of free and conjugated salsolinol are found. The high level of free and conjugated catecholamines found in the umbilical plasma demonstrates that fetal sympathetic nervous system is strongly activated at delivery. Furthermore the presence of sulfoconjugating activity similar to that of the adult is confirmed.     

The thromboplastic activity of lung surfactant in amniotic fluid and its application to prenatal assessment of fetal lung maturity

Based on the fact that both tissue thromboplastin and lung surfactant show lamellar structures under the electron microscope and belong chemically to lipoprotein, the thromboplastic activity of lung surfactant in amniotic fluid was studied by measuring plasma recalcification time. The results obtained were as follows (1) The surfactant fractions isolated from amniotic fluid and rabbit or pig lung showed the thromboplastic activity with dose response. (2) The thromboplastic activity of amniotic fluid increased with advancing gestational age. (3) It was found that the thromboplastic activity determined by plasma recalcification time was parallel with the surfactant concentration of amniotic fluid. (4) The shortening rate of plasma recalcification time in amniotic fluid could estimate well the risk of RDS, and the critical value for RDS was assumed to be about 33%.     

A simple and reliable method for the purification of human alphafetoprotein (AFP) from amniotic fluid and fetal livers.

Highly purified human alphafetoprotein has been isolated from amniotic fluid and fetal livers by a combination of salting-out, and gel filtration, ion exchange, and concanavalin-A affinity chromatography. They yield ranged from 25 to 37%, and purity was demonstrated by radioimmunodiffusion, crossed radioimmunoelectrophoresis, polyacrylamide gel electrophoresis, and comparison with other preparations by radioimmunoassay. Immunochemical potency by weight of alphafetoprotein purified from amniotic fluid was similar to that from fetal liver, with molecular weights of 70 000 and 68 500 respectively. The amino acid and carbohydrate composition is also reported. This physicochemical method is relatively simple and inexpensive and is well suited for large scale production.     

正常足月产妇羊水组织因子水平与妊娠高凝状态、羊水栓塞的关系研究

目的探讨正常足月产妇羊水中的组织因子(TF)水平与妊娠高凝状态及羊水栓塞的关系。方法选取2013年1月至2014年12月该院待产的正常足月妊娠产妇158例,检测产妇血浆、羊水、羊水上清液及羊水沉渣中的TF和组织因子途径抑制物(TFPI)水平。结果羊水沉渣TF水平为(1 409.36±120.34)ng/L,明显高于血浆、羊水及羊水上清液水平,差异有统计学意义(P0.05);血浆TF水平为(30.17±6.49)ng/L,明显低于羊水各标本水平,差异有统计学意义(P0.05);羊水沉渣TFPI水平为(9.46±1.77)g/L,明显低于血浆、羊水及羊水上清液水平,差异有统计学意义(P0.05);血浆TFPI水平为(22.19±5.16)g/L,明显高于羊水各标本(P0.05);羊水及羊水上清液TF和TFPI水平差异无统计学意义(P0.05);羊水、羊水上清液、羊水沉渣中TF与TFPI呈负相关关系(P0.05),其中羊水沉渣相关性最强(r=-0.903,P0.05),血浆标本TF与TFPI无相关性(P0.05)。结论正常足月产妇羊水TF含量较高,而TFPI较低,可能在羊水栓塞的发生机制中起一定的临床作用。     

Studies on serum apolipoproteins and lipids in amniotic fluid and neonatal urine

Amniotic fluid and neonatal urine were examined for the presence of lipids and serum apolipoproteins. Human apolipoproteins A-I, A-II, and ApoD found principally in serum high density lipoproteins were identified in both neonatal urine and amniotic fluid. A lecithin/sphingomyelin ratio of greater than 5 associated with fetal lung maturity was accompanied by the disappearance of A-II from amniotic fluid. Dissimilarities of total fatty acid composition of amniotic fluid when compared to cord serum or neonatal urine indicate other tissue sources for fatty acids found in amniotic fluid. In addition, the presence of serum apolipoproteins and lipids in both amniotic fluid and neonatal urine suggests that a least a portion of these constituents could be derived from fetal urine.     

Prenatal diagnosis of galactosemia and properties of galactose-1-phosphate uridyltransferase in erythrocytes of galactosemic variants as well as in human fetal and adult organs

The kinetic characteristics and isoelectrofocusing patterns of uridyltransferase and the concentrations of galactose-1-phosphate in hemolysates were investigated in a family with compound variants of Duarte and classical galactosemia. There were no significant differences in Km values between the genotypes. However, the isoelectrofocusing study with thin-layer polyacrylamide gels (PAGIF) as well as with agarose gels (AGIF) showed a distinctive difference. The enzyme from the Duarte variant resolved into at least two more activity bands at pH between 5.2 and 5.4. The accumulation of galactose-1-phosphate was observed only in homozygotes for classical galactosemia. Compound heterozygotes (G-D) without any clinical manifestations did not show an accumulation of galactose-1-phosphate. The isoelectrofocusing study of the enzyme in human tissues revealed their activity resolving into multiple bands, 6-8 bands at pH 5.50-6.00 and 1-3 bands at pH 4.9-5.2. No significant differences were found in the patterns between fetal and adult liver except that the intensity of the anodic bands (pH 4.9-5.2) was weaker in fetal tissues. Prenatal diagnosis of classical galactosemia was performed in nine families by measuring the enzyme activity in cultivated amniotic fluid cells. Absence of the enzyme activity in amniotic fluid cells was found in two cases, and in four cases the heterozygosity was diagnosed by a relative low enzyme activity, 30-50% of the activity in control cells cultured in parallel.     

Cocaine and metabolite concentrations in the fetal guinea pig after chronic maternal cocaine administration.

To determine the disposition of cocaine (COC) and metabolites after chronic COC exposure in the late gestation guinea pig, six time-bred Dunkin-Hartley guinea pigs were given 10 daily 6 mg/kg COC s.c. injections from day 50 of gestation. Maternal blood and urine, fetal cord blood, and brain and amniotic fluid were collected 1 hr after the last injection. There was no difference between maternal and fetal plasma COC concentrations. This may be due to the combined effect of lower protein binding and ion trapping of COC in the fetus. Benzoylecgonine was higher in maternal plasma, but benzoylnorecgonine was higher in fetal plasma. COC brain-to-plasma ratios were similar in the dam and fetus. Benzoylecgonine was the only metabolite that could be detected in the brain, but levels were too low to quantitate. COC accumulated 3 to 4 times plasma concentrations in the amniotic fluid and was directly proportional to fetal plasma COC concentrations. Benzoylnorecgonine in amniotic fluid accumulated to 2 times fetal plasma levels. The in vitro half-life of COC in amniotic fluid was 30 times longer than plasma elimination half-life in vivo. The high level and long duration of COC in amniotic fluid serve as a reservoir for prolonged fetal COC exposure.     

High molecular weight angiotensinogen: a pregnancy associated protein

Although it was previously recognized that human amniotic fluid contained appreciable quantities of angiotensinogen, it remained unknown what fraction of the total is high molecular weight angiotensinogen. Fractionation of human amniotic fluid showed that high molecular weight angiotensinogen represents the major component of total angiotensinogen; 58% for 11 normotensive pregnant women and 67% for 12 hypertensive pregnant women. In contrast to plasma where high molecular weight angiotensinogen is elevated in hypertensive pregnant women as compared to normotensive pregnant women, no such difference exists for amniotic high molecular weight angiotensinogen. Serum and amniotic fluid high molecular weight angiotensinogen were compared in six subjects, and no significant correlation was found. In fetal cord blood, high molecular weight angiotensinogen represented only 5.8% of the total angiotensinogen. The site of synthesis of high molecular weight angiotensinogen remains unknown but these data suggest that it is produced in the placenta or in the maternal uterine tissue. Therefore, we propose that human high molecular weight angiotensinogen should be classified as a pregnancy-associated protein.     

外周静脉抗凝血沉淀试验在羊水栓塞早期确诊中的价值

目的 探讨外周静脉抗凝血沉淀试验在羊水栓塞早期确诊中的价值,为复苏与救治提供依据.方法 采用血液沉淀试验,通过回顾分析,对2005年3月～2014年3月发生在蒲城县医院和其他医院的10例羊水栓塞病人（观测组）的静脉抗凝血,离心后取血浆上层及全血底层样本,分别做羊水成分检测,并对同期该院随机抽取的100例健康产妇（对照组）破膜后至产后2h内的抗凝血标本做同样检测.结果 观测组10例,6例（60％）血浆上层和全血底层均检测到脂肪球、毳毛、鳞状上皮细胞、胎粪、黏液和碎片,3例（30％）检出脱落上皮和鳞状上皮,1例（10％）检出脂肪球和毳毛,阳性检出率100％.对照组100例,仅1例（1％）标本有极少量脂肪球,其余均未见任何羊水成分,阳性检出率1％.经统计学处理,两组检出率差异有统计学极显著性意义（X2=192,P＜0.005）.结论 外周静脉抗凝血离心沉淀试验对羊水成分的检出,快速、简单、经济.具有更早期确诊和警示栓塞发生的价值,可在临床中推广.     

Fibronectin in synovial fluid and tissue in rheumatoid arthritis

Fibronectin is a glycoprotein found in body fluids, loose connective tissue matrix and in basement membranes. Fibronectin in rheumatoid arthritis synovial fluid was immunologically indistinguishable from the plasma form, as shown by double-diffusion analysis. Fibronectin isolated from rheumatoid synovial fluid by affinity chromatography on gelatin--Sepharose had a polypeptide pattern similar to that of plasma fibronectin in SDS--polyacrylamide gel electrophoresis. In fifty-one patients with rheumatoid arthritis and related diseases fibronectin concentrations is synovial fluid were 445 +/- 103 micrograms/ml (mean +/- SD) and within normal range, 335 +/- 52 micrograms/ml, in plasma. Immunofluorescence staining showed a prominent increase of fibronectin in the proliferating synovial connective tissue in rheumatoid arthritis as compared to normal synovial membrane. The results suggest an increased local production of fibronectin in rheumatoid synovial tissue.     

Measurement, Characterization, and Source of Somatostatin-like Immunoreactivity in Human Amniotic Fluid

Somatostatin-like immunoreactivity (SLI) is widely distributed in tissues and biological fluids. To determine whether SLI is also present in amniotic fluid, samples obtained by amniocentesis from 30 normal and 27 abnormal pregnancies were studied by radioimmunoassay. Direct incubation of [(125)I-Tyr(1)]tetradecapeptide somatostatin (SRIF) with amniotic fluid resulted in 89% tracer degradation. Damage was reduced to <5% when samples were acidified and boiled before the assay. With this technique, SLI was detectable in all normal amniotic fluid samples; the mean level at 15-20 wk of gestation (320+/-55 pg/ml, n = 15) being 4.5 times higher than the mean at 32-43 wk (70+/-12 pg/ml, n = 15) (P < 0.001). In cases of preeclampsia (n = 6), gestational diabetes (n = 5), anencephaly (n = 1), and meningomyelocele (n = 1), SLI values were in the normal range, but in one juvenile diabetic and one patient with chronic renal failure, SLI was undetectable (<10 pg/ml). In a pair of monochorionic diamniotic twins, SLI levels were very different (33 and 197 pg/ml), which suggests that fetal factors are more important than materno-placental ones in determining amniotic fluid SLI. Serial dilutions of amniotic fluid showed parallelism with standard SRIF. When concentrates of pooled amniotic fluid were chromatographed on Sephadex G-25 columns, all SLI eluted in the void volume ahead of SRIF even after treatment with 8 M urea and dithiothreitol. This "big" SLI incubated in amniotic fluid showed 100% stability over 24 h at 37 degrees C, whereas SRIF was rapidly inactivated (t((1/2)) congruent with 7 min). Extracts of placenta and fetal membranes contained no SLI, but small amounts (6-20% of total amniotic fluid SLI) were found in cells from fresh fluid. Radioimmunoassay of SLI in extracts of seven paired cord arterial and venous plasma samples showed no arteriovenous gradient consistent with fetal origin of cord blood SLI. It is concluded that (a) amniotic fluid contains SLI which is of fetal origin and (b) normal levels vary with gestational age. The SLI has a higher molecular weight (>/=5,000) and is more stable in amniotic fluid than SRIF.     

血浆纤维结合蛋白的动态变化在骨折愈合中的临床意义

采用酶标仪比浊法研究１１８名骨折患者愈合过程中１～７周血浆纤维结合蛋白（Ｆｎ）变化规律及骨折损伤程度与Ｆｎ的关系。实验结果表明，骨折后１～３周，Ｆｎ明显减少，第４～５周Ｆｎ呈缓慢上升，第６周恢复正常。因此看出Ｆｎ与骨折损伤、修复程度有密切关系，并起着十分重要的作用。     

Functional characteristics of cord blood T lymphocytes after lectin and anti-CD3 stimulation

Newborns are more susceptible than adults to infections, which suggests a relative immaturity of the immune system early after birth. Cord blood T cells differ significantly both in surface phenotype and function from adult T cells. We examined the proliferation and expression of activation molecules by lymphocytes isolated from umbilical cord blood or peripheral blood of adults. The lymphocytes were cultured for 5 days in the presence of phytohemagglutinin, concanavalin A, or anti-CD3 monoclonal antibody. Cord blood T cells expressed the CD45RA molecule, while a low proportion expressed the RO isoform, a marker of primed or activated lymphocytes. Furthermore, more than 95% of neonatal lymphocytes bear the CD38 molecule, but do not express the CD57 molecule. After stimulation by phytohemagglutinin or concanavalin A, the lymphocytes from newborns were activated and proliferated as efficiently as adult T cells. Anti-CD3 did not cause neonatal lymphocytes to proliferate, but these cells expressed activation molecules, such as HLA-DR antigens and the receptor for interleukin-2 and transferrin. The relevance of these findings to tolerance induction in immature cord blood T cells is discussed.     

Reactivity of amniotic fluid alpha-fetoprotein with concanavalin A-sepharose in pre-natal diagnosis of fetal malformations

The proportion of concanavalin A-non-reactive alpha-fetoprotein was determined in 215 amniotic fluid samples from second trimester pregnancies. the median percentage for concanavalin A-non-binding alpha-fetoprotein was 35.5% at the 15th week and 32.2% at the 18th gestational week. Nineteen of the 23 pregnancies with various fetal malformations showed highly elevated total alpha-fetoprotein levels. In this group, the value for non-reactive alpha-fetoprotein was below the normal range in 12 out of 13 samples collected at 15-17 weeks of pregnancy and in four out of six samples at 18-19 weeks. Four pathological pregnancies had only moderately elevated total alpha-fetoprotein levels (5.3-7.9 SD above the mean) and two of these samples had a low percentage of the concanavalin A-non-binding fraction. The amniotic fluid alpha-fetoprotein concentration was between 3 and 5 SD and over 5 SD above the mean in four and seven normal pregnancies, respectively. The concanavalin A-fractionation classified correctly 10 out of these 11 cases. The results indicate that the determination of the proportion of concanavalin A-non-binding alpha-fetoprotein is a useful supplementary test to the total alpha-fetoprotein assay.