Collagen gel contraction by fibroblasts requires cellular fibronectin but not plasma fibronectin

Fibroblasts embedded in three-dimensional lattices of collagen fibrils have been known to require serum constituents to induce a cell-mediated contraction of collagen gels. The gel contraction was studied with human skin fibroblasts cultured in the presence of fetal bovine serum (FBS). Removal of bovine serum fibronectin (sFN) from FBS did not affect the extent of gel contraction. Gel contraction occurred in serum-free defined media. Therefore, it is concluded that sFN is not required for gel contraction. That cellular FN (cFN) synthesized and secreted by fibroblasts plays a crucial role in gel contraction was suggested by the following experiments: (1) We obtained monoclonal antibodies (mAb A3A5) against fibroblast surface antigens, which suppressed the fibroblast-mediated gel contraction. Immunoblot analyses showed that mAb A3A5 recognizes cFN secreted by human fibroblasts and human plasma FN (pFN), but not bovine sFN in FBS used for culture. (2) Addition of rabbit antisera, which recognize human cFN, to a serum-free gel culture inhibited contraction. Uninvolvement of human pFN in gel contraction was further confirmed by the fact that neither pretreatment of fibroblasts with excess amounts of human pFN nor the presence of excess amounts of human pFN in gels affected the extent of gel contraction. This study seems to be the first demonstration of functional difference between cFN and pFN (or sFN) and proposes a novel mode of binding of fibroblasts with collagen fibrils via cFN during cell-mediated collagen morphogenesis.     

Assignment of disulphide bonds in synthetic endothelin-1 isomers by fast atom bombardment mass spectrometry.

Endothelin-1 (ET-1), a 21-residue vasoconstrictor peptide possessing four cysteinyl residues at positions 1, 3, 11 and 15, was synthesized by random oxidation of a tetrahydro-ET-1. On reverse-phase high-performance liquid chromatography, crude product was shown to be a mixture of two disulphide isomers. A method was developed to determine the disulphide structure of the isomers. The method consisted of (a) limited digestion with chymotrypsin, (b) cleavage with cyanogen bromide and (c) manual Edman degradation. Through this procedure, each isomer afforded specific fragments containing a single disulphide bond, which were identified by fast atom bombardment mass spectrometry. Isomer 1, the minor component, afforded a fragment containing Cys 3 and Cys 15, and isomer 2, the major component, afforded fragments containing Cys 3 and Cys 11. Since little disulphide exchange was observed, it could be concluded clearly that the disulphide bond pairs in isomer 1 were Cys 1-Cys 11 and Cys 3-Cys 15, while those in isomer 2 were Cys 1-Cys 15 and Cys 3-Cys 11 (the same as natural ET-1). The procedure was successfully applied to two synthetic analogues, [Gly18]-ET-1 and [Pro16]-ET-1.     

Increase in potential activities of protein phosphatases PP1 and PP2A in lymphoid tissues of autoimmune MRL/MpJ-lpr/lpr mice.

The differential assay conditions for protein phosphatases PP1, PP2A, and PP2C were extensively studied by using crude extracts from mouse lymphoid tissues as enzyme sources. Under these conditions, the protein phosphatase activities were measured in MRL/MpJ-lpr/lpr mice (MRL/lpr mice), autoimmune-prone mice, and MRL/MpJ(-)+/+ mice (MRL/+/+ mice) and C3H/HeJ mice as the controls. In MRL/lpr mice, significant alterations in protein phosphatase activities from those in the control mice were demonstrated. In spleen and liver from MRL/lpr mice, potential activities of PP1 and PP2A were distinctly elevated over those of the control mice. These elevations appeared to be due to accumulation of the abnormal lymphocytes that emerged in MRL/lpr mice. Although the PP1 activity in MRL/lpr lymph nodes was lower than those of normal spleen and thymus, it was greatly increased by Co(2+)-trypsin treatment so that the PP1 activity of MRL/lpr lymph nodes was the highest among those of all the tissues examined. In contrast, PP2C activity showed no remarkable alteration in the autoimmune disease model mice as compared with that in the control mice. These results demonstrated a specific elevation in potency of protein dephosphorylation in the tissues of MRL/lpr mice, suggesting a new explanation for the defect in signal transduction in this disease.     

Proteins and Carbohydrates in Xylem Sap from Squash Root

The xylem sap from squash roots was collected from the cut surfaceof stems, and the proteins and carbohydrates in the sap wereanalyzed. The sap contained 18.6 µg ml^–1 proteinand the major polypeptides were as follows: 1) two polypeptides,of 75 and 40 kDa, with high-mannose glycans, the levels of whichincreased for about 24 h after cutting and then decreased; 2)a 32-kDa polypeptide, which appeared soon after cutting, disappearedand then reappeared again 48–64 h after cutting; and 3)a 19-kDa and a 14-kDa polypeptide, which were present constitutively.The carbohydrates contained in the xylem sap were fractionatedinto 80% ethanol-soluble and -insoluble material, and whichwere analyzed by high-performance liquid chromatography, gaschromatography and enzymatic mathods. The former fraction containedconsiderable amounts of myo-inositol and fructose as free sugarsand oligosaccharides composed mainly of galactose, arabinoseand glucose. The latter contained polysaccharides composed mainlyof uronic acids, galactose and arabinose. The possible significanceof these substances, which may mediate the interactions betweenthe root and the aerial organs, is discussed. (Received April 20, 1992; Accepted July 4, 1992)     

Effect of fatigue on maximal inspiratory pressure-flow capacity.

The inspiratory muscles can be fatigued by repetitive contractions characterized by high force (inspiratory resistive loads) or high velocities of shortening (hyperpnea). The effects of fatigue induced by inspiratory resistive loaded breathing (pressure tasks) or by eucapnic hyperpnea (flow tasks) on maximal inspiratory pressure-flow capacity and rib cage and diaphragm strength were examined in five healthy adult subjects. Tasks consisted of sustaining an assigned breathing frequency, duty cycle, and either a "pressure-time product" of esophageal pressure (for the pressure tasks) or peak inspiratory flow rate (for the flow tasks). Esophageal pressure was measured during maximal inspiratory efforts against a closed glottis (Pesmax), maximal transdiaphragmatic pressure was measured during open-glottis expulsive maneuvers (Pdimax), and maximal inspiratory flow (VImax) was measured during maximal inspiratory efforts with no added external resistance before and after fatiguing pressure and flow tasks. The reduction in Pesmax) with pressure fatigue (-25 +/- 7%) was significantly greater than the change in Pesmax with flow fatigue (-8 +/- 8%, P less than 0.01). In contrast, the reductions in Pdimax (-11 +/- 8%) and VImax (-16 +/- 3%) with flow fatigue were greater than the changes in Pdimax (-0.6 +/- 4%, P less than 0.05) or VImax (-3 +/- 4%, P less than 0.05) with pressure fatigue. We conclude that respiratory muscle performance is dependent not only on the presence of fatigue but whether fatigue was induced by pressure tasks or flow tasks. The specific impairment of Pesmax and not of Pdimax or flow with pressure fatigue may reflect selective fatigue of the rib cage muscles.(ABSTRACT TRUNCATED AT 250 WORDS)     

A small GTP-binding protein (G protein) recognized by smg p25A GDP dissociation inhibitor (GDI) in human platelet membranes and GDI for this small G protein in human platelet cytosol

We have recently purified from bovine brain cytosol a novel type of regulatory protein for smg p25A, named smg p25A GDP dissociation inhibitor (GDI), that regulates the GDP/GTP exchange reaction of smg p25A by inhibiting the dissociation of GDP from and thereby the subsequent binding of GTP to it. This smg p25A GDI is inactive for other ras p21/ras p21-like small GTP-binding proteins (G proteins) including c-Ha-ras p21, smg p21, rhoA p21 and rhoB p20. In human platelet membranes, smg p25A was not detected but a G protein with an apparent Mr value of 24,000 (24KG) was recognized by smg p25A GDI and the dissociation of GDP from and the binding of GTP to 24KG were inhibited by smg p25A GDI. The doses of smg p25A GDI necessary for these activities for both 24KG and smg p25A were the same. This 24KG was not recognized by an anti-smg p25A monoclonal antibody. The GDI activity for human platelet 24KG and smg p25A was detected in human platelet cytosol. This human platelet GDI was recognized by an anti-smg p25A GDI polyclonal antibody. These results indicate that there is a 24KG-24KG GDI system similar to a smg p25A-smg p25A GDI system in human platelets.     

Secretion in yeast of mutant human lysozymes with and without glutathione bound to cysteine 95

A mutant human lysozyme C77A, in which Cys-77 is replaced with Ala, was secreted by Saccharomyces cerevisiae as two proteins (C77A-a and C77A-b) with different specific activities. A peptide fragment from Val93 to Ala108 was obtained from C77A-a by pepsin digestion, and examined by fast atom bombardment mass spectrometry and amino acid analysis. The results showed that glutathione was attached to the thiol group of Cys95 of the fragment through a disulfide linkage. This observation was confirmed by quantitative formation of free glutathionesulfonic acid from C77A-a by performic acid treatment. In contrast, there was no modification in the case of C77A-b. These results indicate that C77A-a contained a mixed disulfide with glutathione attached to cysteine residue 95. In C77A-b, there appears to be a free thiol of Cys95 surrounded by many side chains, which was not modified by iodoacetic acid under native conditions, suggesting that the attachment of glutathione occurs during folding. These findings further suggest that in the oxidation step of disulfide bond formation in human lysozyme secreted by yeast, mixed disulfides are formed with glutathione and that posttranslational modification with glutathione can occur even in a protein secreted by yeast.