Solid-Phase Guanidinylation as a Diversification Strategy of Poly-l-proline Type II Peptide Mimic Scaffolds

Glycoprotein 330 (gp330) is a member of a family of receptors related to the low density lipoprotein receptor (LDLR). Although several ligands have been shown to bind gp330 in solid-phase assays, the ability of gp330 to mediate ligand endocytosis has not been demonstrated. To develop a cellular model for gp330 function we screened a variety of cultured cell lines and identified several that expressed this protein, including immortalized rat type II pneumocytes and a human and two rodent tumor cell lines. Using type II pneumocytes, endocytosis of a previously described gp330 ligand, urokinase (uPA) complexed with plasminogen activator inhibitor-1 (uPA:PAI-1) and two new ligands, PAI-1 and pro-uPA, was demonstrated. RAP, the 39 kDa receptor-associated protein known to antagonize ligand binding to gp330 in solid-phase binding assays, completely inhibited both internalization and degradation of the radiolabeled ligands by type II pneumocytes. This suggested that the clearance of these ligands was dependent on either gp330 or the LDLR-related protein (LRP), which shares several ligand-binding characteristics with gp330. By using polyclonal antibodies to gp330, the cellular internalization and degradation of the ligands were inhibited by 30–50%; remaining ligand internalization and degradation activity could be partially inhibited by polyclonal antibodies against LRP. These findings indicate that gp330, like other LDLR family members, mediates endocytosis of its ligands. In addition, gp330 acts in concert with LRP in type II pneumocytes to mediate clearance of a variety of proteins involved in plasminogen activation, including uPA:PAI-1 complexes PAI-1 and pro-uPA.(ABSTRACT TRUNCATED AT 250 WORDS)

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Evaluation of an enzyme immunoassay kit for estrogen receptor measurement--report II.

Clinical Chemistry ◽

10.1093/clinchem/34.10.2053 ◽

1988 ◽

Vol 34 (10) ◽

pp. 2053-2057 ◽

Cited By ~ 2

Author(s):

S Raam ◽

D M Vrabel

Keyword(s):

Enzyme Immunoassay ◽

Binding Sites ◽

Solid Phase ◽

Functional Characterization ◽

Quantitative Agreement ◽

Type I ◽

Type Ii ◽

Immunoreactive Protein ◽

Estrogen Binding

Abstract We present evidence to show that monoclonal antibodies to estrogen receptors (ER) in solid phase recognize the secondary estrogen binding sites with moderate to low affinity for estradiol (E2). An excellent quantitative agreement was found in five cytosols between the ER values obtained by the enzyme immunoassay (ER-EIA) and the amount of secondary estrogen binding sites measured by the assay involving dextran-coated charcoal (Clin Chem 1986;32:1496). The immunoreactive protein recognized by the antibody-coated beads, when allowed to react with ER(+) cytosols, is shown to bind [3H]estradiol only when the ligand concentration exceeds 8 nmol/L. Further biochemical and functional characterization of the immunoreactive protein is required to establish similarities/dissimilarities between this protein, high-affinity Type I ER sites, and the secondary sites such as Type II sites.

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Type IIA Procollagen Containing the Cysteine-rich Amino Propeptide Is Deposited in the Extracellular Matrix of Prechondrogenic Tissue and Binds to TGF-β1 and BMP-2

The Journal of Cell Biology ◽

10.1083/jcb.144.5.1069 ◽

1999 ◽

Vol 144 (5) ◽

pp. 1069-1080 ◽

Cited By ~ 191

Author(s):

Yong Zhu ◽

Anush Oganesian ◽

Douglas R. Keene ◽

Linda J. Sandell

Keyword(s):

Extracellular Matrix ◽

Solid Phase ◽

Protein Domain ◽

Collagen Molecule ◽

Endochondral Bone Formation ◽

Type Ii ◽

Chondroprogenitor Cells ◽

Recombinant Type ◽

Form Type ◽

Tgf Β1

Type II procollagen is expressed as two splice forms. One form, type IIB, is synthesized by chondrocytes and is the major extracellular matrix component of cartilage. The other form, type IIA, contains an additional 69 amino acid cysteine-rich domain in the NH2-propeptide and is synthesized by chondrogenic mesenchyme and perichondrium. We have hypothesized that the additional protein domain of type IIA procollagen plays a role in chondrogenesis. The present study was designed to determine the localization of the type IIA NH2-propeptide and its function during chondrogenesis. Immunofluorescence histochemistry using antibodies to three domains of the type IIA procollagen molecule was used to localize the NH2-propeptide, fibrillar domain, and COOH-propeptides of the type IIA procollagen molecule during chondrogenesis in a developing human long bone (stage XXI). Before chondrogenesis, type IIA procollagen was synthesized by chondroprogenitor cells and deposited in the extracellular matrix. Immunoelectron microscopy revealed type IIA procollagen fibrils labeled with antibodies to NH2-propeptide at ∼70 nm interval suggesting that the NH2-propeptide remains attached to the collagen molecule in the extracellular matrix. As differentiation proceeds, the cells switch synthesis from type IIA to IIB procollagen, and the newly synthesized type IIB collagen displaces the type IIA procollagen into the interterritorial matrix. To initiate studies on the function of type IIA procollagen, binding was tested between recombinant NH2-propeptide and various growth factors known to be involved in chondrogenesis. A solid phase binding assay showed no reaction with bFGF or IGF-1, however, binding was observed with TGF-β1 and BMP-2, both known to induce endochondral bone formation. BMP-2, but not IGF-1, coimmunoprecipitated with type IIA NH2-propeptide. Recombinant type IIA NH2-propeptide and type IIA procollagen from media coimmunoprecipitated with BMP-2 while recombinant type IIB NH2-propeptide and all other forms of type II procollagens and mature collagen did not react with BMP-2. Taken together, these results suggest that the NH2-propeptide of type IIA procollagen could function in the extracellular matrix distribution of bone morphogenetic proteins in chondrogenic tissue.

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A Two-Site, Monoclonal Antibody-Based Immunoassay for von Willebrand Factor -Demonstration that vWF Function Resides in a Conformational Epitope

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661555 ◽

1986 ◽

Vol 55 (03) ◽

pp. 318-324 ◽

Cited By ~ 19

Author(s):

S Chand ◽

A McCraw ◽

R Hutton ◽

E G D Tuddenham ◽

A H Goodall

Keyword(s):

Monoclonal Antibody ◽

Von Willebrand Factor ◽

Solid Phase ◽

Fluid Phase ◽

Conformational Epitope ◽

Type Ii ◽

Normal Individuals ◽

Von Willebrand ◽

Willebrand Factor ◽

Umbilical Cord Vein

SummaryTwo monoclonal antibodies (RFF-VIII: R/l and RFF-VIII:R/ 2) which recognise the same epitope on von Willebrand factor (vWF) have been used in a simple, two-site, solid-phase immunoradiometric (IRMA) or enzyme-linked assay (ELISA) to analyse vWF in plasma from normal individuals and from patients with von Willebrand’s disease (vWD). Results obtained confirm our previous findings (using RFF-VIII :R/2 in a one-site, fluid-phase IRMA) that the MAbs detect the presence of an epitope on the vWF molecule that reflects its function. This epitope is involved in vWF binding to the GPIb protein on platelets. It is reduced in all types of vWD, including type II (or variant) vWD. It is present in normal plasma, in vWF released from normal platelets and from cultured umbilical cord vein endothelial cells. The epitope is, however, found to be reduced in serum. Studies on SDS-treated vWF prove that this GPIb-binding site is dependent on the conformation of the vWF multimers.

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Bile salt activation of cerebroside sulphate sulphohydrolase

Biochemical Journal ◽

10.1042/bj1890045 ◽

1980 ◽

Vol 189 (1) ◽

pp. 45-49 ◽

Cited By ~ 6

Author(s):

A L Fluharty ◽

R L Stevens ◽

R T Miller ◽

H Kihara

Keyword(s):

Bile Salt ◽

Metachromatic Leukodystrophy ◽

Solid Phase ◽

Type I ◽

Type Ii ◽

Salt Activation ◽

Arylsulphatase A ◽

High Concentrations ◽

Activation Type ◽

Putative Mutant

The cholate and taurodeoxycholate activations of cerebroside sulphate sulphohydrolase (cerebroside-3-sulphate 3-sulphohydrolase, EC 3.1.6.8) activity of arylsulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) were compared. Taurodeoxycholate caused a sharp peak of response at a concentration of 1.25 mg/ml (type-I activation). Cholate also showed type-I activation but, in addition, it evoked a second, higher, response plateau at concentrations between 5 and 10 mg/ml (type-II activation). At the pH of the reaction, cholate is converted largely to the sparingly soluble free aicd, so at the high concentrations associated with type-II activation, copious precipitates were formed. It was found that the precipitated material was essential for the type-II activation. Type-I activation appears to involve bile salt interaction with substrate, while type-II activation appears to involve enzyme interaction with solid-phase cholic acid. the putative mutant arylsulphatase A in an unusual form of metachromatic leukodystrophy hydolysed cerebroside sulphate only in the presence of high levels of cholate. The type-II activation may thus be simulating a physiological desulphation reaction.

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THERMODYNAMICS OF TYPE II KEROGEN TRANSFORMATION

Geology and Geochemistry of Combustible Minerals ◽

10.15407/ggcm2019.03.025 ◽

2019 ◽

Vol 3 (180) ◽

pp. 25-40

Author(s):

Yuri Khokha ◽

Oleksandr Lyubchak ◽

Myroslava Yakovenko

Keyword(s):

Molecular Dynamics ◽

Solid Phase ◽

High Energy ◽

Type Ii ◽

Distribution Of Elements ◽

Gas System ◽

Hydrocarbon Gas ◽

Molecular Dynamics Methods ◽

Transformation Of Organic Matter ◽

Physical And Chemical

The article reviews the chemical structure of type II kerogen. The changes that occur with the structure of type II kerogen as it passes through the stages of catagenesis from immature to post-mature are evaluated. Structural models of type II kerogen at different stages of catagenesis are presented: both obtained empirically after studying the structure by physical and chemical methods and the results of modelling by molecular dynamics method. Methods of equilibrium thermodynamics are used to calculate the composition of the kerogen–gas system for crust sections in the range of 1–20 km with a heat flux of 40 to 100 mW/m2. The composition of kerogen/fluid geochemical system is calculated using the E. T. Jaynes formalism. It boils down to determining the optimal distribution of 5 elements (C, H, O, N, S) among the 44 additive constituents of the solid phase (i. e., type II kerogen) and other individual components that are included in the system (CO2, H2O, H2S, NH3, CH4, C2H6, C3H8, i-C4H10, n-C4H10, i-C5H12, neo-C5H12, n-C5H12). Comparison with the experiments showed that the results of the calculations do not contradict the experiments, with study the structure and changes in type II kerogen with increasing degree of catagenesis. In the analysis of changes in the concentrations of water, carbon dioxide and hydrogen sulfide, it is founded that kerogen could be not only a donor of atoms for gas components, but also their acceptor in contact with a high-energy fluid stream. It is shown that the determination of sulfur-containing atomic groups of kerogen by thermodynamic modelling yields gives more reliable results than molecular dynamics methods. Established is that the concept of “methane-graphite death”, which takes place in the state of thermodynamic equilibrium in the transformation of organic matter, is erroneous. The calculation shows that the composition of the kerogen–gas system, in addition to methane and carbon, includes solid-phase heteroatom groups, various additive components of aromatic structures and gases, both organic and inorganic. The distribution of elements between the additive components of kerogen and gases in this system controls the pressure and temperature in a complex way. The nature of changes in hydrocarbon gas concentrations in equilibrium with type II kerogen indicates the presence of an “oil window” in low-warmed zones within 2–4 km depths.

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Solid-Phase Synthesis of 1,3,5-Trisubstituted 1,4-Diazepin-2-one Peptide Mimic

Advances in Experimental Medicine and Biology - Peptides for Youth ◽

10.1007/978-0-387-73657-0_98 ◽

2009 ◽

pp. 213-214

Author(s):

Hassan S. Iden ◽

William D. Lubell

Keyword(s):

Solid Phase Synthesis ◽

Solid Phase ◽

Phase Synthesis ◽

Peptide Mimic

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Heparin-Associated Thrombocytopenia: The Antibody Is Not Heparin Specific

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648491 ◽

1992 ◽

Vol 67 (05) ◽

pp. 545-549 ◽

Cited By ~ 141

Author(s):

A Greinacher ◽

I Michels ◽

C Mueller-Eckhardt

Keyword(s):

Platelet Activation ◽

Dextran Sulfate ◽

Dermatan Sulfate ◽

Solid Phase ◽

Binding Capacity ◽

Low Grade ◽

Type Ii ◽

Pentosan Polysulfate ◽

Heparin Administration

SummaryIn this study the hypothesis was assessed whether heparin-associated thrombocytopenia (HAT) may be caused by an antibody dependent on polysulfated oligosaccharide epitopes, present not only on heparin but also on different polysulfated substances such as dextran sulfate and pentosan polysulfate. We found that the major factor for eliciting platelet activation with sera of HAT type II patients is neither the structure nor the AT III binding capacity of an oligosaccharide, but rather its grade of sulfation. This was shown by in vitro crossreactivity studies with 40 sera of HAT type II patients using unfractionated heparins, LMW heparins (Fragmin, Fraxiparin), enoxaparin, LMW heparinoid (Org 10172 and its subfractions), de-N-sulfated heparin, dermatan sulfate, dextran sulfate, pentosan polysulfate and dextran. Platelet activation was measured by the heparin induced platelet activation (HIPA) assay and the serotonin release assay (SRA). The platelet activating factor was isolated with the IgG fraction, but did not bind to heparin and dextran sulfate fixed to a solid phase. By isoimmune fixation electrophoresis a monoclonal gammopathy was ruled out in the three sera assessed. The in vivo effect of different LMW heparins and the heparinoid Org 10172 was observed in 10 patients with HAT type II. In a prospective study, a compatible heparin-like anticoagulant was selected for 10 HAT patients for whom further parenteral anticoagulation was required. The only substance that showed no crossreactivity in vitro was the LMW heparinoid Org 10172, which differs from heparin and LMW heparins by its low-grade sulfation. Upon treatment with the heparinoid, all 10 patients had a good clinical outcome, even if they had previously developed thromboembolic complications under LMW heparin administration. As Org 10172 contains a small amount of a LMW heparin-like substance (3%) this heparinoid should not be used in HAT patients without prior in vitro testing. We conclude that heparin-associated thrombocytopenia is not caused by a heparin-specific antibody and that a major factor contributing to the pathomechanism is the high grade of sulfation present in a variety of polysulfated oligosaccharides.

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