scholarly journals Acquisition of hyaluronate-binding affinity in vivo by newly synthesized cartilage proteoglycans

1989 ◽  
Vol 258 (3) ◽  
pp. 875-880 ◽  
Author(s):  
J D Sandy ◽  
J R O'Neill ◽  
L C Ratzlaff
Keyword(s):  
Binding Affinity ◽  
Gradient Centrifugation ◽  
The Other ◽  
Binding Properties ◽  
High Affinity ◽  
Cscl Gradient ◽  
Cartilage Proteoglycans ◽  
Relationship Of ◽  
Total Tissue

We have studied the hyaluronate-binding properties of aggregating cartilage proteoglycans synthesized in vivo by immature (6-week), mature (25-week) and aged (75-week) rabbits. Precursor isotope (35SO4) was given by intra-articular injection and articular cartilage was removed from rabbits after periods ranging from 1.5 h to 168 h. Proteoglycans were extracted with 4 M-guanidinium/HCl and monomers were isolated by CsCl gradient centrifugation under dissociative conditions. The percentages of both radiolabelled and total tissue monomers with a high affinity for hyaluronate [that is, capable of forming aggregates on Sepharose CL-2B in the presence of 0.8% (w/w) hyaluronate] were then determined. For all samples about 30% of the tissue monomers were high-affinity; however, less than 5% of the radiolabelled monomers were high-affinity at 1.5 h after injection, and this figure increased gradually with time in vivo. The increase was rapid in immature rabbits, such that after 24 h, about 30% of the radiolabelled monomers were high-affinity; on the other hand for mature and aged rabbits the increase was markedly slower such that 30% high-affinity was attained only after about 72 h. The results show that aggregating cartilage proteoglycans are secreted in vivo in a ‘precursor’ form with a low affinity for hyaluronate, and suggest that conversion of these monomers to a form with a higher binding affinity occurs with a half-time of about 12 h in immature cartilages but greater than 24 h in mature cartilages. The possible relationship of these findings to the process of proteoglycan aggregation in vivo is discussed.

scholarly journals Biosynthesis of respiratory-tract mucins. Incorporation of radioactive precursors into glycoproteins by canine tracheal explants in vitro

1973 ◽  
Vol 136 (4) ◽  
pp. 837-844 ◽  
Author(s):  
Daniel B. Ellis ◽  
Glenn H. Stahl
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sialic Acids ◽  
Equilibrium Density ◽  
Agarose Gels ◽  
Cscl Gradient ◽  
Tracheal Explants

1. Canine tracheal explants, cultured in medium 199, actively incorporated radioactive precursors into secreted macromolecules in vitro. 2. Puromycin, 6-diazo-5-oxo-l-norleucine and ouabain markedly inhibited the incorporation of these precursors. 3. Exogenous glucosamine at concentrations above 20mm caused a greater than 50% inhibition of the incorporation of l-[G-3H]fucose and l-[U-14C]serine. 4. Carbohydrate content of the purified secretions was approximately 50% and consisted principally of galactose, N-acetylglucosamine, N-acetylgalactosamine, fucose and sialic acids. 5. Chromatography on DEAE-cellulose and Bio-Gel A-150m and equilibrium density-gradient centrifugation in a CsCl gradient confirmed the presence of mucous glycoproteins. 6. Electrophoresis on 1% agarose gels gave profiles that were identical with canine respiratory mucus obtained in vivo. 7. These results support the utility of the explant system for studies of respiratory secretions.

scholarly journals Aptamer-modified nanomaterials: principles and applications

2016 ◽  
Vol 18 (1-2) ◽  
Author(s):  
Katharina Urmann ◽  
Julia Modrejewski ◽  
Thomas Scheper ◽  
Johanna-G. Walter
Keyword(s):  
Chemical Synthesis ◽  
Binding Affinity ◽  
Nanostructured Materials ◽  
Nanostructured Material ◽  
Solid Support ◽  
Binding Properties ◽  
Promising Alternative ◽  
Nanostructured Surfaces ◽  
High Affinity

AbstractAptamers are promising alternative binders that can substitute antibodies in various applications. Due to the advantages of aptamers, namely their high affinity, specificity and stability, along with the benefits originating from the chemical synthesis of aptamers, they have attracted attention in various applications including their use on nanostructured material. This necessitates the immobilization of aptamers on a solid support. Since aptamer immobilization may interfere with its binding properties, the immobilization of aptamers has to be investigated and optimized. Within this review, we give general insights into the principles and factors controlling the binding affinity of immobilized aptamers. Specific features of aptamer immobilization on nanostructured surfaces and nanoparticles are highlighted and a brief overview of applications of aptamer-modified nanostructured materials is given.

Alternative splicing of the mouse profilin II gene generates functionally different profilin isoforms

2000 ◽  
Vol 113 (21) ◽  
pp. 3795-3803 ◽  
Author(s):  
A. Di Nardo ◽  
R. Gareus ◽  
D. Kwiatkowski ◽  
W. Witke
Keyword(s):  
Alternative Splicing ◽  
Cell Motility ◽  
Hela Cells ◽  
The Other ◽  
Actin Dynamics ◽  
High Affinity ◽  
Other Hand ◽  
The Brain

Profilins are a conserved family of proteins participating in actin dynamics and cell motility. In the mouse, two profilin genes are known. Profilin I is expressed universally at high levels, while profilin II is expressed mainly in the brain. Here we describe the occurrence of two mouse profilin II isoforms, A and B, which are derived by alternative splicing. They are identical through residue 107 of the protein, but then have distinct C-terminal sequences. Profilin IIA binds to poly-L-proline and actin with high affinity similar to profilin I. Profilin IIB on the other hand does not bind to actin and the affinity for poly-L-proline is greatly diminished. However, tubulin was found to bind to GST-profilin IIB, and in vivo GFP-profilin IIB was recruited to spindles and asters during mitosis in HeLa cells. Our results indicate unexpected diversity in the functions of the profilin family of proteins, and suggest that in mouse profilin IIA is intimately involved in actin dynamics, while profilin IIB associates with other cytoskeletal components.

scholarly journals Noncontiguous regions in the extracellular domain of EGF receptor define ligand-binding specificity.

1991 ◽  
Vol 2 (5) ◽  
pp. 337-345 ◽  
Author(s):  
I Lax ◽  
R Fischer ◽  
C Ng ◽  
J Segre ◽  
A Ullrich ◽  
...  
Keyword(s):  
Binding Energy ◽  
Ligand Binding ◽  
Binding Site ◽  
Binding Affinity ◽  
Egf Receptor ◽  
Binding Properties ◽  
Amino Terminal ◽  
High Affinity ◽  
Domain Iii ◽  
Domain I

Murine epidermal growth factor (EGF) binds with approximately 250-fold higher binding affinity to the human EGF receptor (EGFR) than to the chicken EGFR. This difference in binding affinity enabled the identification of a major ligand-binding domain for EGF by studying the binding properties of various chicken/human EGFR chimera expressed in transfected cells lacking endogenous EGFR. It was shown that domain III of EGFR is a major ligand-binding region. Here, we analyze the binding properties of novel chicken/human chimera to further delineate the contact sequences in domain III and to assess the role of other regions of EGFR for their contribution to the display of high-affinity EGF binding. The chimeric receptors include chicken EGFR containing domain I of the human EGFR, chicken receptor containing domain I and III of the human EGFR, and two chimeric chicken EGFR containing either the amino terminal or the carboxy terminal halves of domain III of human EGFR, respectively. In addition, the binding of various human-specific anti-EGFR monoclonal antibodies that interfere with EGF binding is also compared. It is concluded that noncontiguous regions of the EGFR contribute additively to the binding of EGF. Each of the two halves of domain III has a similar contribution to the binding energy, and the sum of both is close to that of the entire domain III. This suggests that the folding of domain III juxtaposes sequences that together constitute the ligand-binding site. Domain I also provides a contribution to the binding energy, and the added contributions of both domain I and III to the binding energy generate the high-affinity binding site typical of human EGFR.

scholarly journals Self-renaturing fractions in the separated strands of mouse satellite deoxyribonucleic acid

1972 ◽  
Vol 128 (2) ◽  
pp. 193-198 ◽  
Author(s):  
W. D. Sutton ◽  
P. M. B. Walker
Keyword(s):  
Satellite Dna ◽  
Deoxyribonucleic Acid ◽  
Gradient Centrifugation ◽  
High Thermal Stability ◽  
The Self ◽  
The Other ◽  
Cscl Gradient ◽  
Dna Strands ◽  
Variable Extent ◽  
Mouse Satellite Dna

Pyrimidine- and purine-rich strands of Mus musculus satellite DNA prepared by alkaline CsCl-gradient centrifugation can self-renature to a variable extent to give partial duplexes with high thermal stability. These duplexes were purified by treatment with nuclease S1 followed by hydroxylapatite chromatography, and have been shown by pyrimidine-tract analysis to be very similar in sequence to total reassociated satellite DNA. It is believed that the self-renaturing fractions result from variable contamination of each strand with fragments of the other, rather than from molecular inversions. The predominantly single-stranded properties of these fractions may be partly due to the ability of mouse satellite DNA strands to reassociate in non-stoicheiometric proportions.

scholarly journals Distribution of proteins between nucleus and cytoplasm of amoeba proteus

1981 ◽  
Vol 88 (3) ◽  
pp. 516-525 ◽  
Author(s):  
L Goldstein ◽  
C Ko
Keyword(s):  
Amoeba Proteus ◽  
Cellular Protein ◽  
The Other ◽  
Nuclear Transplantation ◽  
Main Function ◽  
Functional Sites ◽  
Protein Mass ◽  
High Affinity ◽  
Nuclear Binding

By transplanting nuclei between labeled and unlabeled cells, we determined the localization of the major proteins of amebas and described certain features of their intracellular distributon. We identified approximately 130 cellular proteins by fluorography of one-dimensional polyacrylamide electrophoretic gels and found that slightly less than half of them (designated NP, for nuclear proteins) are almost exclusively nuclear. About 95 percent of the other proteins (designated CP for cytoplamsic proteins) are roughly equally concentrated in nucleus and cytoplasm, but - because the cytoplasm is 50 times larger than the nucleus - about 98 percent of each of the latter is in the cytoplasm. Of the CP, roughly 5 percent are not detectable in the nucleus. Assuming that these are restricted to the cytoplasm only because, for example, they are in structures too large to enter the nucleus and labeled CP readily exit a nucleus introduced into unlabeled cytoplasm, we conclude that the nuclear envelope does not limit the movement of any nonstructural cellular protein in either direction between the two compartments. Some NP are not found in the cytoplasm (although ostensibly synthesized there) presumably because of preferential binding within the nucleus. Almost one half of the protein mass in nuclei in vivo is CP and apparently only proteins of that group are lost from nuclei when cells are lysed. Thus, while an extracellular environment allows CP to exit isolated nuclei, the nuclear binding affinities for NP are retained. Further examination of NP distribution shows that many NP species are, in fact, detectable in the cytoplasm (although at only about 1/300 the nuclear concentration), apparently because the nuclear affinity is relatively low. These proteins are electrophoretically distinguishable from the high-affinity NP not found in the cytoplasm. New experiments show that an earlier suggestion that the nuclear transplantation operation causes an artifactual release of NP to the cytoplasm is largely incorrect. Moreover, we show that cytoplasmic "contamination" of nuclear preparations is not a factor in classifying proteins by these nuclear transplantation experiments. We speculate the no mechanism has evolved to confine most CP to the cytoplasm (where they presumably function exclusively) because the cytoplasm's large volume ensures that CP will be abundant there. Extending Bonner's idea of "quasi-functional nuclear binding sites" for NP, we suggest that a subset of NP usually have a low affinity for available intranuclear sites because their main function(s) occurs at other intranuclear sites to which they bind tightly only when particular metabolic conditions demand. The other NP (those completely absent from cytoplasm) presumable always are bound with high affinity at their primary functional sites.

scholarly journals Modular Structure of PACT: Distinct Domains for Binding and Activating PKR

2001 ◽  
Vol 21 (6) ◽  
pp. 1908-1920 ◽  
Author(s):  
Gregory A. Peters ◽  
Rune Hartmann ◽  
Jun Qin ◽  
Ganes C. Sen
Keyword(s):  
Protein Domain ◽  
Affinity Binding ◽  
Translation Inhibition ◽  
High Affinity ◽  
Cellular Apoptosis ◽  
Domain 3 ◽  
Function Relationship ◽  
Relationship Of

ABSTRACT PACT is a 35-kDa human protein that can directly bind and activate the latent protein kinase, PKR. Here we report that PKR activation by PACT causes cellular apoptosis in addition to PKR autophosphorylation and translation inhibition. We analyzed the structure-function relationship of PACT by measuring its ability to bind and activate PKR in vitro and in vivo. Our studies revealed that among three domains of PACT, the presence of either domain 1 or domain 2 was sufficient for high-affinity binding of PACT to PKR. On the other hand, domain 3, consisting of 66 residues, was absolutely required for PKR activation in vitro and in vivo. When fused to maltose-binding protein, domain 3 was also sufficient for efficiently activating PKR in vitro. However, it bound poorly to PKR at the physiological salt concentration and consequently could not activate it properly in vivo. As anticipated, activation of PKR by domain 3 in vivo could be restored by attaching it to a heterologous PKR-binding domain. These results demonstrated that the structure of PACT is modular: it is composed of a distinct PKR-activation domain and two mutually redundant PKR-interacting domains.

Inhibition of 2,3-oxidosqualene: β-amyrin-cyclase, S-adenosyl-l-methionine: cycloartenol C-24-methyltransferase and cycloeucalenol: obtusifoliol isomerase by rationally designed molecules containing a tertiary amine function

1983 ◽  
Vol 11 (5) ◽  
pp. 537-543 ◽  
Author(s):  
ALAIN RAHIER ◽  
PIERRETTE BOUVTER ◽  
LUIGI CATTEL ◽  
ACHARAN NARULA ◽  
PIERRE BENVENISTE
Keyword(s):  
Tertiary Amine ◽  
Inhibitory Action ◽  
Plant Sterol ◽  
The Other ◽  
Maize Seedlings ◽  
Higher Plant ◽  
High Affinity ◽  
Amine Function

25-Azacycloartanol (I), 2-aza-2-dihydrosqualene (II) and Tridemorph (2,6-dimethyl-N-tridecylmorpholine)(III) are potent inhibitors of higher plant sterol biosynthesis. The first two molecules have been designed using rational enzymological concepts. I, II and III were shown to inhibit the S-adenosyl-l-methionine: cycloartenol C-24-methyltransferase, the 2,3-oxidosqualene: β- amyrin-cyclase and the cycloeucalenol: obtusifoliol isomerase, respectively. Inhibition was demonstrated either in vivo on bramble cell suspensions or in vitro on microsomes from maize seedlings. Each inhibitor has been shown to have a high affinity for its presumed enzymic target and only negligible inhibitory action on the other two enzymes. The applications of these results to further physiological studies are discussed.

Synthesis and biological activity of histogranin and related peptides

1995 ◽  
Vol 73 (2) ◽  
pp. 209-214 ◽  
Author(s):  
Jyoti A. Prasad ◽  
Vijay K. Shukla ◽  
Simon Lemaire
Keyword(s):  
Nmda Receptor Antagonist ◽  
The Other ◽  
Peptide Fragments ◽  
Antagonist Activity ◽  
Brain Membrane ◽  
High Affinity ◽  
Aspartate Receptor ◽  
Vitro Binding

Histogranin (HN) was first isolated from bovine adrenal medulla and shown to be a pentadecapeptide displaying N-methyl-D-aspartate (NMDA) receptor antagonist activity. To determine the active pharmacophore of HN, fragments of the peptide were synthesized and their structure–activity relationships studied by measuring their ability to displace the binding of [125I][Ser1]HN to rat brain membrane preparations and to block NMDA-induced convulsions in mice. In the binding assay, only the full length peptide HN and HN(1–10) displayed a high affinity (Ki of 72 and 162 nM, respectively). All other tested fragments with deletions at the N- and (or) C-terminals of the molecule showed large (16- to 2500-fold) decreases in potency. The least active peptide fragment tested was HN(6–10) (Ki of 164 μM). In vivo, HN and HN(2–15) (100 nmol; i.c.v.) produced 94 and 40% protection against NMDA-induced convulsions in mice, respectively. None of the other peptide fragments displayed significant anticonvulsant activity. The protective activity of HN (60 and 100 nmol) was markedly antagonized by coadministration of HN(1–10) (100 nmol). The results indicate that the in vivo anti-NMDA and in vitro binding activities of HN and related peptides, with the exception of HN(1–10), depend upon the integrity of the molecule. On the other hand, the high affinity of HN(1–10) for HN binding sites correlates well with its antagonist effects towards the activity of the parent peptide.Key words: histogranin, peptide receptor, N-methyl-D-aspartate receptor, anticonvulsant.

scholarly journals IMMUNE RESPONSES IN VITRO

1972 ◽  
Vol 136 (5) ◽  
pp. 1241-1257 ◽  
Author(s):  
Robert E. Click ◽  
Loretta Benck ◽  
Barbara J. Alter ◽  
Judith C. Lovchik
Keyword(s):  
Immune Responses ◽  
The Other ◽  
Dilution Technique ◽  
Antibody Synthesis ◽  
Different Strains ◽  
Relationship Of ◽  
The Relationship ◽  
Limiting Dilution

The finding that the relationship of the in vitro and in vivo responses of different strains of mice is under genetic control indicates that at least two mechanisms must operate under in vivo conditions to control 19S antibody synthesis. One is involved in the termination of 19S antibody synthesis; the other has a regulatory role on the magnitude of the response. In light of these findings, various concepts based on other genetically controlled immune responses and on the limiting dilution technique should be reassessed. Furthermore, the suppressive in vivo mechanism may be an important type of control in the resistance or susceptibility to the establishment or maintainance of neoplasms.

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