A bone matrix calcification-initiator noncollagenous protein

1977 ◽  
Vol 232 (3) ◽  
pp. C115-C127 ◽  
Author(s):  
M. R. Urist ◽  
A. J. Mikulski ◽  
M. Nakagawa ◽  
K. Yen
Keyword(s):  
Calcium Binding ◽  
Binding Capacity ◽  
Bone Matrix ◽  
Bone Collagen ◽  
Chemical Extraction ◽  
Insoluble Residue ◽  
Affinity Constant ◽  
Sequential Chemical Extraction

When completely demineralized, the densely packed structure of bone matrix does not recalcify, neither in physiologic solutions in vitro nor in implants in vivo. Even when inorganic and organic calcification inhibitors (which normally are stored in bone matrix) are removed first by autolytic digestion in neutral buffers at 37C and then by sequential chemical extraction, implants of the EDTA insoluble residue will not recalcify after as long as 4 wk in a muscle pouch. However, if first demineralized in cold dilute HCl, second, extracted and autodigested in buffers solution at 37C, and then further extracted in EDTA and other solutions at 2C, a calcification initiator protein (Cp) is unmasked, and the residue will invariable recalcify. CIP, isolated by gel filtration and column chromatography, is a disulfide-bonded glycoprotein aggregate composed of subunites of a moleclar mass of 55,000. CIP is composed of a large proportion of acidic amino acids and has a calcium binding capacity of about 1.8 times greater than albumin. The affinity constant CaCIP, calculated by ultrafiltration of physiologic solutions of Ca2+, is log K, 2.9. Observations on implants of residues that containe a) CIP but not a bone morphogenetic property (BMP), B) BMP accompanied by CIP activity, or c) neither BMP nor CIP activity suggested that BMP covers CIP and that the two are attached to bone collagen in tandem. Whether CIP plays a part in calcification of the normal skeleton requires further investigation.

Activity-Related Calcium Dynamics in Motoneurons of the Nucleus Hypoglossus From Mouse

1999 ◽  
Vol 82 (6) ◽  
pp. 2936-2946 ◽  
Author(s):  
Mario B. Lips ◽  
Bernhard U. Keller
Keyword(s):  
Quantitative Analysis ◽  
Action Potential ◽  
Calcium Binding ◽  
Calcium Influx ◽  
Binding Capacity ◽  
Calcium Transients ◽  
Calcium Dynamics ◽  
The Brain

A quantitative analysis of activity-related calcium dynamics was performed in motoneurons of the nucleus hypoglossus in the brain stem slice preparation from mouse by simultaneous patch-clamp and microfluorometric calcium measurements. Motoneurons were analyzed under in vitro conditions that kept them in a functionally intact state represented by rhythmic, inspiratory-related bursts of excitatory postsynaptic currents and associated action potential discharges. Bursts of electrical activity were paralleled by somatic calcium transients resulting from calcium influx through voltage-activated calcium channels, where each action potential accounted for a calcium-mediated charge influx around 2 pC into the somatic compartment. Under in vivo conditions, rhythmic-respiratory activity in young mice occurred at frequencies up to 5 Hz, demonstrating the necessity for rapid calcium elevation and recovery in respiratory-related neurons. The quantitative analysis of hypoglossal calcium homeostasis identified an average extrusion rate, but an exceptionally low endogenous calcium binding capacity as cellular parameters accounting for rapid calcium signaling. Our results suggest that dynamics of somatic calcium transients 1) define an upper limit for the maximum frequency of respiratory-related burst discharges and 2) represent a potentially dangerous determinant of intracellular calcium profiles during pathophysiological and/or excitotoxic conditions.

scholarly journals Isolated osteoclasts resorb the organic and inorganic components of bone.

1986 ◽  
Vol 102 (4) ◽  
pp. 1164-1172 ◽  
Author(s):  
H C Blair ◽  
A J Kahn ◽  
E C Crouch ◽  
J J Jeffrey ◽  
S L Teitelbaum
Keyword(s):  
Bone Matrix ◽  
Attachment Site ◽  
Organic Matrix ◽  
Total Radioactivity ◽  
Maximum Activity ◽  
Bone Collagen ◽  
Collagenolytic Activity ◽  
Maximal Rate

Osteoclasts are the principal resorptive cells of bone, yet their capacity to degrade collagen, the major organic component of bone matrix, remains unexplored. Accordingly, we have studied the bone resorptive activity of highly enriched populations of isolated chicken osteoclasts, using as substrate devitalized rat bone which had been labeled in vivo with L-[5-3H]proline or 45Ca, and bone-like matrix produced and mineralized in vitro by osteoblast-like rat osteosarcoma cells. When co-cultured with a radiolabeled substrate, osteoclast-mediated mineral mobilization reached a maximal rate within 2 h, whereas organic matrix degradation appeared more slowly, reaching maximal rate by 12-24 h. Thereafter, the rates of organic and inorganic matrix resorption were essentially linear and parallel for at least 6 d when excess substrate was available. Osteoclast-mediated degradation of bone collagen was confirmed by amino acid analysis. 39% of the solubilized tritium was recovered as trans-4-hydroxyproline, 47% as proline. 10,000 osteoclasts solubilized 70% of the total radioactivity and 65% of the [3H]-trans-4-hydroxyproline from 100 micrograms of 25-50 micron bone fragments within 5 d. Virtually all released tritium-labeled protein was of low molecular weight, 99% with Mr less than or equal to 10,000, and 65% with Mr less than or equal to 1,000. Moreover, when the 14% of resorbed [3H]proline-labeled peptides with Mr greater than or equal to 2,000 were examined for the presence of TCA and TCB, the characteristic initial products of mammalian collagenase activity, none was detected by SDS PAGE. In addition, osteoclast-conditioned medium had no collagenolytic activity, and exogenous TCA and TCB fragments were not degraded by osteoclasts. On the other hand, osteoclast lysates have collagenolytic enzyme activity in acidic but not in neutral buffer, with maximum activity at pH 4.0. These data indicate that osteoclasts have the capacity to resorb the organic phase of bone by a process localized to the osteoclast and its attachment site. This process appears to be independent of secretion of neutral collagenase and probably reflects acid protease activity.

scholarly journals Promoting the Calcium-Uptake Bioactivity of Casein Phosphopeptides in vitro and in vivo

2021 ◽  
Vol 8 ◽  
Author(s):  
Guo Liu ◽  
Baoyan Guo ◽  
Shengwei Sun ◽  
Minna Luo ◽  
Fei Liu ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Calcium Transport ◽  
Calcium Uptake ◽  
Calcium Absorption ◽  
Serum Alkaline Phosphatase ◽  
Binding Capacity ◽  
Animal Experiments ◽  
Casein Phosphopeptides

Casein phosphopeptides have been studied widely for their ability to chelate calcium. However, systematic studies on the effects of casein phosphopeptides (CPP) on calcium absorption in vitro and in vivo are scarce. The purities of two commercially available products, CPP1 and CPP2, are 18.37 and 25.12%, respectively. Here, the in vitro calcium binding capacity of CPP2 was 142.56 ± 7.39 mg/g, which was higher than that of CPP1 (107.15 ± 6.27 mg/g). The calcium transport results in a Caco-2 monolayer model indicated that, relative to controls, CPP1 and CPP2 increased calcium transport by 21.78 and 53.68%, respectively. Subsequent animal experiments showed that the CPP2-Ca-H group (1% Ca, 0.4% CPP2) had significant increases in the femur index, serum Ca2+ and serum osteocalcin levels, and femoral Ca content. The CPP2-Ca-H animal also had decreased serum alkaline phosphatase levels, parathyroid hormone content, and urinary pyridinoline content. Overall, our results demonstrated that CPP2 had stronger effects on promoting calcium uptake than CPP1.

IN VIVO AND IN VITRO STUDIES ON IONIZED VERSUS TOTAL SERUM CALCIUM IN HYPERPARATHYROIDISM

1973 ◽  
Vol 74 (3) ◽  
pp. 501-510 ◽  
Author(s):  
F. Lindgärde
Keyword(s):  
Calcium Binding ◽  
Binding Capacity ◽  
Total Serum ◽  
Total Serum Calcium ◽  
Point Of Intersection ◽  
Straight Lines ◽  
And Control ◽  
The Relationship

ABSTRACT Measurements of ionized (Ca+ + ) calcium and total (Cat) calcium have been performed in sera from hyperparathyroid, parathyroidectomized and control subjects. The relationship between Ca++ and Cat has been analysed statistically. It was found that two straight lines with the point of intersection at Ca++ =2.75 and Cat = 5.55 mEq./l, and slopes of 0.628 and 0.447, respectively, would describe the data more accurately than one single line. However, when the calcium level was varied by the addition of calcium chloride in vitro in individual or pooled sera, the Ca++–Cat relationship appeared to be linear. The results suggest that sera from hyperparathyroid subjects have an increased calcium binding capacity.

Age-related changes in composition and Ca2+-binding capacity of canine cortical bone extracts

1988 ◽  
Vol 255 (1) ◽  
pp. H101-H110 ◽  
Author(s):  
M. R. Pinto ◽  
J. P. Gorski ◽  
J. T. Penniston ◽  
P. J. Kelly
Keyword(s):  
Calcium Binding ◽  
Organic Phosphorus ◽  
Binding Capacity ◽  
Exchange Process ◽  
Age Groups ◽  
Binding Studies ◽  
Exchangeable Calcium ◽  
Age Related

The variable of age was used to study macromolecules in bone that may mediate in part the in vivo readily exchangeable calcium-binding capacity (VCa2+D). Organic components were extracted from nonmineralized bone with 4 M guanidine-HCl and from both nonmineralized and mineralized bone with 0.1 M EDTA. The composition of pup bone extracts demonstrated an enrichment in protein, hexuronate, sialic acid, organic phosphorus, and bound sulfate when compared with other age groups. In vitro calcium-binding studies identified low-affinity (Kd congruent to 10(-3) M) sites in both types of extracts; high-affinity sites (Kd congruent to 10(-5) M) were only evident in EDTA extracts of bone. Readily exchangeable calcium-binding capacity in vivo was found to decrease from pup (40.7 mM) to adolescent (11.1 mM) to the mature/old groups (2.6/1.2 mM); however, a large difference in low-affinity site number was only observed between pup and adolescent bone extracts. The overall organic composition of EDTA and guanidine-HCl extracts generally reflected the composition of total bone, which dropped dramatically on a dry weight basis from pup to adolescent groups. A similar pattern was observed with the number of low-affinity binding sites measured in vitro. In vitro binding data indicate that nonmineralized matrix of pup bone, extractable by 4 M guanidine-HCl, possesses enough capacity to accommodate approximately 40% of the readily exchangeable pool. As age progresses, other components of the blood-bone exchange process, such as vascularity, may reduce the readily exchangeable calcium pool size below the amount of low-affinity sites measured in vitro.

Biological and biomedical applications of collagenous biomaterials

1990 ◽  
Vol 48 (3) ◽  
pp. 856-857
Author(s):  
Yasushi P. Kato ◽  
Michael G. Dunn ◽  
Frederick H. Silver ◽  
Arthur J. Wasserman
Keyword(s):  
Biomedical Applications ◽  
Soft Tissues ◽  
Collagen Fibers ◽  
Bone Collagen ◽  
Cover Slip ◽  
Fibroblast Migration ◽  
Cellular Expression ◽  
Defect Repair

Collagenous biomaterials have been used for growing cells in vitro as well as for augmentation and replacement of hard and soft tissues. The substratum used for culturing cells is implicated in the modulation of phenotypic cellular expression, cellular orientation and adhesion. Collagen may have a strong influence on these cellular parameters when used as a substrate in vitro. Clinically, collagen has many applications to wound healing including, skin and bone substitution, tendon, ligament, and nerve replacement. In this report we demonstrate two uses of collagen. First as a fiber to support fibroblast growth in vitro, and second as a demineralized bone/collagen sponge for radial bone defect repair in vivo.For the in vitro study, collagen fibers were prepared as described previously. Primary rat tendon fibroblasts (1° RTF) were isolated and cultured for 5 days on 1 X 15 mm sterile cover slips. Six to seven collagen fibers, were glued parallel to each other onto a circular cover slip (D=18mm) and the 1 X 15mm cover slip populated with 1° RTF was placed at the center perpendicular to the collagen fibers. Fibroblast migration from the 1 x 15mm cover slip onto and along the collagen fibers was measured daily using a phase contrast microscope (Olympus CK-2) with a calibrated eyepiece. Migratory rates for fibroblasts were determined from 36 fibers over 4 days.

In Vitro and in Vivo Comparison of Binding of 99m-Tc-Iabeled Anti-CEA MAb F33-104 with 99m-Tc-labeled Anti-CEA MAb BW431/26

1999 ◽  
Vol 38 (04) ◽  
pp. 115-119
Author(s):  
N. Oriuchi ◽  
S. Sugiyama ◽  
M. Kuroki ◽  
Y. Matsuoka ◽  
S. Tanada ◽  
...  
Keyword(s):  
Nude Mice ◽  
Binding Capacity ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Cell Binding ◽  
Vitro Binding ◽  
Labeling Method ◽  
Human Colon Adenocarcinoma

Summary Aim: The purpose of this study was to assess the potential for radioimmunodetection (RAID) of murine anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) F33-104 labeled with technetium-99m (99m-Tc) by a reduction-mediated labeling method. Methods: The binding capacity of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA by means of in vitro procedures such as immunoradiometric assay and cell binding assay and the biodistribution of 99m-Tc-labeled anti-CEA MAb F33-104 in normal nude mice and nude mice bearing human colon adenocarcinoma LS180 tumor were investigated and compared with 99m-Tc-labeled anti-CEA MAb BW431/26. Results: The in vitro binding rate of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA in solution and attached to the cell membrane was significantly higher than 99m-Tclabeled anti-CEA MAb BW431/261 (31.4 ± 0.95% vs. 11.9 ± 0.55% at 100 ng/mL of soluble CEA, 83.5 ± 2.84% vs. 54.0 ± 2.54% at 107 of LS 180 cells). In vivo, accumulation of 99m-Tc-labeled anti-CEA MAb F33-104 was higher at 18 h postinjection than 99m-Tc-labeled anti-CEA MAb BW431/26 (20.1 ± 3.50% ID/g vs. 14.4 ± 3.30% ID/g). 99m-Tcactivity in the kidneys of nude mice bearing tumor was higher at 18 h postinjection than at 3 h (12.8 ± 2.10% ID/g vs. 8.01 ± 2.40% ID/g of 99m-Tc-labeled anti-CEA MAb F33-104, 10.7 ± 1.70% ID/g vs. 8.10 ± 1.75% ID/g of 99m-Tc-labeled anti-CEA MAb BW431/26). Conclusion: 99m-Tc-labeled anti-CEA MAb F33-104 is a potential novel agent for RAID of recurrent colorectal cancer.

Preparation of dextran–casein phosphopeptide conjugates, evaluation of its calcium binding capacity and digestion in vitro

2021 ◽  
pp. 129332
Author(s):  
Lan Jiang ◽  
Shuhong Li ◽  
Nan Wang ◽  
Shuang Zhao ◽  
Yue Chen ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Binding Capacity ◽  
Casein Phosphopeptide

scholarly journals Exosomal S100A4 derived from highly metastatic hepatocellular carcinoma cells promotes metastasis by activating STAT3

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Haoting Sun ◽  
Chaoqun Wang ◽  
Beiyuan Hu ◽  
Xiaomei Gao ◽  
Tiantian Zou ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cancer Progression ◽  
Calcium Binding ◽  
Tumor Metastasis ◽  
Metastatic Potential ◽  
Functional Roles ◽  
Stat3 Phosphorylation ◽  
Hcc Cells

AbstractIntercellular cross-talk plays important roles in cancer progression and metastasis. Yet how these cancer cells interact with each other is still largely unknown. Exosomes released by tumor cells have been proved to be effective cell-to-cell signal mediators. We explored the functional roles of exosomes in metastasis and the potential prognostic values for hepatocellular carcinoma (HCC). Exosomes were extracted from HCC cells of different metastatic potentials. The metastatic effects of exosomes derived from highly metastatic HCC cells (HMH) were evaluated both in vitro and in vivo. Exosomal proteins were identified with iTRAQ mass spectrum and verified in cell lines, xenograft tumor samples, and functional analyses. Exosomes released by HMH significantly enhanced the in vitro invasion and in vivo metastasis of low metastatic HCC cells (LMH). S100 calcium-binding protein A4 (S100A4) was identified as a functional factor in exosomes derived from HMH. S100A4rich exosomes significantly promoted tumor metastasis both in vitro and in vivo compared with S100A4low exosomes or controls. Moreover, exosomal S100A4 could induce expression of osteopontin (OPN), along with other tumor metastasis/stemness-related genes. Exosomal S100A4 activated OPN transcription via STAT3 phosphorylation. HCC patients with high exosomal S100A4 in plasma also had a poorer prognosis. In conclusion, exosomes from HMH could promote the metastatic potential of LMH, and exosomal S100A4 is a key enhancer for HCC metastasis, activating STAT3 phosphorylation and up-regulating OPN expression. This suggested exosomal S100A4 to be a novel prognostic marker and therapeutic target for HCC metastasis.

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