scholarly journals Enzymatic tracers in the study of vascular permeability.

1979 ◽  
Vol 27 (8) ◽  
pp. 1120-1130 ◽  
Author(s):  
N Simionescu
Keyword(s):  
Vascular Permeability ◽  
Molecular Size ◽  
Molecular Shape ◽  
Enzyme Concentration ◽  
Gel Chromatography ◽  
Microvascular Endothelium ◽  
Tracer Solution ◽  
Molecular Radius

Elucidation of the ultrastructural basis of vascular permeability was aided by the development of cytochemical techniques for visualizing the distribution, within the vessel wall, of intravenously injected peroxidatic enzymes of varying molecular size. Tracer enzymes available range from 10 A (hemeoctapeptide) to 52 A (catalase) effective molecular radius. The use of enzymatic probe molecules assumes a thorough characterization of: (a) the molecular charge (isoelectric point of the native enzyme, and when feasible, its polyanionic and polycationic derivatives; (b) effective molecular radius (ae); (c) peroxidase activity (to detect by spectrophotometry of DAB-oxidizing activity, the optimal pH, temperature, and enzyme concentration to be employed in the cytochemical procedure). Molecular shape and state of dispersion of the enzymatic probes should be determined by gel chromatography and spectrophotometry of both the tracer solution and aliquots of blood plasma collected after i.v. injection of the tracer. Conditions required for the probe administration include: (a) the investigation of potential side effects (tests for toxicity and vascular leakage) and (b) estimation of the tracer volume and concentration which does not affect significantly the blood volume and osmotic pressure. Determination in vitro of the crosslinking of tracer molecules induced by the aldehyde fixative to be employed, also gives an indication on potential diffusion artifacts. Based on the information thus obtained, the design of the cytochemical procedure should also take into account the possible use of methods for enhancing the peroxidatic reaction product: nitrogenous ligands (imidazole, diaminopyrimidine, histidine) or polyphenolic mordants (galloylglucoses). The usefulness of peroxidatic tracers in the investigation of vascular permeability is exemplified by some results obtained on the microvascular endothelium in vivo (trasncytosis, intercellular pathway, etc.), and on endothelial cells isolated from heart microvasculature.

Molecular Size Distribution of Fibrinogen Derivatives Formed in Vitro and in Vivo: a Chromatographic Study

1976 ◽  
Vol 36 (01) ◽  
pp. 014-026 ◽  
Author(s):  
M. B Donati ◽  
R Verhaeghe ◽  
D. E Culasso ◽  
J Vermylen
Keyword(s):  
Biological Fluids ◽  
Gel Filtration ◽  
Molecular Size ◽  
Gel Chromatography ◽  
Chromatographic Study ◽  
Elution Volume ◽  
Human Fibrinogen ◽  
Elution Pattern

SummaryUsing gel chromatography, fibrinogen derivatives present in purified systems or in biological fluids were separated and partially characterized. Eight groups of fibrinogen derivatives could be separated by gel filtration through 6% agarose in large columns, four with an elution volume smaller and four groups with an elution volume larger than that of fibrinogen. Careful calibration of the column allowed estimation of the diffusion coefficients of some of the derivatives and, thus, comparison with derivatives previously identified. Three, rather than two, groups of intermediate derivatives were observed during the degradation of human fibrinogen by plasmin in vitro or in vivo. One of these had a marked tendency to polymerize.A rather distinct difference in elution pattern was found between plasma obtained during streptokinase administration and from patients with intravascular coagulation.

scholarly journals The characterization of a protein–polysaccharide isolated from Kurloff cells of the guinea pig

1970 ◽  
Vol 118 (5) ◽  
pp. 783-790 ◽  
Author(s):  
M. F. Dean ◽  
Helen Muir
Keyword(s):  
Chondroitin Sulphate ◽  
Molecular Size ◽  
Gel Chromatography ◽  
Oestrogen Treatment ◽  
Molar Ratios ◽  
Low Concentrations ◽  
Cell Induction ◽  
Toxic Constituent

Kurloff cells of guinea pigs increase in number and accumulate in the spleen on oestrogen treatment. Because they contain metachromatic inclusions and are considered to be lymphocytes they were examined as a possible model for mucopolysaccharidoses like Hurler's syndrome, where some lymphocytes are also metachromatic. Oestrogen treatment produced a large increase in a glycosaminoglycan resembling chondroitin 4-sulphate in chemical analysis, chromatographic behaviour and i.r. spectrum but with an additional strong band at 805cm−1. Material isolated without proteolysis behaved on gel chromatography as a multiple-chain protein–polysaccharide whose molecular size was decreased by proteolysis. It contained xylose and galactose in molar proportions with serine, compatible with the presence of the same linkage region as in cartilage chondroitin 4-sulphate proteins and which likewise underwent alkaline β-elimination. Kurloff glycosaminoglycan chains were significantly longer than chondroitin sulphate chains of cartilage protein–polysaccharides as assessed by gel chromatography and the molar ratios of galactosamine to xylose or to serine. Kurloff cells thus contain intact rather than partially degraded protein–polysaccharide and hence are not analogous to Hurler cells, and their electron micrographs were also different. The purified Kurloff protein–polysaccharide and glycosaminoglycan isolated here has been shown by Marshall, Swettenham, Vernon-Roberts & Revell (1970) to be toxic specifically to macrophages at extremely low concentrations in vitro, unlike chondroitin sulphate of protein–polysaccharides from cartilage. The toxic constituent may account for the i.r.-absorption band at 805cm−1. Although active incorporation of [35S]sulphate occurs at early stages of Kurloff-cell induction (Marshall et al. 1970), the fully developed Kurloff cell studied here showed very low incorporation in vitro and in vivo, suggesting that the inclusions are specialized for the storage of the toxic material.

scholarly journals ANTI-INFLAMMATORY ACTIVITY OF CURCUMIN AND CAPSAICIN AUGMENTED IN COMBINATION

2017 ◽  
Vol 9 (6) ◽  
pp. 145
Author(s):  
Thriveni Vasanth Kumar ◽  
Manjunatha H. ◽  
Rajesh Kp
Keyword(s):  
Acetic Acid ◽  
Protective Effect ◽  
Vascular Permeability ◽  
Diclofenac Sodium ◽  
Significant Inhibition ◽  
Inflammatory Activity ◽  
Anti Inflammatory ◽  
The Individual

Objective: Dietary curcumin and capsaicin are well known for their health beneficial potencies. The current study was done to assess the anti-inflammatory activity of curcumin, capsaicin and their combination by employing in vitro and in vivo models.Methods: We investigated the protective effect of curcumin, capsaicin and their combination using in vitro heat induced human red blood cell (HRBC) membrane stabilisation, in vivo 3% agar induced leukocyte mobilisation and acetic acid induced vascular permeability assay.Results: Curcumin, capsaicin and their combination exhibited concentration dependent protective effect against heat-induced HRBC membrane destabilisation, while combined curcumin and capsaicin restored 87.0±0.64 % membrane stability and it is found to be better than curcumin, capsaicin and diclofenac sodium (75.0±0.25. 72±0.9 and 80.0±0.31 %) protective effect. In agar suspension induced leukocyte mobilization assay, the combined curcumin and capsaicin had shown 39.5±1.58 % of inhibition compared to individual curcumin and capsaicin, which showed moderate inhibition of 16.0±3.14 and 21.6±2.17 % respectively. Besides, the combined curcumin and capsaicin had shown highly significant inhibition of acetic acid-induced vascular permeability in rats (62.0±3.14 %), whereas individual curcumin and capsaicin showed moderate inhibition of vascular permeability with 36.0±2.41 and 43.0±1.92 % respectively.Conclusion: This study demonstrates the significant anti-inflammatory property of combined curcumin and capsaicin at half of the individual concentration of curcumin and capsaicin.

scholarly journals Treatment with monocyte-derived partially purified GM-CSF but not G-CSF aborts the development of transplanted chloroleukemia in rats

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 1077-1080 ◽  
Author(s):  
JJ Jimenez ◽  
AA Yunis
Keyword(s):  
Conditioned Medium ◽  
Molecular Size ◽  
Myelogenous Leukemia ◽  
Rat Lung ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

We have previously demonstrated that cultured rat chloroleukemia cells, MIA C51, will terminally differentiate to macrophages when treated with rat lung-conditioned medium in vitro and in vivo. In the present study we fractionated rat monocyte-conditioned medium by ultrafiltration according to molecular size. The fraction with molecular weight (mol wt) 30 to 50 Kd containing partially purified granulocyte-macrophage colony-stimulating factor (GM-CSF) activity caused the differentiation of C51 cells to macrophages in vitro and in diffusion chambers in vivo. Treatment of young rats with this fraction aborted the development of chloroleukemia from transplanted C51 cells. In contrast, the fraction with mol wt 10 to 30 Kd containing virtually all the G-CSF activity exhibited no differentiation activity either in vitro or in vivo. It is concluded that in this rat myelogenous leukemia model partially purified GM-CSF but not G-CSF contains the effector molecule(s) causing terminal differentiation of C51 cells and tumor cell rejection.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (11) ◽  
pp. 5796-5805
Author(s):  
P Orlean
Keyword(s):  
Molecular Size ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Nonpermissive Temperature ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Covalent Modifications

Glycosyl phosphatidylinositol (GPI) anchoring, N glycosylation, and O mannosylation of protein occur in the rough endoplasmic reticulum and involve transfer of precursor structures that contain mannose. Direct genetic evidence is presented that dolichol phosphate mannose (Dol-P-Man) synthase, which transfers mannose from GDPMan to the polyisoprenoid dolichol phosphate, is required in vivo for all three biosynthetic pathways leading to these covalent modifications of protein in yeast cells. Temperature-sensitive yeast mutants were isolated after in vitro mutagenesis of the yeast DPM1 gene. At the nonpermissive temperature of 37 degrees C, the dpm1 mutants were blocked in [2-3H]myo-inositol incorporation into protein and accumulated a lipid that could be radiolabeled with both [2-3H]myo-inositol and [2-3H]glucosamine and met existing criteria for an intermediate in GPI anchor biosynthesis. The likeliest explanation for these results is that Dol-P-Man donates the mannose residues needed for completion of the GPI anchor precursor lipid before it can be transferred to protein. Dol-P-Man synthase is also required in vivo for N glycosylation of protein, because (i) dpm1 cells were unable to make the full-length precursor Dol-PP-GlcNAc2Man9Glc3 and instead accumulated the intermediate Dol-PP-GlcNAc2Man5 in their pool of lipid-linked precursor oligosaccharides and (ii) truncated, endoglycosidase H-resistant oligosaccharides were transferred to the N-glycosylated protein invertase after a shift to 37 degrees C. Dol-P-Man synthase is also required in vivo for O mannosylation of protein, because chitinase, normally a 150-kDa O-mannosylated protein, showed a molecular size of 60 kDa, the size predicted for the unglycosylated protein, after shift of the dpm1 mutant to the nonpermissive temperature.

scholarly journals Increased Carrier Peptide Stability through pH Adjustment Improves Insulin and PTH(1-34) Delivery In Vitro and In Vivo Rather than by Enforced Carrier Peptide-Cargo Complexation

Pharmaceutics ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 993
Author(s):  
Mie Kristensen ◽  
Ragna Guldsmed Diedrichsen ◽  
Valeria Vetri ◽  
Vito Foderà ◽  
Hanne Mørck Nielsen
Keyword(s):  
Oral Delivery ◽  
Molecular Size ◽  
Therapeutic Peptide ◽  
Enzymatic Stability ◽  
Therapeutic Peptides ◽  
Ph Dependent ◽  
Pharmacologically Active ◽  
High Degree

Oral delivery of therapeutic peptides is hampered by their large molecular size and labile nature, thus limiting their permeation across the intestinal epithelium. Promising approaches to overcome the latter include co-administration with carrier peptides. In this study, the cell-penetrating peptide penetratin was employed to investigate effects of co-administration with insulin and the pharmacologically active part of parathyroid hormone (PTH(1-34)) at pH 5, 6.5, and 7.4 with respect to complexation, enzymatic stability, and transepithelial permeation of the therapeutic peptide in vitro and in vivo. Complex formation between insulin or PTH(1-34) and penetratin was pH-dependent. Micron-sized complexes dominated in the samples prepared at pH-values at which penetratin interacts electrostatically with the therapeutic peptide. The association efficiency was more pronounced between insulin and penetratin than between PTH(1-34) and penetratin. Despite the high degree of complexation, penetratin retained its membrane activity when applied to liposomal structures. The enzymatic stability of penetratin during incubation on polarized Caco-2 cell monolayers was pH-dependent with a prolonged half-live determined at pH 5 when compared to pH 6.5 and 7.4. Also, the penetratin-mediated transepithelial permeation of insulin and PTH(1-34) was increased in vitro and in vivo upon lowering the sample pH from 7.4 or 6.5 to 5. Thus, the formation of penetratin-cargo complexes with several molecular entities is not prerequisite for penetratin-mediated transepithelial permeation a therapeutic peptide. Rather, a sample pH, which improves the penetratin stability, appears to optimize the penetratin-mediated transepithelial permeation of insulin and PTH(1-34).

Cortactin Depletion Increases Pulmonary Vascular Permeability In Vitro And In Vivo

2011 ◽  
Author(s):  
Patrick Belvitch ◽  
Mary E. Brown ◽  
Sara M. Camp ◽  
Anjali Desai ◽  
Liliana Moreno-Vinasco ◽  
...  
Keyword(s):  
Vascular Permeability ◽  
Pulmonary Vascular Permeability

scholarly journals Tumor Uptake of Triazine Dendrimers Decorated with Four, Sixteen, and Sixty-Four PSMA-Targeted Ligands: Passive versus Active Tumor Targeting

Biomolecules ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 421 ◽  
Author(s):  
Jongdoo Lim ◽  
Bing Guan ◽  
Kien Nham ◽  
Guiyang Hao ◽  
Xiankai Sun ◽  
...  
Keyword(s):  
Specific Binding ◽  
Quantitative Imaging ◽  
Molecular Size ◽  
Tumor Uptake ◽  
Competitive Binding Assay ◽  
The Core ◽  
Positron Emission ◽  
Pentanedioic Acid

Various glutamate urea ligands have displayed high affinities to prostate specific membrane antigen (PSMA), which is highly overexpressed in prostate and other cancer sites. The multivalent versions of small PSMA-targeted molecules are known to be even more efficiently bound to the receptor. Here, we employ a well-known urea-based ligand, 2-[3-(1,3-dicarboxypropyl)-ureido] pentanedioic acid (DUPA) and triazine dendrimers in order to study the effect of molecular size on multivalent targeting in prostate cancer. The synthetic route starts with the preparation of a dichlorotriazine bearing DUPA in 67% overall yield over five steps. This dichlorotriazine reacts with G1, G3, and G5 triazine dendrimers bearing a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) group for 64Cu-labeling at the core to afford poly(monochlorotriazine) intermediates. Addition of 4-aminomethylpiperidine (4-AMP) and the following deprotection produce the target compounds, G1-(DUPA)4, G3-(DUPA)16, and G5-(DUPA)64. These targets include 4/16/64 DUPA groups on the surface and a DOTA group at the core, respectively. In vitro cell assay using PC3-PIP (PSMA positive) and PC3-FLU (PSMA negative) cells reveals that G1-(DUPA)4 has the highest PC3-PIP to PC3-FLU uptake ratio (10-fold) through the PSMA-mediated specific uptake. While G5-(DUPA)64 displayed approximately 12 times higher binding affinity (IC50 23.6 nM) to PC3-PIP cells than G1-(DUPA)4 (IC50 282.3 nM) as evaluated in a competitive binding assay, the G5 dendrimer also showed high non-specific binding to PC3-FLU cells. In vivo uptake of the 64Cu-labeled dendrimers was also evaluated in severe combined inmmunodeficient (SCID) mice bearing PC3-PIP and PC3-FLU xenografts on each shoulder, respectively. Interestingly, quantitative imaging analysis of positron emission tomograph (PET) displayed the lowest tumor uptake in PC3-PIP cells for the midsize dendrimer G3-(DUPA)16 (19.4 kDa) (0.66 ± 0.15%ID/g at 1 h. p.i., 0.64 ± 0.11%ID/g at 4 h. p.i., and 0.67 ± 0.08%ID/g at 24 h. p.i.). Through the specific binding of G1-(DUPA)4 to PSMA, the smallest dendrimer (5.1 kDa) demonstrated the highest PC3-PIP to muscle and PC3-PIP to PC3-FLU uptake ratios (17.7 ± 5.5 and 6.7 ± 3.0 at 4 h p.i., respectively). In addition, the enhanced permeability and retention (EPR) effect appeared to be an overwhelming factor for tumor uptake of the largest dendrimer G5-(DUPA)64 as the uptake was at a similar level irrelevant to the PSMA expression.

STRUCTURE ACTIVITY RELATIONSHIPS OF DERMATAN SULFATE : EFFECT OF MOLECULAR SIZE AND CARBOXYL GROUP ESTERIFICATION ON HEPARIN C0FACT0R-II ACTIVATION

1987 ◽  
Author(s):  
J Mardiguian ◽  
M Corgier ◽  
M Jouany
Keyword(s):  
Molecular Weight ◽  
Dermatan Sulfate ◽  
Methyl Esters ◽  
Molecular Size ◽  
Carboxyl Groups ◽  
Gel Chromatography ◽  
Carboxyl Group ◽  
Heparin Cofactor Ii ◽  
High Affinity

Dermatan is a high molecular weight glycosaminoglycan which has been shown to enhance the inhibition of thrombin by heparin-cofactor II. The aim of this study was to establish the influence of the molecular size and the role of the carboxyl group on the in vitro activity of Dermatan Sulfate. Pig skin Dermatan Sulfate was fractionated according to molecular size by gel-chromatography on Ultrogel Ac 44. Each fraction was characterized by its sulfur content and by its mean molecular weight measured on a TSK - 4000 column in reference to standard heparin fractions. Methyl esters of the unfractionated Dermatan Sulfate with varying degree of esterification, where prepared via activation of the carboxyl groups with a carbodiimide and reaction with methanol. The results of this study show that the heparin - cofactor II mediated anti-thrombin activity of Dermatan Sulfate is increasing with the molecular weight and is abolished by esterification of the carboxyl groups. Moreover, it can be speculated that each fraction contains the same amount of high affinity fraction and that, like heparin, the potency of the high affinity component is increasing with the molecular weight.

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