Barley β-glucan effects on emulsification and in vitro lipolysis of canola oil are modulated by molecular size, mixing method, and emulsifier type

2020 ◽  
Vol 103 ◽  
pp. 105643 ◽  
Author(s):  
Honglei Zhai ◽  
Purnima Gunness ◽  
Michael J. Gidley
Keyword(s):  
Canola Oil ◽  
Molecular Size ◽  
In Vitro Lipolysis ◽  
Mixing Method

In vitro Lipolysis as a Tool for the Establishment of IVIVC for Lipid-Based Drug Delivery Systems

2019 ◽  
Vol 16 (8) ◽  
pp. 688-697
Author(s):  
Ravinder Verma ◽  
Deepak Kaushik
Keyword(s):  
Drug Delivery ◽  
Ph Value ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Rapid Screening ◽  
Resonance Spectroscopy ◽  
Fed State ◽  
In Vitro Lipolysis ◽  
Ph Stat

: In vitro lipolysis has emerged as a powerful tool in the development of in vitro in vivo correlation for Lipid-based Drug Delivery System (LbDDS). In vitro lipolysis possesses the ability to mimic the assimilation of LbDDS in the human biological system. The digestion medium for in vitro lipolysis commonly contains an aqueous buffer media, bile salts, phospholipids and sodium chloride. The concentrations of these compounds are defined by the physiological conditions prevailing in the fasted or fed state. The pH of the medium is monitored by a pH-sensitive electrode connected to a computercontrolled pH-stat device capable of maintaining a predefined pH value via titration with sodium hydroxide. Copenhagen, Monash and Jerusalem are used as different models for in vitro lipolysis studies. The most common approach used in evaluating the kinetics of lipolysis of emulsion-based encapsulation systems is the pH-stat titration technique. This is widely used in both the nutritional and the pharmacological research fields as a rapid screening tool. Analytical tools for the assessment of in vitro lipolysis include HPLC, GC, HPTLC, SEM, Cryo TEM, Electron paramagnetic resonance spectroscopy, Raman spectroscopy and Nanoparticle Tracking Analysis (NTA) for the characterization of the lipids and colloidal phases after digestion of lipids. Various researches have been carried out for the establishment of IVIVC by using in vitro lipolysis models. The current publication also presents an updated review of various researches in the field of in vitro lipolysis.

scholarly journals Enzymic characterization in vitro of recombinant proprotein convertase PC4

1999 ◽  
Vol 343 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Ajoy BASAK ◽  
Bakary B. TOURÉ ◽  
Claude LAZURE ◽  
Majambu MBIKAY ◽  
Michel CHRÉTIEN ◽  
...  
Keyword(s):  
Synthetic Peptides ◽  
Serine Proteases ◽  
Active Form ◽  
Molecular Size ◽  
Maximal Activity ◽  
Transition Metal Ions ◽  
Proprotein Convertase ◽  
Major Band ◽  
Insulin Growth Factor

Proprotein convertase PC4A, a member of the subtilisin/kexin family of serine proteases, was obtained in enzymically active form following expression of vaccinia virus recombinant rat (r)PC4A in GH4C1 cells. It displayed maximal activity at pH 7.0 and a Ca2+ concentration of 2.0 mM. Using PC4-specific antibodies, Western blot analysis of the medium revealed a major band at ≈ 54 kDa, corresponding to the molecular size of mature rPC4A. Among the various peptidyl-[4-methylcoumarin 7-amide (MCA)] substrates tested, the one that was preferred the most by rPC4A was acetyl (Ac)-Arg-Lys-Lys-Arg-MCA, which is cleaved 9 times faster (as judged from Vmax/Km measurements) than the best furin and PC1 substrate, pGlu-Arg-Thr-Lys-Arg-MCA. Recombinant rPC4A, along with human (h)furin and hPC1, cleaved a 17-amino-acid synthetic peptide, YQTLRRRVKR↓ SLVVPTD (where ↓ denotes site of cleavage, and the important basic residues are shown in bold), encompassing the junction between the putative pro-segment of rPC4A and the active enzyme, suggesting a possible auto-activation of the enzyme. In an effort to identify potential physiological substrates for PC4, studies were performed with pro-[insulin-growth-factor (IGF)]-derived synthetic peptides, namely Ac-PAKSAR↓ SVRA (IGF-I66-75) and Ac-PAKSER↓ DVST (IGF-II63-72), as well as two lysine mutants [(IGF-I66-75Lys70) and (IGF-II63-72Lys67)]. Unlike PC1 and furin, rPC4A cleaved efficiently both IGF-I66-75 and IGF-II63-72, suggesting a possible role of PC4 in the maturation of IGF-I and -II. In contrast, the peptides with a position 2 (P2) lysine mutation, IGF-I66-75Lys70 and IGF-II63-72Lys67, were cleaved more efficiently by PC1 and furin compared with rPC4A. Furthermore, using synthetic peptides containing the processing sites of pituitary adenylate-cyclase-activating polypeptide (PACAP)-38, we were able to confirm that, of the two testicular enzymes PC4 and PC7, PC4 is the best candidate enzyme for maturation of PACAP. Our data suggest that rPC4A is a functionally active convertase, with a substrate specificity somewhat different from that of other convertases, namely KXXR↓ (where X denotes any other residue). As expected, p-chloromercuribenzoic acid and metal chelators such as EDTA, EGTA and trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid inhibit the proteolytic activity of rPC4A, whereas it is activated by dithiothreitol. PC4A was also inhibited by transition-metal ions (Cu2+>Hg2+>Zn2+ Ni2+>Co2+), as well as by small peptide semicarbazones (SCs), such as Arg-Lys-Lys-Arg-SC (Ki 0.75 μM) and Arg-Ser-Lys-Arg-SC (Ki 11.4 μM).

Ficoll and dextran vs. globular proteins as probes for testing glomerular permselectivity: effects of molecular size, shape, charge, and deformability

2005 ◽  
Vol 288 (4) ◽  
pp. F605-F613 ◽  
Author(s):  
Daniele Venturoli ◽  
Bengt Rippe
Keyword(s):  
Hard Spheres ◽  
Molecular Size ◽  
Globular Proteins ◽  
Molecular Probes ◽  
Glomerular Permeability ◽  
Ample Evidence ◽  
Deformable Structure ◽  
Glomerular Barrier ◽  
Size Asymmetry

Polydisperse mixtures of dextran or Ficoll have been frequently used as molecular probes for studies of glomerular permselectivity because they are largely inert and not processed (reabsorbed) by the proximal tubules. However, dextrans are linear, flexible molecules, which apparently are hyperpermeable across the glomerular barrier. By contrast, the Ficoll molecule is almost spherical. Still, there is ample evidence that Ficoll fractional clearances (sieving coefficients) across the glomerular capillary wall (GCW) are markedly higher than those for neutral globular proteins of an equivalent in vitro Stokes-Einstein (SE) radius. Physical data, obtained by “crowding” experiments or measurements of intrinsic viscosity, suggest that the Ficoll molecule exhibits a rather open, deformable structure and thus deviates from an ideally hard sphere. This is also indicated from the relationship between (log) in vitro SE radius and (log) molecular weight (MW). Whereas globular proteins seem to behave in a way similar to hydrated hard spheres, polydisperse dextran and Ficoll exhibit in vitro SE radii that are much larger than those for compact spherical molecules of equivalent MW. For dextran, this can be partially explained by a high-molecular-size asymmetry. However, for Ficoll the explanation may be that the Ficoll molecule is more flexible (deformable) than are globular proteins. An increased compressibility of Ficoll and an increased deformability and size asymmetry for dextran may be the explanation for the fact that the permeability of the GCW is significantly higher when assessed using polysaccharides such as Ficoll or dextran compared with that obtained using globular proteins as molecular size probes. We suggest that molecular deformability, besides molecular size, shape, and charge, plays a crucial role in determining the glomerular permeability to molecules of different species.

scholarly journals A macromolecular inhibitor of the antizyme to ornithine decarboxylase

1982 ◽  
Vol 204 (3) ◽  
pp. 647-652 ◽  
Author(s):  
K Fujita ◽  
Y Murakami ◽  
S Hayashi
Keyword(s):  
Ornithine Decarboxylase ◽  
Rat Liver ◽  
Molecular Size ◽  
Antizyme Inhibitor ◽  
Heat Labile

A macromolecular factor that inhibits the activity of the antizyme to ornithine decarboxylase (ODC) was found in rat liver extracts. The factor, ‘antizyme inhibitor’, was heat-labile, non diffusable and of similar molecular size to ODC. The antizyme inhibitor re-activated ODC that had been inactivated by antizyme, apparently by replacing ODC in a complex with antizyme. Therefore the antizyme inhibitor can be used to assay the amount of inactive ODC-antizyme complex formed in vitro. When assayed by this method, the complex was shown to be eluted before ODC from a Sephadex G-100 column. Significant increase in ODC activity was observed when the antizyme inhibitor was added to crude liver extracts from rats that had been injected with 1,3-diaminopropane to cause decay of ODC activity, suggesting the presence of inactive ODC-antizyme complex in the extracts.

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.

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