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PubMed1977

Beta-lipoprotein cholesterol quantitation with polycations.

We compared direct determination of beta-lipoprotein cholesterol after selective extraction of very-low-density and high-density lipoproteins from serum with poly(ethyleneimine) and a cation-exchange resin with the classical quantitation after lipoprotein fractionation with the ultracentrifuge. At beta-lipoprotein cholesterol concentrations between 1.50 and 5.00 g/liter the correlation is linear (r = 0.95). The precision for the extraction procedure is as good (CV 2.4-2.8%) as for the quantitation by ultracentrifugation (CV 3.2-6.0%). From solutions of isolated lipoproteins, very-low-density lipoproteins are 93% extracted and high-density lipoproteins 60%, but low-density-lipoproteins only 5%. The molecular mechanism of the extraction is supposed to be due to both hydrophobic interaction of long-chain fatty acid residues and ionic interaction of phospholipids located at the surface of very low-density and high-density lipoproteins and the lipophilic polycation.

Cholesterol↗

PubMed1991

Selective transport of an anti-transferrin receptor antibody through the blood-brain barrier in vivo.

The brain capillary endothelium, which makes up the blood-brain barrier (BBB) in vivo, expresses high concentrations of transferrin receptor, and recent studies show that an antitransferrin receptor monoclonal antibody may function as a BBB drug transport vector. The present report examines the pharmacokinetics of clearance of radiolabeled antitransferrin receptor monoclonal antibody from the bloodstream in rats in vivo, and also assesses the extent to which brain selectively extracts the antibody from the blood compared to other peripheral organs such as liver, kidney, myocardium, or lung. [125I]Mouse immunoglobulin G2a control antibody was cleared monoexponentially with a half-time of 9.8 +/- 2.3 h. The clearance of the [3H]OX-26 antitransferrin receptor antibody from blood was biexponential with half-times of 2.2 +/- 0.8 min (61 +/- 10% of clearance) and 3.9 +/- 0.2 h (39 +/- 4% of clearance). The OX-26 antibody was rapidly taken up by liver during the first 60 min after injection, but this uptake reached rapid saturation, and hepatic OX-26 content actually declined subsequent to the first hour after injection. In contrast, brain continuously extracted the OX-26 antibody from the bloodstream, and the brain volume of distribution of OX-26 reached a value 18-fold greater than the volume of distribution of the mouse immunoglobulin G2a at 5 h after injection. There was no specific uptake of the OX-26 by myocardium or lung, and minor uptake by kidney was observed that also reached saturation within the first 60 min after injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

[Detection of nuclear matrix and intermediate filament scaffold in Crypthecodinium cohnii].

Nuclear matrix (NM) and intermediate filament (IF) scaffold in primitive eukaryote Crypthecodinium cohnii were shown using selective extraction together with embedment-free electron microscopy, whole mount cell preparation and immunoblot techniques. There exists a delicate NM-IF network spreading over cytoplasm and nucleus in dinoflagellate cells, however, nuclear lamina is undeveloped. The diameter of NM fiber is about 3-5 nm and IF is 10 nm. Chromosomes are connected with NM filament network. Immunoblot analysis showed that dinoflagellate contained keratin-like polypeptides (63 kD and 67 kD) while mammalian lamin antibodies did not crossreact with dinoflagellate total protein. Our experiment results demonstrated that a framework similar to NM-IF scaffold in mammalian cell appeared in primitive eukaryote. We propose that: (1) NM-IF scaffold is not restrict to vertebrate cell, and it may be originated from early stages of eukaryote evolution; (2) Keratin is probably very conservative; (3) Compared with IF, lamina might appear late in evolution, and some of primitive characteristics of dinoflagellate nucleus may be related to the lack of lamina.

Animals↗

PubMed1991

HPLC determination of polyamines in urine.

Determination of polyamines (putrescine, spermidine and spermine) in urine samples of patients was worked out using high performance liquid chromatography (HPLC) and pre-column derivatization. For HPLC separation, a method of reversed-phase chromatography with gradient elution of water-organic mobile phases, UV and fluorescence detection were applied. Special care was devoted to the preseparation step, the hydrolysis, derivatization and selective extraction of clinical samples. The optimal HPLC conditions were recommended for simultaneous determination of putrescine, spermidine and spermine in a minimal analysis time (about 15 min) and suitable chromatographic resolution values (Rij greater than 1.5). Detection limits for all polyamines were evaluated (40 pmol for putrescine, 25 pmol for spermidine and 20 pmol for spermine) for the analysis of polyamines in the urine. The urine samples of healthy people and the cancer patients were compared and the procedure or the complete analysis has been described.

Chromatography, High Pressure Liquid↗

PubMed1990

New approach of three-dimensional crystallization of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Selective extraction procedure was applied for obtaining different proteins from sarcoplasmic reticulum vesicles following the main steps of previous observations. High concentration of a nonionic detergent, such as polyoxyethylene-10-lauryl ether (C12E10) prevented the formation of Ca(2+)-ATPase crystals. It has been observed that only the 300 kDa protein could induce crystallization from among proteins being undissolved from of membrane. A modification of MacLennan's procedure--applied for ATPase precipitation from deoxycholate solubilized sarcoplasmic reticulum--has been described and an ammonium acetate precipitated Ca(2+)-ATPase was used in the experiments for increase of Ca(2+)-ATPase concentration in the crystallization process. The repeated supplementation of purified and C12E10 solubilized Ca(2+)-ATPase with ammonium acetate precipitated Ca(2+)-ATPase made possible a formation of larger and larger crystals with different periodicity.

Animals↗

PubMedDate not supplied

[Isolation and some properties of exoribonuclease from the cell nuclei of the rat liver].

Exoribonuclease from rat liver cell nuclei was isolated and purified using selective extraction at pH 8.0, salting out with ammonium sulfate (30-50% saturation) and chromatography on DEAE-Sephadex A-50. The enzyme has the pH optimum 7.4-7.6, it requires Mg-2+. It catalyses the gradual removal of ribonucleoside-5'-monophosphates from synthetic polynucleotides and RNA; it preferably attacks single-stranded polynucleotides and is less efficient when hydrolysing poly-stranded helical polymers. It is supposed that exoribonuclease can participate in the destruction of non-informative regions in new-formed hyper-molecular RNA of cell nuclei.

Animals↗

PubMed1989

Mouse heart laminin. Purification of the native protein and structural comparison with Engelbreth-Holm-Swarm tumor laminin.

Laminin was selectively extracted from different mouse tissues using EDTA-containing buffer. By immunoblotting with an antiserum raised against mouse Engelbreth-Holm-Swarm (EHS) tumor laminin, such extracts could be shown to contain laminin-like molecules with a low apparent proportion of A chain to B chains. Native laminin was purified from mouse heart tissue and was shown to have an aberrant polypeptide composition as compared to mouse EHS tumor laminin. Most prominently, mouse heart laminin contains an Mr 300,000 polypeptide which is not antigenically related to the A or the B chains. Furthermore, nonreducible polypeptide components were seen with apparent Mr values of 600,000 and 900,000. The Mr 600,000 component contains epitopes shared with both EHS tumor laminin and the Mr 300,000 polypeptide and possibly represents a covalently cross-linked complex of an A or B chain with the Mr 300,000 chain.

Animals↗

PubMed1989

Effect of dexamethasone on prostaglandin synthesis and on lipocortin status in human endothelial cells. Inhibition of prostaglandin I2 synthesis occurring without alteration of arachidonic acid liberation and of lipocortin synthesis.

Glucocorticoids have been shown to decrease prostaglandin I2 synthesis in human endothelial cells, suggesting the possible involvement of lipocortin in the inhibition of arachidonic acid liberation achieved by phospholipase A2 (De Caterina, R., and Weksler, B. B. (1986) Thromb. Haemostasis 55, 369-374). To test this hypothesis, human endothelial cells labeled with [14C]arachidonic acid were stimulated with thrombin (2 units/ml, 10 min), resulting in the secretion of free arachidonic acid together with various 14C-labeled metabolites, mainly 6-keto-prostaglandin F1 alpha, the stable derivative of prostaglandin I2. Under conditions where prior incubation of cells with dexamethasone reduced by 51% 6-keto-prostaglandin F1 alpha production, phospholipid hydrolysis induced by thrombin remained unaffected. Using three rabbit polyclonal antibodies directed against endonexin I, lipocortin I, and lipocortin II, evidence was obtained for the presence in human endothelial cells of equivalent amounts of lipocortin I and an immunologically unrelated 33-kDa protein, together with lower quantities of 67-kDa calelectrin/calcimedin. These Ca2+- and phospholipid-binding proteins were selectively extracted with [ethylene-bis(oxyethylene-nitrilo)]tetraacetic acid (EGTA) from cell membranes precipitated in the presence of Ca2+, and they displayed an inhibitory activity against pig pancreas phospholipase A2. However, the amounts of the three proteins were not changed by cell treatment with 2.5 microM dexamethasone, as detected upon polyacrylamide gel electrophoresis by silver staining, immunoblotting, or autoradiography following [35S]methionine in vivo labeling. Since the antiphospholipase A2 activity of EGTA extracts was hardly modified, it was concluded that an increased synthesis of lipocortin cannot account for the inhibition of prostaglandin synthesis brought about by dexamethasone, suggesting other biological functions for these proteins.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1989

F0 "proton channel" of rat liver mitochondria. Rapid purification of a functional complex and a study of its interaction with the unique probe diethylstilbestrol.

The F0 portion of the rat liver mitochondrial ATP synthase (F0F1-ATPase) has been purified by a rapid, high yield procedure. F0 is selectively extracted from inner membrane vesicles with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) after prior treatment of the vesicles with guanidine HCl to remove F1. The resultant F0 is functional in proton translocation assays and separates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis into four major and three minor Coomassie-stainable bands, all with apparent molecular masses below 30 kDa. This CHAPS-purified F0 preparation was characterized in detail for its capacity to interact with the unique probe diethylstilbestrol (DES) which, depending on conditions, has been shown to interact with rat liver F0F1 to either inhibit or promote ATP hydrolysis (McEnery, M. W., and Pedersen, P.L. (1986) J. Biol. Chem. 261, 1745-1752). DES-inhibitory sensitivity could be conferred on F1-ATPase activity with the same concentration dependence on F0 as conferral of oligomycin sensitivity. DES was shown also to inhibit the magnitude of valinomycin induced proton influx, while initiating proton efflux in asolectin vesicles reconstituted with F0 and loaded with K+. The potency of DES in producing the latter effects was shown to be highly dependent on hydroxyl groups in "para" positions of the two benzene rings within the DES molecule. Finally, in the absence of F0, DES was shown to act as a catalyst of proton influx in K+-loaded asolectin vesicles upon addition of valinomycin. A model based on the structure of DES is presented to account for both the inhibitory and uncoupling properties of this compound.

Animals↗

PubMed1989

Induction of mineral deposition by primary cultures of chicken growth plate chondrocytes in ascorbate-containing media. Evidence of an association between matrix vesicles and collagen.

A serum-free primary culture system for chicken growth plate chondrocytes has been developed which consistently undergoes mineral deposition. Upon attainment of confluency, the chondrocytes develop locally into multilayer cellular nodules leading to matrix calcification. Mineralization first occurs in matrix vesicles (MV) that are abundant in the extraterritorial matrix between the hypertrophic cells. Studies with 45Ca reveal that significant accumulation of Ca2+ occurs as early as day 12, continuing progressively throughout the culture period. By day 24, the nodules become densely calcified. Fourier transform infrared spectroscopy reveals the mineral to be similar to apatite, with features essentially identical to those of mineral formed by MV in vitro. The presence of ascorbate is critical to the culture system; in its absence, calcification is rarely observed. Ascorbate stimulates MV formation and synthesis of cellular protein, alkaline phosphatase, and especially types II and X collagens. In addition, there is strong evidence that the types II and X collagens are associated with MV. 1) Electron microscopy reveals MV embedded in a type II collagenous network; 2) Western blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of MV using monospecific antibodies to types X and II collagen indicate that both collagens are present in specific MV fractions; 3) sucrose gradient purification of MV does not remove associated collagens; 4) graded salt extraction selectively releases type II collagen from MV; and 5) incubation of radiolabeled types II and X collagens with MV leads to their cosedimentation upon subsequent centrifugation. Taken together, the data suggest that coordinated synthesis of the collagens, alkaline phosphatase, MV formation, and Ca2+ accumulation by the cultures combine to induce mineral deposition in the multilayer nodules.

Alkaline Phosphatase↗

PubMed1989

Risks and safety practices in hemapheresis procedures.

In hemapheresis procedures carried out for the collection of blood components, healthy donors are subjected to the old familiar risks of blood donation, plus those specifically relating to the use of blood cell separators and to the selective extraction (and loss to the donor) of individual blood components. The responsible physician must actively control these procedures, from the point of view of effective component collection and protection of the donor from undesirable procedural reactions, as well as potentially dangerous blood component depletion. The application of similar hemapheretic techniques to patients is quite a different matter. Patients do not have the same tolerance of blood manipulative techniques as do healthy donors. Furthermore, therapeutic component depletions are usually much more radical than the procedures applied to donors. The physician responsible for such therapeutic operations must play an active, consultative role with the clinical physician in discussing the likely efficacy of such treatment, the identification of risk factors, obtaining informed consent, deciding on a schedule of operations, replacement solutions, supplementary therapy, and overall supervision of procedures.

Blood Component Removal↗

PubMed1989

Lipid content and lipid type as determinants of the epidermal permeability barrier.

During terminal differentiation, mammalian epidermal lipids undergo striking changes in both composition and distribution. Phospholipids and neutral lipids are replaced by a mixture of ceramides and neutral lipids organized in intercellular lamellar bilayers. Whether all of these lipids and/or whether specific lipid classes regulate permeability barrier function is not known. When hairless mice were treated with acetone, the degree of barrier perturbation (measured as transepidermal water loss, TEWL) increased linearly with the amount of lipid removed. Moreover, virtually all lipid species appeared to be removed by acetone treatment. In contrast, the nonpolar organic solvent, petroleum ether, while removing greater amounts of lipids, provoked lesser barrier abnormalities. As determined by both quantitative thin-layer chromatography and histochemistry, petroleum ether selectively extracted nonpolar lipids leaving sphingolipids and free sterols in place. In petroleum ether-treated animals, subsequent acetone treatment removed additional sphingolipids and produced a dramatic increase in TEWL. A linear relationship existed for the quantities of sphingolipid removed and degree of barrier disruption in acetone-treated, but not petroleum ether-treated animals. These results support a relationship between the total lipid content of the stratum corneum and barrier function. Secondly, although the results demonstrate the participation of the total lipid mixture in the barrier, removal of nonpolar species alone appears to cause only a modest level of barrier disruption, while removal of sphingolipids and free sterols leads to a more profound level of barrier perturbation.

Acetone↗

PubMed1985

[Lectin immunoradiometric assay versus improved radioimmunoassay: a comparison of methods for determination of desialylated forms of human chorionic gonadotropin].

Two different assay systems have been developed for the measurement of asialo hCG(ashCG). One of them is an immunoradiometric assay which utilizes peanut lectin (PNA) to selectively extract ashCG and then directly quantify it with purified radiolabeled antiserum which is generated against carboxy terminal peptide of hCG beta (beta-CTP). PNA is highly specific for the terminal carbohydrate linkage Gal beta-(1----3)-Gal NAc which is only exposed to hCG when the O-serine linked oligosaccharide of its unique beta-CTP region is deficient in sialic acid. The other is an improved RIA using a specific antiserum to as beta-CTP, in which extraction of hCG with monoclonal antibody-Sepharose 4B conjugate is used to obtain increased sensitivity with retention of specificity. The ashCG concentration can be measured as low as 0.01 and 0.05 pmoles/ml in the presence of an excess amount of hCG in each of these assays. These assays were applied to detect ashCG in the urine of patients with trophoblastic tumors and normal pregnant women. The proportion of ashCG to total hCG in choriocarcinoma was significantly higher than that in normal pregnancy in both assays. However, the proportion was dependent upon the amount of total hCG and it decreased as total hCG increased.

Asialoglycoproteins↗

PubMed1987

Identification and purification of a kidney membrane protein which specifically binds the amino-terminal domain of native parathyroid hormone.

Nitrocellulose blots of bovine kidney membrane proteins were prepared from denaturing polyacrylamide gels. Strips of the blots were incubated with parathyroid hormone (PTH), washed, and then incubated with antisera against the hormone. Exposure to horseradish peroxidase-linked second antibody led to staining of a 51-kDa protein. No staining was observed in blots not incubated with PTH. Fragments 35-84 and 19-84 of PTH reacted strongly with the antisera, but did not lead to staining of the 51-kDa protein on the blots. Staining was visible, but greatly reduced, when fragment 9-84 was used. Oxidation of the native hormone at positions 8 and 18 led to reductions in staining of the band which were quantitatively similar to the reductions in biological activity induced by such oxidations. These properties suggested that the 51-kDa protein recognizes the amino-terminal portions of PTH, which is the segment of the molecule required for its biological activities. Several micrograms of the 51-kDa protein were purified to homogeneity by selective extraction from the membranes with detergent and by elution from multiple two-dimensional gels. The purified protein retained its PTH-dependent staining and specificity. This protein may be a PTH receptor or a fragment of a PTH receptor from kidney.

Animals↗

PubMed1985

Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum.

Phospholamban, the putative protein regulator of the Ca2+ pump of cardiac sarcoplasmic reticulum, was purified to apparent homogeneity from canine cardiac sarcoplasmic reticulum vesicles by selective extraction with sodium cholate, followed by adsorption to calcium oxalate, solubilization in Zwittergent 3-14, and specific elution from p-hydroxymercuribenzoate-agarose. Phospholamban, isolated in the dephosphorylated state, was purified 80-fold in 15% yield (approximately 2 mg of phospholamban/g of sarcoplasmic reticulum protein). Nondissociated phospholamban exhibited an apparent Mr = 25,000 in sodium dodecyl sulfate-polyacrylamide gels. Partially dissociated phospholamban, induced by boiling in sodium dodecyl sulfate, exhibited five distinct mobility forms in sodium dodecyl sulfate-polyacrylamide gels, of apparent molecular weights between 5,000-6,000 and 25,000. Phospholamban was phosphorylated to a level of 190 nmol of Pi/mg of protein by cAMP-dependent protein kinase, consistent by minimum stoichiometry with a subunit molecular weight of approximately 5,000. Phospholamban prepared by the present method was different in several respects from the proteins that have been isolated in other laboratories. Pure phospholamban was cysteine rich, containing 6 residues/100 amino acid residues. Dephosphorylated phospholamban was strongly basic with a pI = 10; phosphorylation decreased the pI to approximately 6.7. Pure phospholamban (and phospholamban present in sarcoplasmic reticulum vesicles) was not readily extracted into acidified chloroform/methanol, suggesting that the protein does not behave as an acidic proteolipid. The purified protein was highly antigenic. Phospholamban was localized by immunochemical methods to cardiac membranes enriched in sarcoplasmic reticulum, but was absent from sarcoplasmic reticulum membranes prepared from fast skeletal muscle. The method described for isolation of cardiac phospholamban is highly reproducible and relatively simple, and should be useful for further detailed studies designed to probe the molecular structure of the protein.

Adenosine Triphosphatases↗

PubMed1987

Characterization of water-insoluble proteins in human lens nuclei.

Water-insoluble proteins from the nuclei of human normal and cataractous lenses were analyzed by solubilizing the lens material in various organic solvent/water mixtures. Gel permeation chromatography with TSK-3000 and TSK-4000 columns and a high performance liquid chromatography system were utilized to fractionate the water-insoluble proteins. The effective solvent systems were a) acetonitrile:tetrahydrofuran: water = 20:60:20, v/v%; b) tetrahydrofuran:water = 50:50, v/v%; and c) methanol:water = 25:75, v/v%. Different protein fractions could be selectively extracted with different solvent mixtures. The fractions obtained were partially identified using antibodies made against the soluble alpha-, beta-, and gamma-crystallins. Protein profiles obtained from cataractous nuclei differed markedly from those obtained from normal nuclei of the same age. In particular, an approximately 350K protein aggregate was found to increase with age in cataractous nuclei. This 350K aggregate is only a minor component in the nuclei of normal lenses.

Aged↗

PubMed1986

Use of electrical methods in the study of hepatocytic autophagy.

Electropermeabilisation--i.e. subjecting cells to a short series of high-voltage pulses- has been used to make isolated rat hepatocytes transiently permeable to small molecules. The permeabilised cells can be loaded with [14C]sucrose at 0 degrees C and resealed by a brief incubation at 37 degrees C. The electrical treatment does not impair cellular function, since autophagic sequestration of sucrose occurs at the same rate as in untreated cells. The latter are permeable to sucrose immediately after isolation, due to the presence of hyperpermeable surface blebs similar to those formed in response to electrical treatment. In order to separate [14C]sucrose-sequestering organelles from cytosol, the cells are broken up by electrodisruption--i.e. a single high-voltage pulse given in a non-ionic medium (isotonic sucrose)--and the resulting cell corpses are sedimented through a dense metrizamide/sucrose cushion to form a cytosol-free pellet containing intact lysosomes. Since mitochondria also take up sucrose, the radioactivity in autophagosomes/lysosomes must be selectively extracted with a low concentration of digitonin. The resulting procedure provides a specific bioassay of autophagic sequestration. The sequestration is virtually completely inhibited by the autophagy inhibitor 3-methyladenine, and at temperatures below 20 degrees C. The sequestration step is energy-dependent, and can probably account for the known energy requirement of autophagic-lysosomal protein degradation.

Animals↗

PubMed1986

Development and application of a specific radioimmunoassay for trifluoperazine N4'-oxide to a kinetic study in humans.

A hapten derivative of prochlorperazine N4'-oxide, 7-(3-carboxy-propionyl)-prochlorperazine N4'-oxide, was synthesized and coupled to bovine serum albumin. Immunization of New Zealand White rabbits with this conjugate generated antibodies which were utilized to develop a radioimmunoassay (RIA) for the quantitation of trifluoperazine N4'-oxide (TFPNO). This assay enabled for the first time the quantitation of TFPNO in plasma, and 20 pg can be measured in a 200-microliter plasma sample with a coefficient of variation less than 5%. Statistically, indistinguishable results were obtained by the assay procedure with or without the selective extraction of TFPNO, and also in the presence or absence of an excess of trifluoperazine (TFP) and other major metabolites, such as trifluoperazine sulfoxide and 7-hydroxytrifluoperazine. This RIA and a previously reported RIA for TFP were used to directly determine plasma concentrations of TFP and TFPNO after administration of a single 5-mg p.o. dose of TFP to six healthy male volunteers. The mean +/- S.D. for the peak concentration, time to peak concentration, area under the curve from 0 to 72 hr and the elimination T1/2 for the terminal portion of the plasma concentration vs. time curve for TFPNO were found to be 3.7 +/- 0.7 ng/ml, 3.2 +/- 0.4 hr, 51.1 +/- 9.8 ng X hr/ml and 26.9 +/- 14.0 hr, respectively, whereas the corresponding values for TFP were found to be 2.7 +/- 0.7, 3.1 +/- 0.8, 42.2 +/- 6.9 and 21.9 +/- 6.5, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗