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PubMed1983

Radioenzymatic assay for direct measurement of plasma pyridoxal 5'-phosphate.

A radioenzymatic assay for measurement of pyridoxal 5'-phosphate (PLP) is described, based on the incubation of L-[3H]tyrosine (10(6) cpm, spec. acty. 1.88 Ci/mol) in the presence of the apoenzyme tyrosine decarboxylase (EC 4.1.1.25) from Streptococcus faecalis and PLP in phosphate buffer (0.1 mol/L, pH 5.5) at 37 degrees C for 60 min. The decarboxylated metabolite formed, [3H]tyramine, was selectively extracted into ethyl acetate, and the tritium radioactivity in the sample was determined by liquid scintillation counting. As little as 0.5 nmol of PLP can be detected per liter. The assay is specific, no cross reactivity having been noted for several compounds closely related to PLP. With this we could directly measure the concentrations of PLP in plasma without prior deproteinization and ether washing of the samples. Using the assay to determine plasma concentrations of PLP in healthy adult populations, we found results that were comparable with previously reported data.

Apoenzymes↗
PubMed1980

Identification and partial purification of human tumor nucleolar antigen 54/6.3.

The present study was designed to characterize the human tumor nucleolar antigens found first in the HeLa cells and subsequently in a broad range of human cancers. For visualization of the antigens, HeLa cell nucleolar or nuclear protein fractions were analyzed on 4% polyacrylamide isoelectric focusing gels. The gels were incubated with rabbit antisera to HeLa cell nucleoli and then with fluorescein- or peroxidase-conjugated goat anti-rabbit immunoglobulin G. With this technique, two major nucleolar antigens (focusing at pH 6.3 and pH 6.1) were identified. These antigens were also found in the Namalwa cell, but not in human liver cells. Purification of the antigen(s) was achieved by selective extraction of Namalwa cell nuclei with 10 mM Tris-HCl (pH 8), 40 to 100% ammonium sulfate precipitation, diethylaminoethyl cellulose chromatography in which the antigen was eluted with 0.15 M NaCl buffer (DE-0.15M fraction), and use of isoelectric focusing gels. The immunostained bands (HuAg 6.3 and HuAg 6.1) and the bright nucleolar immunofluorescence of the HeLa cells were not observed after the antisera were preabsorbed with the DE-0.15M fraction. The immunostained bands (HuAg 6.3 and HuAg 6.1) and the nucleolar immunofluorescence of the HeLa cells were also observed when isoelectric focusing gels were incubated with antiserum from rabbits immunized with the DE-0.15M fraction. On the sodium dodecyl sulfate second dimension of the two-dimensional polyacrylamide gel electrophoresis, the antigen(s) migrated as single spots with appraent molecular weights of 54,000.

Antigens, Neoplasm↗
PubMedDate not supplied

[Interpretation of the pictures of meridional diffraction on X-rays of membrane-free rabbit spinal muscles in a state of rigor and their dependence on physico-chemical parameters of rigorising solution].

Results of the studies of structural and mechanical properties of rabbit psoas muscle by means of X-ray diffraction and physiological technique are summarised along the following lines: localisation of minor proteins in the thick filaments and the origin of meridional reflections in the X-ray diffraction patterns of rigorised rabbit psoas muscle; molecular basis of the effects of pH, ionic strength, pyrophosphate, ethyleneglycol and pyrophosphate-ethyleneglycol mixture on the value of rigor tension and their dependence on the sarcomerès length and concentration of Mg2+; effect of selective extraction of minor proteins from M- and P-zones of the thick filaments on meridional reflections; structure of the thick filaments in the state of "Ca2+-free rigor"; attachment of cross-bridges to actin filaments in relaxing solution (is the model of "sterick brocking" correct?); role of structural transitions in S-2-segments of myosin in force generation.

Animals↗
PubMed1981

Methylation of membrane proteins in human erythrocytes. Identification and characterization of polypeptides methylated in lysed cells.

An in vitro system was developed for studying protein methylation reactions in human red blood cells. Packed erythrocytes were lysed by freeze-thawing in the presence of S-adenosyl[methyl-3H]methionine. Specific incorporation of base-labile methyl groups into the band 3 anion transport protein and the major sialoglycoprotein (glycophorin, periodic acid-Schiff reagent-1) was demonstrated by dodecyl sulfate gel electrophoresis at pH 2.4, selective extractions with Triton X-100 and lithium diiodosalicylate, and protease sensitivity. Two other unidentified intrinsic membrane proteins with Mr = 96,000 and 23,500 were also methylated. Little radioactivity was incorporated into membrane proteins when membranes were incubated with S-adenosyl-L-[methyl-3H]methionine in the absence of cytosol. No evidence was obtained for incorporation of methyl label into extrinsic proteins including bands 1, 2, 2.1, 4, 5, 6, or in zone 4.5. Proteolytic digestion of intact cells and isolated membranes revealed that one site of methylation on the band 3 polypeptide may be at the inner surface of the membrane near the junction of the cytoplasmic domain and the membrane domain. The rates of hydrolysis of the incorporated methyl groups were characterized over a range of pH values. These rates were compared to those of methyl esterified amino acids and peptides, including aspartic acid beta-methyl ester which has been isolated from proteolytic digests of methylated erythrocyte membranes (Janson, C. A., and Clarke, S. (1980) J. Biol. Chem. 225, 11640-11643). We find that the rates of base-catalyzed hydrolysis of beta-methyl esters of aspartic acid and gamma-methyl esters of glutamic acid are highly sensitive to the presence of substituents on the alpha-carboxyl and alpha-amino groups. The rate of hydrolysis of the membrane-incorporated methyl groups are consistent with those of internal aspartic acid and glutamic acid methyl ester residues.

Chymotrypsin↗
PubMed1981

Purification and initial characterization of intrinsic membrane-bound alkaline phosphatase from chicken epiphyseal cartilage.

Alkaline phosphatase has been purified from microsomes of chicken epiphyseal cartilage by first selectively extracting certain adventitious proteins with 0.25 M trichloroacetate. The membrane-bound enzyme was then solubilized by 1% cholate in buffered 33% saturated ammonium sulfate and purified by column chromatography on Bio-Gel A-5m, extraction with 1-butanol, and ion exchange chromatography on DEAE-Bio-Gel A. The purified alkaline phosphatase from the cartilage membrane had a subunit molecular weight of 53,000 and a holoenzyme weight of 207,000-220,000, indicating a tetramer. The pH optima for p-nitrophenylphosphate, ATP, and pyrophosphate hydrolysis were 10.3, 9.0, and 8.5, respectively. Values of Vmax (in micromoles/min/mg) were 220, 3.1, and 0.8, respectively. Substrate inhibition was pronounced at values of pH below 8.5. Inhibition of p-nitrophenylphosphate hydrolysis at pH 10.3 showed that phosphate and arsenate were competitive inhibitors (KI = 1.88 and 0.15 mM, respectively) and levamisole was an uncompetitive inhibitor (KI = 0.32 mM), while L-phenylalanine and ZnCl2 were mixed inhibitors (KI = 15.8 and 0.02 mM, respectively). Inhibition by preincubation in 1 mM EDTA was reversible by readdition of 0.25 mM MgCl2 nd 20 microM ZnCl2. The data indicate that this membrane-bound alkaline phosphatase from chicken epiphyseal cartilage is a Zn2+ and possibly Mg2+-containing enzyme. While the subunit molecular weight and kinetic properties of the enzyme are quite typical of vertebrate alkaline phosphatases, the tightness of binding to the membrane lipids, the extreme sensitivity to substrate inhibition, and the tetrameric conformation of the holoenzyme are unusual.

Alkaline Phosphatase↗
PubMed1978

Dextromethorphan: radioimmunoassay and pharmacokinetics in the dog.

A specific radioimmunoassay (RIA) for the determination of the widely used non-narcotic antitussive agent, dextromethorphan (DM) in plasma and urine has been developed using an antiserum to DM which was obtained from rabbits following immunization with an albumin conjugate of (+)-3-methoxymorphinan-17-succinyloxyethyl. Specificity of the RIA was achieved by selective extraction of DM from its known metabolites into hexane at pH 6.5 with 86% recovery. Employing tritium labelled-DM as the radioligand the RIA had a limit of sensitivity of about 4 ng/ml of plasma using a 0.5 ml sample. Following I.V. administration of 2 mg/kg of DM to two dogs, the plasma levels of intact DM declined biexponentially; the terminal exponential phase having a half-life of 2.5-3.9 hr. The volume of distribution of the central compartment ranged from 5-6.4 l/kg while the metabolic clearance rate ranged from 2.5-2.6 1/hr/kg. Oral administration of 10 mg/kg of DM in gelatin capsule resulted in peak plasma levels of 50 and 92 ng/ml at 2 hr post-drug. Following both routes of administration, less than 3.5% of the dose was excreted in the urine as intact DM indicating almost complete biotransformation and/or alternate routes of excretion. In one human subject who received 0.5 mg/kg of DM P.O. in a syrup, the plasma levels were less than 1 ng/ml.

Animals↗
PubMedDate not supplied

Metabolism and disposition studies of 9-hydroxyellipticine and 2-methyl-9-hydroxyellipticinium acetate in animals.

Radiolabeled 9-hydroxyellipticine (iv and ip routes) and the corresponding radioactive quaternary salt, 2-methyl-9-hydroxyellipticinium acetate (iv route), were administered to mice. Tissue distribution was then followed up to 64 hours by means of autoradiography. Both drugs accumulated in the kidneys, lungs, and liver; 9-hydroxyellipticine also accumulated in the spleen and bone marrow. The quaternary salt was concentrated in the gastrointestinal walls and the salivary and thyroid glands; none was found in the brain. Metabolic studies after administration of these antitumor drugs to rats and mice showed that 9-hydroxyellipticine is extensively metabolized, mainly to its glucuronide. Under our experimental conditions, the ellipticinium derivative was excreted unchanged in the bile (70%) and in the urine (30%). Pharmacokinetic studies in mice using either radioactivity measurements or selective extraction followed by spectrofluorometric quantitation have shown that blood levels drop very rapidly during the distribution phase followed by a much slower disposition phase, with a half-life of about 30 hours for the quaternary salt.

Alkaloids↗
PubMed1981

Immunoregulatory plasma low density lipoprotein: the biologic activity and receptor-binding specificity is independent of neutral lipids.

A normal human plasma low density lipoprotein subfraction (LDL-In), recovered within the intermediate density lipoprotein subclass of density range 1.006 to 1.019 g/ml, has previously been demonstrated to bind to a specific cell surface receptor independent of the LDL receptor and to regulate inductive events in the triggering of lymphocytes. The constituents of LDL-In that are responsible for the biologic activity and receptor-binding specificity were examined with respect to the role of sterols and other neutral lipids. Partial delipidization of LDL-In with heptane in the presence of starch permitted selective extraction of greater than 98% of the neutral lipids from the native lipoprotein particle. These partially delipidated polar lipid:protein particles were compared with native LDL-In with respect to charge, apoprotein content, suppressive activity for lymphocytes, and specific binding to the lymphocyte LDL-In receptor. The cholesterol- and glyceride-depleted LDL-In retained all characteristics of the native lipoprotein. These observations indicate that the interaction of LDL-In particles with the lymphocyte and the suppression of lymphocyte function are independent of the neutral lipid content of this bioregulatory lipoprotein, and that mechanisms other than cholesterol loading of the cell or sterol exchange with the plasma membrane must be operative.

Binding Sites↗
PubMed1995

Optimization of antisense oligodeoxynucleotide structure for targeting bcr-abl mRNA.

Antisense oligodeoxynucleotides targeted to bcr-abl are potential ex vivo purging agents for use with autologous bone marrow transplantation in the treatment of chronic myeloid leukemia (CML). We investigated, in a cell-free system, the activity and nuclease resistance of phosphodiester, phosphorothioate, chimeric methylphosphonate/phosphodiester, and chimeric methylphosphonate/phosphorothioate antisense octadecamers directed against either b2a2 or b3a2 bcr-abl breakpoint RNAs. Certain chimeric compounds were shown to possess targeted activity broadly equal to the parent phosphodiester or phosphorothioate forms and greater resistance to the nucleases present in cell extracts. Selected chimeric structures were compared with phosphodiester and phosphorothioate analogues for antisense activity in human CML cells containing either b2a2 or b3a2 bcr-abl breakpoint mRNAs. We present results showing that all four structures can suppress bcr-abl mRNA level in vivo. The rank of in vivo activity is chimeric methylphosphonate/phosphodiester > or = phosphodiester > phosphorothioate > methylphosphonate/phosphorothioate. We show that b2a2 breakpoint RNAs can be more effectively targeted than b3a2 sequence RNAs both in vitro and in vivo and suggest that RNA secondary structure may be a possible explanation for this phenomenon.

Base Sequence↗
PubMed1993

Sustained effect of angiotensin II on tyrosine phosphorylation of annexin I in glomerular mesangial cells.

By means of selective extraction in a Ca(2+)-chelating medium and immunoblotting, four annexins (I, II, V, and VI) were identified in both isolated rat renal glomeruli and rat glomerular mesangial cells. Upon 32P labeling of these cells in culture, annexin I was immunoprecipitated using a specific polyclonal antibody and was found to incorporate radioactivity in a constitutive manner. However, as with epidermal growth factor (200 ng/ml), addition of angiotensin II (10(-7) M), arginine-vasopressin (10(-7) M), or endothelin I (10(-7) M) resulted in a 2-3-fold stimulation of annexin I phosphorylation. The basal phosphorylation as well as the stimulating effect of angiotensin II were also detected by immunoblotting annexin extracts using an antiphosphotyrosine antibody. In addition, among various phosphotyrosyl proteins isolated from EGTA extracts by adsorption onto an anti-phosphotyrosine antibody, annexin I was specifically recognized by Western blotting using a monoclonal anti-annexin I antibody, and displayed the same increase upon cell stimulation with angiotensin II. Moreover, thin layer chromatographic analysis of phosphoamino acids present in immunoprecipitated [32P]annexin I showed an exclusive labeling of phosphotyrosine residue(s). Finally, the effect of angiotensin II was detectable after 10 min, maximal at 6 h, and present until 12 h of incubation. Using 12-h stimulation, tyrosine phosphorylation of annexin I displayed a maximum at 10(-7) to 10(-6) M angiotensin II. These data report for the first time the stimulation of annexin I tyrosine phosphorylation by biologically active peptides acting via receptors belonging to the superfamily of seven hydrophobic domain, G-protein-linked receptors, which lack an intrinsic protein tyrosine kinase. This suggests a possible role of annexin I in the mitogenic effect of angiotensin II, arginine-vasopressin, and endothelin I, which was previously observed on rat glomerular mesangial cells as well as on other cells.

Angiotensin II↗
PubMed1994

Native cartilage matrix protein (CMP). A compact trimer of subunits assembled via a coiled-coil alpha-helix.

Cartilage matrix protein (CMP), a major component of many types of cartilage, is a noncollagenous glycoprotein with a molecular mass of 148 kDa consisting of three identical subunits. With the aim of performing a more comprehensive characterization, we purified CMP in a native conformation from fetal bovine rib cartilage avoiding the denaturing solvents previously used. CMP could be selectively extracted with EDTA-containing buffer which indicates a divalent cation-dependent anchorage in the cartilage matrix. Determination of the amino-terminal sequence of the bovine protein confirmed its identity when compared with published cDNA sequences of chicken and human CMP. Electron microscopy revealed the presence of three ellipsoid subunits which are connected at one end. Sequence analysis indicated the presence of a coiled-coil alpha-helical assembly domain formed by the COOH-terminal end of the subunits. The trimeric structure was retained after complete reduction under native conditions which shows that the coiled-coil domain is stable also in the absence of interchain disulfide bonds.

Amino Acid Sequence↗
PubMed1994

An information model for medical events.

Information gathered during the healthcare process is lost when forced into rigidly structured record-oriented databases. By contrast, content can be difficult to manipulate if stored as unstructured text. Spurred by the requirements of electronic publishing, military procurement and the Internet, new robust standards for structuring documents have been developed and deployed. These standards can provide a foundation for a document-based Electronic Medical Record System. In order to fully exploit this added flexibility, an information model is necessary to define both the direct and contextual content of documents. Once context, as well as fact, are recorded in formal structures, inferential techniques can either selectively extract knowledge and data from documents or aggregate data to create summaries so that all interested and authorized parties have a better chance of meeting their information needs from a single, permanent data source.

Humans↗
PubMed1994

[Investigation of nuclear lamina in Tetrahymena thermophila].

The nuclear lamina in the nuclei of Tetrahymena thermophila was investigated using the techniques of selective extraction and embedment-free electron microscopy, as well as immunofluorescence staining. By means of immunoblotting and protein isolating, the lamina was found to consist mainly of a 66 KD polypeptide. While rabbit-anti-lamins polyclonal antibodies crossreacted with this protein, neither murine-anti-lamin A&C mAB C23 nor murine-anti-lamin III mAB 223 showed positive reaction with it. It was also demonstrated that the lamin is not so rich as that in mammalian cells. The existence of lamina in T. thermophila, a unicellular organism, seems to show that the lamina is a basic characteristic structure of eukaryotic cells, and that the function of the lamina may not be limited to the process of breakdown and reconstitution of nuclear envelope during mitosis.

Animals↗
PubMed1993

[Nuclear matrix-intermediate filament scaffold of cybrid cells crossed between rabbit reticulocytes and K562 cells].

To elucidate the role of the mammalian erythroid cytoplasmic factor on erythroid cell denucleation, cybridization of neo gene transferred rabbit reticulocytes with human K562 erythroleukemia cells was performed, the structure and composition of the nuclear matrix-intermediate filaments (NM-IF) system of the reticulocytes, K562 cells and the cybrids between them, the K-RRneo Cells, were studied by employing the techniques of selective extraction and whole mount electron microscopy. It was shown that regional condensation of nuclear matrix with a thinned nuclear lamina were seen in the cybrids, while the intermediate filaments exhibited a network pattern reorganization comparatively similar with those of reticulocytes but different from the radiation pattern of the K562 cells. SDS-PAGE and western blot analysis also revealed that the proteins affinity to vimentin antibody in the cybrids migrate in a pattern similar with that of the rabbit reticulocytes, both of them with only residual depolymerized vimentin complex but not the polymerized vimentin band of 55 kd. These results suggested that the factors in the cytoplasm of the rabbit reticulocytes might function through a mechanism of vimentin depolymerization or blockage of gene expression, which finally resulted in destruction of intermediate filaments that facilitates the expellation of nucleus from cytoplasm.

Animals↗
PubMed1975

Modification of Escherichia coli membranes in the prereplicative phase of phage T4 infection. Specificity of association and quantitation of bound phage proteins.

Reinfection of Escherichia coli with the bacterial virus T4 causes modifications of the properties of the host cell envelope during the preeplicative phase of the lytic cycle. These changes include altered densities cell enveloped and their subfractions, morphological modifications of membrane vesicles, and association of newly synthesized proteins with the host cell envelope. Polypeptide analysis by high resolution electrophoresis on polyacrylamide slab gels in dodecyl sulfate revealed that most of some 30 prereplicative phage-coded polypeptides are attached to this structure. Different means of cell disruption and selective extraction procedures, such as variations of ionic strength, removal of divalent cations, and the addition of chaotropic agents or detergents were used to study the characteristics of these attachments. Many proteins appeared to be artifactually absorbed or weakly bound to the envelope, Separation of cell walls from plasma membranes showed that all of the tightly bound proteins were associated withthe cell membrane fraction. The partitioning of phage proteins between the different fractions was monitored using 12 polypeptides which were identified as products of distinct phage genes. Of these, 8 were eliminated as potential membrane markers. Four polypeptides, the products of genes rIIA, rIIB, 39, and 52 were operationally defined as membrane proteins. The number of molecules of the 12 identified phage gene products, synthesized during a single lytic cycle, was determined. The results allowed the estimation of the concentration in the membrane of those proteins which were found to be quantitatively associated with that structure. Association of phage proteins with the cell envelope was found to be unaffected by mutations in any of the identified phage polypeptides.

Amino Acids↗
PubMed1993

Development of a sensitive and specific radioimmunoassay for benztropine.

A benztropine RIA based on polyclonal antisera raised in New Zealand white rabbits has been developed. The drug-protein conjugates employed had a variety of moles of benztropine hemisuccinate coupled per mole of protein (bovine serum albumin or bovine thyroglobulin). Six antisera were developed and the one with the highest titer was further evaluated for its cross reactivity to N-desmethylbenztropine (4%) and the antipsychotic agents fluphenazine, flupenthixol, chlorpromazine, and haloperidol (all < 1%). The selected antiserum demonstrated sufficient sensitivity to measure benztropine from 0.156 to 100 ng/mL plasma in a 200-microL plasma sample, with a mean CV of < 6%. The RIA was applied to the analysis of steady-state plasma samples obtained from patients undergoing treatment with benztropine and plasma samples obtained from human volunteers and dogs orally dosed with the drug. Both the human and dog plasma samples, when analyzed after hydrolysis with beta-glucuronidase/sulfatase, demonstrated increments in benztropine concentrations, suggesting the drug may be undergoing biotransformation to phase II metabolite(s). In addition, when benztropine was selectively extracted from the unhydrolyzed plasma samples, there was a significant decrease in drug level, which further suggested that the antiserum cross reacted with phase II metabolite(s). The shape of the plasma concentration versus time profile obtained from the dog studies suggested that the drug might also undergo enterohepatic recycling.

Animals↗
PubMed1993

Microtubule network and microtubule-associated proteins in leukemic T lymphocytes.

Cytoskeletal changes have been known to occur in cell transformation. Vinca alkaloids which bind to the tubulin dimer and inhibit microtubule (MT) assembly as well as disrupting the MT network and mitotic spindle, have been used as cancer chemotherapeutic agents. It has been proposed that apart from their anti-mitotic activity, these drugs act on the peripheral MT of malignant cells to produce their cytolytic effects. In this paper we demonstrate the presence of an altered cytoplasmic MT network in MOLT-4 and HuT-78 leukemic cells (human T-cell leukemic lines) compared to normal human peripheral blood lymphocytes stimulated with mitogens. In addition, using a selective extraction protocol we have compared microtubule-associated proteins (MAPs) profiles of G1/S synchronized leukemic human T-cells and 20 h mitogen-stimulated human peripheral blood T-cells. We observed a dramatic decrease in the expression of a MAP of apparent molecular weight 52 kDa and pI 5.2 in the leukemic cells synchronized at the G1/S border of the cell cycle. These results suggest that altered MT network morphology and MAP synthesis may be components of the malignant phenotype in the T-lymphocytic leukemias studied here.

Cell Cycle↗
PubMed1977

Interference by p-hydroxyphenobarbital in the 125I-radioimmunoassay of serum and urinary phenobarbital.

A radioimmunoassay for barbiturates is shown to be equally sensitive to phenobarbital and its major urinary metabolite, p-hydroxyphenobarbital, in serum and urine. Interference by the metabolite can be essentially eliminated by selectively extracting phenobarbital into chloroform. The extraction efficiency of the method for phenobarbital was 93+/-2% (SD) over the concentration range studied. Although cross reactivity between barbiturates and their metabolites may be less important in determining cases of barbiturates abuse or overdose, it may be extremely important if data on serum or urine are required for accurate estimates of drug disposition, or in establishing dose/response relationships.

Cross Reactions↗