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PubMed1995

Vascular effects of selected antihypertensive drugs derived from traditional medicinal herbs.

1. The pharmacological actions of the active ingredients extracted or purified from two selected traditional Chinese medicinal plants on vascular smooth muscles are briefly reviewed. The active ingredients of these herbal drugs include tetrandrine (TET) and total ginseng saponins (TGS). These natural products have been clinically used in China for the treatment of cardiovascular diseases due to their vasodilatory and antihypertensive actions. 2. Studies from this laboratory have confirmed previously reported characteristics of these drugs as Ca2+ antagonists in vascular tissues. On the other hand, they also elicited inhibitory effects in response to a wide variety of receptor stimulations as indicated by contractility studies using isolated vascular tissues and radioligand binding studies using isolated subcellular membranes. 3. TET has been demonstrated as an effective but not very selective Ca2+ antagonist. Other than the vasodilatory action on arteries and veins, TET also shows a vasoconstrictive effect in veins. 4. TGS from panax notoginseng may be acting as a novel and selective Ca2+ antagonist that does not interact with the L-type Ca2+ channel (e.g. in KCl-induced contraction) but may interact with the putative receptor operated Ca2+ channel (e.g. in phenylephrine-induced contraction). TGS from panax quinquefolium, on the other hand, enhanced the vasoconstrictor effect produced by phenylephrine, but not KCl.

Alkaloids↗

PubMed1999

ATPase activity of purified and reconstituted multidrug resistance protein MRP1 from drug-selected H69AR cells.

The ATP-binding cassette transporter protein, multidrug resistance protein MRP1, was purified from doxorubicin-selected H69AR lung tumor cells which express high levels of this protein. A purification procedure comprised of a differential two-step solubilization of MRP1 from plasma membranes with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate followed by immunoaffinity chromatography using the MRP1-specific monoclonal antibody QCRL-1 was developed. Approximately 300 microgram of MRP1 was obtained from 6 mg of plasma membranes at 80-90% purity, as indicated by silver staining of protein gels. After reconstitution of purified MRP1 into proteoliposomes, kinetic analyses indicated that its K(m) for ATP hydrolysis was 104+/-22 microM with maximal activity of 5-10 nmol min(-1) mg(-1) MRP1. MRP1 ATPase activity was further characterized with various inhibitors and exhibited an inhibition profile that distinguishes it from P-glycoprotein and other ATPases. The ATPase activity of reconstituted MRP1 was stimulated by the conjugated organic anion substrates leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) with 50% maximal stimulation achieved at concentrations of 150 nM and 1.6 microM, respectively. MRP1 ATPase was also stimulated by glutathione disulfide but not by reduced glutathione or unconjugated chemotherapeutic agents. This purification and reconstitution procedure is the first to be described in which the ATPase activity of the reconstituted MRP1 retains kinetic characteristics with respect to ATP-dependence and substrate stimulation that are very similar to those deduced from transport studies using MRP1-enriched plasma membrane vesicles.

ATP-Binding Cassette Transporters↗

PubMed2001

Highly selective water channel activity measured by voltage clamp: analysis of planar lipid bilayers reconstituted with purified AqpZ.

Aquaporins are membrane channels selectively permeated by water or water plus glycerol. Conflicting reports have described ion conductance associated with some water channels, raising the question of whether ion conductance is a general property of the aquaporin family. To clarify this question, a defined system was developed to simultaneously measure water permeability and ion conductance. The Escherichia coli water channel aquaporin-Z (AqpZ) was studied, because it is a highly stable tetramer. Planar lipid bilayers were formed from unilamellar vesicles containing purified AqpZ. The hydraulic conductivity of bilayers made from the total extract of E. coli lipids increased 3-fold if reconstituted with AqpZ, but electric conductance was unchanged. No channel activity was detected under voltage-clamp conditions, indicating that less than one in 10(9) transport events is electrogenic. Microelectrode measurements were simultaneously undertaken adjacent to the membrane. Changes in sodium concentration profiles accompanying transmembrane water flow permitted calculation of the activation energies: 14 kcal/mol for protein-free lipid bilayers and 4 kcal/mol for lipid bilayers containing AqpZ. Neither the water permeability nor the electric conductivity exhibited voltage dependence. This sensitive system demonstrated that AqpZ is permeated by water but not charged ions and should permit direct analyses of putative electrogenic properties of other aquaporins.

Aquaporins↗

PubMed1982

Expression of adenovirus-2 early region 4: assignment of the early region 4 polypeptides to their respective mRNAs, using in vitro translation.

Adenovirus-2 early region 4 (E4; map positions 91.3 to 99.1) encodes six 5' and 3' coterminal, differently spliced mRNAs, which are 2.5, 2.1, 1.8, 1.5, 1.2, and 0.8 kilobases (kb) long. Hybridization selection with five cloned viral DNA fragments that hybridize with subsets of E4 mRNAs was used to purify these six mRNAs and a previously unreported 3.0-kb mRNA from virus-infected cells. E4 mRNAs which were purified by hybridization selection with cloned EcoRI fragment C (map positions 89.7 to 100) were also fractionated by size. The purified mRNAs were then translated in rabbit reticulocyte or wheat germ lysate systems. The full complement of E4 mRNAs specified as many as 16 different polypeptides, with molecular weights ranging from 24,000 (24K) to 10K. The most abundant E4 mRNA, which was 2.1 kb long, specified an 11K polypeptide. The 1.5-kb mRNA, which differed from the 2.1-kb mRNA only by deletion of a second intron from the 3' untranslated region, also specified an 11K polypeptide. The second most abundant mRNA, which was 1.8 kb long, and the 1.2-kb mRNA, which had an intron deleted from the 3' untranslated region, specified a 15K polypeptide. This polypeptide was labeled more intensely with [5,6-(3)H]leucine than with [35S]methionine. The 3.0- and 2.5-kb mRNAs specified four polypeptides (24K, 22K, 19K, and 17K). Translation of E4 mRNAs with a mean size of 0.8 kb, which accumulated preferentially in the presence of cycloheximide, yielded at least 10 polypeptides that migrated in polyacrylamide gels with apparent molecular weights ranging from 21,800 to 10,000. On the basis of translation in wheat germ lysates and the distribution of polypeptides encoded by size-fractionated mRNAs, we concluded that the 0.8-kb mRNA size class includes a heterogeneous mixture of mRNAs which are probably formed as the result of utilization of alternate splice acceptor and donor sites during removal of the second intron. Our polypeptide assignments for the 2.1-, 1.8-, 1.5-, and 1.2-kb mRNAs are compatible with locations of two open coding regions in the DNA sequence (Herisse et al., Nucleic Acids Res. 9:4023-4042, 1981). The relationship between the four polypeptides encoded by the 3.0- and 2.5-kb mRNAs and the two open coding regions is discussed. The production of multiple polypeptides from a heterogenous mixture of mRNAs in the 0.8-kb size class is compatible with two large open coding regions in that part of the sequence. Thus, nearly all of the potential coding information in the leftward strand of E4 is expressed in translatable form during infection. Moreover, alternate splicing of the 0.8-kb mRNA size class can produce multiple polypeptides with common amino-terminal and different carboxy-terminal amino acid sequences, which may have the same function but different specificities.

Adenoviruses, Human↗

PubMed2005

Fractionation of bovine serum albumin and monoclonal antibody alemtuzumab using carrier phase ultrafiltration.

Protein transmission and hence selectivity of separation can be significantly affected by solution pH and ionic strength in protein fractionation using ultrafiltration. Using parameter scanning ultrafiltration, the transmission of bovine serum albumin (BSA) and monoclonal antibody alemtuzumab (Campath-1H) through 300 kDa polyethersulfone (PES) ultrafiltration membranes were studied over a range of pH and salt concentrations, with focus on the likely conditions for achieving "reverse selectivity," i.e., obtaining purified alemtuzumab (approximately 155 kDa) in the permeate. Experimental results demonstrate that the reverse selectivity could be obtained by manipulating the operating conditions such as the solution pH, ionic strength, permeate flux, and system hydrodynamics. With a two-stage batch ultrafiltration process under suitable conditions, the monoclonal antibody alemtuzumab with a purity of > 98% was obtained in the permeate from a feed solution initially containing 0.50 g/l each of BSA and alemtuzumab. Further purity can be expected by selecting more suitable membranes and optimizing operating conditions.

Alemtuzumab↗

PubMed1998

Autologous CD34-selected blood progenitor cell transplants for patients with advanced multiple myeloma.

Fifty-five patients with advanced multiple myeloma received purified CD34-selected peripheral blood progenitor cell transplants following myeloablative chemotherapy. A median of 4.1 x 10(6) CD34 cells/kg (range 1.2-30.7) were infused after busulfan (14 mg/kg) and cyclophosphamide (120 mg/kg); granulocyte-macrophage colony-stimulating factor was used until hematopoietic recovery. Median time to neutrophils >0.5 x 10(9)/l and platelets >20 x 10(9)/l were 12 days (range 10-16 and 8-184 days, respectively). Median follow-up of survivors from the time of transplantation is 33 months (range 7 to 44 months). Thirty-one patients are alive, 19 progression-free. Median progression-free survival is 14 months. Actuarial 3-year progression-free and overall survival are 29+/-14% and 47+/-17%. CD34-selection of peripheral blood progenitor cells provides effective hematopoietic support with significant progression-free and overall survival.

Adult↗

PubMed1997

Serine phospholipid-specific phospholipase A that is secreted from activated platelets. A new member of the lipase family.

Rat platelets secrete two types of phospholipases upon stimulation; one is type II phospholipase A2 and the other is serine-phospholipid-selective phospholipase A. In the current study we purified serine-phospholipid-selective phospholipase A and cloned its cDNA. The final preparation, purified from extracellular medium of activated rat platelets, gave a 55-kDa protein band on SDS-polyacrylamide gel electrophoresis. [3H]Diisopropyl fluorophosphate, an inhibitor of the enzyme, labeled the 55-kDa protein, suggesting that this polypeptide possesses active serine residues. The cDNA for the enzyme was cloned from a rat megakaryocyte cDNA library. The predicted 456-amino acid sequence contains a putative short N-terminal signal sequence and a GXSXG sequence, which is a motif of an active serine residue of serine esterase. Amino acid sequence homology analysis revealed that the enzyme shares about 30% homology with mammalian lipases (lipoprotein lipase, hepatic lipase, and pancreatic lipase). Regions surrounding the putative active serine, histidine, and aspartic acid, which may form a "lipase triad," were highly conserved among these enzymes. The recombinant protein, which we expressed in Sf9 insect cells using the baculovirus system, hydrolyzed a fatty acyl residue at the sn-1 position of lysophosphatidylserine and phosphatidylserine, but did not appreciably hydrolyze phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, and triglyceride. The present enzyme, named phosphatidylserine-phospholipase A1, is the first phospholipase that exclusively hydrolyses the sn-1 position and has a strict head group specificity for the substrate.

Amino Acid Sequence↗

PubMed2005

Selective antiproliferative and apoptotic effects of flavonoids purified from Rhus verniciflua Stokes on normal versus transformed hepatic cell lines.

Considerable attention is being concentrated on dietary flavonoids in developing novel cancer-preventive approaches due to their potential ability to induce selective apoptosis of cancer cells. In this study, we prepared a flavonoid-containing fraction from a crude acetone extract of Rhus verniciflua Stokes (RVS), traditionally used as a food additive and as an herbal medicine, and named RVS chloroform-methanol fraction (RCMF). We evaluated the effects of RCMF on proliferation and apoptosis using mouse embryonic primary hepatic cells (MPHC), embryonic normal hepatic cell line (BNL CL.2), and its SV40-mediated transformed cell line (BNL SV A.8). We also investigated the effects of RCMF on the antioxidant defense system in those cells. This study demonstrated that RCMF exhibited a selective growth inhibition and apoptosis induction on transformed cells. BNL SV A.8 cells were more sensitive to RCMF-mediated cytotoxicity than were MPHC or BNL CL.2. RCMF-mediated reduction of MnSOD activity and glutathione (GSH) content in BNL SV A.8 cells is thought to be associated with RCMF-induced apoptosis. Our findings suggest that RCMF is an agent which may be capable of inducing growth inhibition and apoptosis of hepatic tumor cells.

Animals↗

PubMed1993

Reconstitution of a purified mu-opioid binding protein in liposomes: selective, high affinity, GTP gamma S-sensitive mu-opioid agonist binding is restored.

An opioid binding protein (OBP) purified to homogeneity from bovine striatal membranes has been reconstituted in liposomes. The liposomes were produced by PEG-precipitation of OBP in the presence of a CHAPS extract of bovine striatum, devoid of opioid binding. High affinity mu-agonist binding was restored. The binding was selective for mu-agonists, stereospecific and inhibited by GTP gamma S. These results demonstrate that there is recoupling of OBP with G-protein and confirm our earlier evidence that the purified OBP is a mu-opioid binding site.

Animals↗

PubMed1990

Selective purification of sex-influenced esterase from rat serum by immunoaffinity chromatographies.

Sex-influenced esterase (ES-SI) is a female-specific serum esterase of rats. There are two alleles at the Es-Si locus, Es-Sia coding for ES-SI and Es-Sib, a silent allele. ES-SI was purified to electrophoretic homogeneity from the serum of adult female SI3 rats by a four-step purification. First, albumin was removed from the serum by Blue-Sepharose CL-6B affinity chromatography. Sera of rats carrying Es-Sia and Es-Sib were utilized to make two novel immunoaffinity columns. Rat anti-(ES-SI) alloantibody was obtained after the immunization of rats carrying Es-Sib with rat ES-SI-positive serum. ES-SI bound to the gel in this antibody-conjugated affinity chromatography. Simultaneously, rabbit anti-(rat serum protein lacking ES-SI) antibody was raised against ES-SI-negative rat serum and used to exclude other serum proteins. ES-SI was recovered in the flow-through fractions in this antibody-conjugated immunoaffinity chromatography, whereas the other serum proteins bound to the gel. ES-SI was selectively purified about 240-fold by combining these immunoaffinity chromatographies. This combination is a very powerful technique for purification of the protein. Finally, ES-SI was purified by preparative polyacrylamide gel electrophoresis after which it appeared as a single protein band (using silver staining) on SDS/polyacrylamide gel electrophoresis with a molecular mass of 72 kDa. In the ordinary estimation of enzyme purification, the purification of ES-SI was 30-fold at most, because the specific activity of the enzyme was calculated from the bulky esterase activity. However, ES-SI was selectively purified 2280-fold comparing its recovery to that of total protein. ES-SI exhibited a pI of 4.6 and optimum pH of 6.7. The activity of ES-SI was completely inhibited by diisopropyl fluorophosphate at 10 nM but not at all by eserine.

Animals↗

PubMed2001

An anti-leukemic single chain Fv antibody selected from a synthetic human phage antibody library.

The display of human antibody repertoire on the cell surface of the filamentous bacteriophage has offered a novel strategy for selecting antibodies to a diverse range of purified targets. However, the selection of antibodies with biological functions has not yet been fully investigated. To select phage antibodies with therapeutic potential, a synthetic human single chain Fv (scFv) phage antibody library was panned on whole premyelocytic leukemia cell line (HL60). Phages binding to common receptors and undesirable phages were subtracted by incubating the library with human glioma cells. High affinity binding phages to HL60 cells were enriched by fluorescence-activated cell sorting. After the 6th round of selection, 50% of the selected phage antibodies showed significant binding to HL60 cells, whereas none of the analyzed phage antibodies bound to human pre-B cells (Nalm-6). In addition to binding, one scFv antibody inhibited HL60 cell proliferation by 90% compared to irrelevant scFv antibodies. Taken together the data demonstrate that specific scFv antibodies with biological functions can be isolated by using whole cells as affinity matrix.

Antibody Specificity↗

PubMed2002

Coronary no-reflow is caused by shedding of active tissue factor from dissected atherosclerotic plaque.

Defined angiographically, no-reflow (NR) manifests as an acute reduction in coronary flow in the absence of epicardial vessel obstruction. One candidate protein to cause coronary NR is tissue factor (TF), which is abundant in atherosclerotic plaque and a cofactor for activated plasma coagulation factor VII. Scrapings from atherosclerotic carotid arteries contained TF activity (corresponding to 33.03 +/- 13.00 pg/cm(2) luminal plaque surface). Active TF was sedimented, indicating that TF was associated with membranes. Coronary blood was drawn from 6 patients undergoing coronary interventions with the distal protection device PercuSurge GuardWire (Traatek, Miami, FL). Fine particulate material that was recovered from coronary blood showed TF activity (corresponding to 91.1 +/- 62.16 pg/mL authentic TF). To examine the role of TF in acute coronary NR, blood was drawn via a catheter from coronary vessels in 13 patients during NR and after restoration of flow. Mean TF antigen levels were elevated during NR (194.3 +/- 142.8 pg/mL) as compared with levels after flow restoration (73.27 +/- 31.90 pg/mL; P =.02). To dissect the effects of particulate material and purified TF on flow, selective intracoronary injection of atherosclerotic material or purified relipidated TF was performed in a porcine model. TF induced NR in the model, thus strengthening the concept that TF is causal, not just a bystander to atherosclerotic plaque material. The data suggest that active TF is released from dissected coronary atherosclerotic plaque and is one of the factors causing the NR phenomenon. Thus, blood-borne TF in the coronary circulation is a major determinant of flow.

Angioplasty↗

PubMed2004

Effect of selection on the topology of genealogical trees.

Selection is one of the factors that most influence the shape of genealogical trees. Here we report results of simulations of the infinite-sites version of Moran's model of population genetics aiming at quantifying how the presence of selection affects the branching pattern (topology) of binary genealogical trees. In particular, we consider a scenario of purifying or negative selection in which all mutations are deleterious and each new mutation reduces the fitness of the individual by the same fraction. Analysis of five statistical measures of tree balance or symmetry borrowed from taxonomy indicates that the genealogical trees of samples of populations in which selection is actuating are in the average more asymmetric than neutral trees and that this effect is enhanced by increasing the sample size. However, a quantitative evaluation of the power of these balance measures to detect a tree topology significantly distinct from the neutral one indicates that they are not useful as tests of neutrality of mutations.

Animals↗

PubMed1996

Selection and characterization of a mutant of feline immunodeficiency virus resistant to 2',3'-dideoxycytidine.

We have selected and plaque purified a mutant of feline immunodeficiency virus (FIV) that is resistant to 2',3'-dideoxycytidine (ddC). This mutant was selected in cultured cells in the continuous presence of 25 microM ddC. The mutant, designated DCR-5c, was fourfold resistant to ddC, threefold resistant to 2',3'-dideoxyinosine, and more than fourfold resistant to phosphonoformic acid. DCR-5c displayed little or no resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine, 3'-azido-3'-deoxythymidine, or 9-(2-phosphonylmethoxyethyl) adenine. Reverse transcriptase purified from DCR-5c was less susceptible to inhibition by ddCTP, phosphonoformic acid, ddATP, or azido-dTTP than the wild-type FIV reverse transcriptase. Sequence analysis of DCR-5c revealed a single base change (G to C at nucleotide 2342) in the reverse transcriptase-encoding region of FIV. This mutation results in substitution of His for Asp at codon 3 of FIV reverse transcriptase. The role of this mutation in ddC resistance was confirmed by site-directed mutagenesis.

Amino Acid Sequence↗

PubMed2006

The Bead blot: a method for identifying ligand-protein and protein-protein interactions using combinatorial libraries of peptide ligands.

Small molecules that bind proteins can be used as ligands for protein purification and for investigating protein-protein and protein-drug interactions. Unfortunately, many methods used to identify new ligands to desired proteins suffer from common shortcomings, including the requirement that the target protein be purified and/or the requirement that the ligands be selected under conditions different from those under which it will be used. We have developed a new method called the Bead blot that can (i) select ligands to unpurified proteins, including trace proteins, present in complex materials (e.g., unfractionated plasma); (ii) select ligands to multiple proteins under a variety of conditions in a single experiment; and (iii) be used with libraries of different types of ligands. In the Bead blot, a library of ligands, synthesized on chromatography resin beads, is incubated with a starting material containing a target protein for which a ligand is sought. The proteins in the material bind to their complementary ligands according to specific affinity interactions. Then the protein-loaded beads are immobilized in a porous matrix, and the proteins are directionally eluted from the beads and captured on a membrane superimposed on the beads. The location of the target protein on the membrane is determined, and because the position of the protein(s) on the membrane reflects the position of the bead(s) in the matrix, the bead that originally bound the protein is identified, with subsequent elucidation of the ligand sequence. Ligands to several targets can be identified in one experiment. Here we demonstrate the broad utility of this method by the selection of ligands that purify plasma protein complexes or that remove pathogens from whole blood with very high affinity constants. We also select ligands to a protein based on competitive elution.

Amino Acid Sequence↗

PubMed2004

[Studies on purification of polyprenol from Ginkgo biloba L. leaves].

OBJECTIVE: To research the methods of purifying polyprenol from leaves of Ginkgo biloba L.. METHODS: The purity of polyprenol was determined by HPLC to select the optimal purifying conditions. RESULTS: The optimal conditions were degreased by 160 times of petroleum ether-ethyl acetate (9:1) firstly, then through a silica gel column (100-140 mesh) and eluted with petroleum etherethyl acetate (19:1). CONCLUSION: The methods are feasible to purify polyprenol from leaves of Ginkgo biloba L. and could increase the purity of polyprenol obviously.

Chromatography, High Pressure Liquid↗

PubMed1985

Hybrid selection of messenger ribonucleoprotein for serum albumin: analysis of specific message-bound proteins.

The messenger RNA for chicken serum albumin together with its specific binding proteins was purified by hybrid selection using filter-bound cloned albumin cDNA. Under conditions where hybridization of the protein-mRNA complex was specific for the immobilized cDNA sequence, no dissociation of the complex into its protein and RNA components was apparent. Electrophoretic analysis of albumin mRNA-specific binding proteins showed three major bands. Two of these were prominent in total poly(A) messenger ribonucleoprotein. The third band was of much greater relative intensity in the albumin mRNA-specific proteins than in total poly(A) messenger ribonucleoprotein. The results suggest that the proteins bound to albumin mRNA represent only a subset of the total population of poly(A) mRNA-associated protein.

Animals↗

PubMed1987

Identification of an estrogen-responsive element from the 5'-flanking region of the rat prolactin gene.

The DNA sequences which interact with the estrogen receptor and which mediate the estrogenic regulation of prolactin gene transcription have been investigated by the use of receptor-DNA-binding experiments and gene transfer studies. Nitrocellulose filter binding assays using highly purified estrogen receptor and cloned fragments of the 5'-flanking region of the rat prolactin gene demonstrate that the receptor selectively binds to DNA sequences located between nucleotides -1713 and -1532 with respect to the transcription initiation site. The binding of the estrogen receptor to this region of the prolactin gene was strongly dependent on receptor concentration, suggesting that receptor dimers may be important in DNA binding. These data demonstrate that the selective binding of purified estrogen receptor to specific sequences of the rat prolactin gene is an intrinsic property of the receptor and is not due to the interaction of receptor with other proteins. The role of specific prolactin gene sequences in mediating the estrogenic regulation of prolactin gene transcription was confirmed by the use of prolactin-chloramphenicol acetyltransferase fusion genes. These studies demonstrated that sequences upstream of position -1532 are required for estrogen responsiveness. Furthermore, the region of the prolactin gene at -1713 to -1495 was able to confer estrogen responsiveness on the thymidine kinase promoter. Exonuclease III protection experiments further localized the receptor-binding sequences to positions -1587 to -1563. Comparison of the nucleotide sequence of the region of the prolactin gene which binds the estrogen receptor with the sequence of other estrogen-responsive genes suggested the presence of the conserved sequence [sequence in text], which shows similarity to sequences thought to mediate glucocorticoid receptor effects on transcription.

Acetyltransferases↗