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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of beta-glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.
Plant Mol Biol 1990 Dec
PMID:Optimization of delivery of foreign DNA into higher-plant chloroplasts. 210 74

Chromosomes were isolated in a preparative scale by synchronisation of CHO cells with a double Thymidine block followed by an arrest in the metaphase by addition of Colcemid. Under proper cultivation conditions a mitotic index of 77% total cells could be routinely achieved. Bulk chromosome preparations free of nuclei and other subcellular particles have been obtained by low speed centrifugation followed by a 60 transfer countercurrent distribution using aqueous two phase systems composed of polyethylenglycol and dextran. The partition of CHO chromosomes previously purified in aqueous two phase systems were studied further to develop a protocol for the separation and isolation of individual chromosomes. Partition experiments with chromosomes changing the electrostatic phase potential by addition of charged PEG-derivatives suggest the existence of relatively highly charged chromosome groups. Most promising results with regard to separation were obtained using two PEG-derivatives, which interact specifically with the bases in DNA. For this affinity partitioning a GC- and AT-specific macroligand were employed. Comparing CCD's using each of these ligands information on the GC and AT content of exposed DNA in the chromosomes groups could be derived, demonstrating that specific sequences of DNA are accessible at the surface of metaphase chromosomes.
Mol Cell Biochem 1986 Feb
PMID:Isolation and fractionation of CHO chromosomes in aqueous two phase systems using charged polymers and base specific macroligands. 242 Nov 51

A modified procedure in two versions (micro, for 10 ml of phage lysate, and macro, for 200-500 ml) is described for preparing lambda phage DNA. The main advantage of the modified method is that it gives a possibility to isolate high-quality DNA from lambda phage lysates in 2-3 hrs. Only standard solutions (TE, NaCl, SDS, MgCl2, EDTA, RNAse A) were used throughout the whole protocol. Incubation with DNAse I and proteinase K was omitted and in microvariant concentration of the phage by PEG 6000 was excluded. Digestion by RNAse A was performed in solution with EDTA and SDS and leads to RNA degradation. The yields of DNA (0.5-2 micrograms per ml of L-broth) are similar to those obtained by other methods. DNA quality is better than in the samples of DNA prepared by other express-methods and practically the same as after CsCl centrifugation. DNA can be used for splitting by restriction enzymes, cloning and gene library construction.
Mol Biol (Mosk)
PMID:[Rapid isolation of phage lambda DNA]. 285 52

The binding characteristics of the beta-adrenergic receptor in the rat ventral prostate homogenate have been studied using the highly potent beta-adrenergic antagonist [125I]cyanopindolol (CYP) as ligand. The bound ligand was separated from the free moiety by precipitation with polyethylene glycol (PEG-6000). This technique is simple, accurate, fast and more advantageous than filtration of the hormone-receptor complex on glass fiber filters or direct centrifugation. [125I]CYP binds to a single class of high affinity sites at an apparent KD value of 23 pM. Using 0.1 microM (-)propranolol to determine non-specific binding, a number of sites of 600 fmol/mg protein were measured. The observed order of potency of adrenergic agonists (KD values) in competing for [125I]CYP binding was: (-)isoproterenol (25 nM) greater than (-)epinephrine (74 nM) much greater than (-)norepinephrine (1900 nM). Detailed study of the binding potency of a large series of beta 1- and beta 2-adrenergic agonists and antagonists showed the presence of a typical beta 2-subtype adrenergic receptor in the rat ventral prostate. The best estimate indicates that the proportion of beta 2-adrenergic receptors in rat ventral prostate is more than 95% of the total population of beta-adrenergic receptors in this tissue. The high selectivity and density of beta 2-adrenergic receptors in rat ventral prostate suggest a physiological role of circulating and/or locally secreted catecholamines in the control of prostatic growth and function.
Mol Cell Endocrinol 1986 Nov
PMID:Characteristics of the beta-adrenergic receptor in the rat ventral prostate using [125I]cyanopindolol. 287 9

A relatively rapid procedure is described for the isolation of the fourth component of complement (C4) from ovine plasma. The method, which recovers approximately 30% C4, is based upon DEAE Sephacel anion exchange chromatography of PEG precipitated plasminogen depleted plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration. SDS-PAGE of purified ovine C4 under reducing conditions revealed a complex pattern of bands which was interpreted on the basis of a three polypeptide chain structure for each of two distinct species, or isotypes, of C4 molecule herein termed C4A and C4B. Each isotype differs in the mol. wt of the alpha chain--108 and 95 K respectively. Nucleophilic substitution of immunoprecipitated ovine C4 with radiolabelled methylamine revealed that both C4 species contained a reactive thiol ester site and that each could be cleaved into an activated form (presumably C4b) characterised by a truncated alpha' chain some 8 K lower in mol. wt. A comparison of the isotype composition of purified C4 with that of immunoprecipitated C4 from the same animal indicated that the purification procedure favoured isolation of the C4B isotype. The mol. wts of both the alpha and beta chains were lowered following digestion of ovine C4 with neuraminidase.
Mol Immunol 1988 Jun
PMID:Purification and characterisation of ovine C4: evidence for two molecular forms in ovine plasma. 317 57

The binding of 125I-labelled human C1q to insoluble rabbit IgG:ovalbumin immune complexes was enhanced by polyethylene glycol (PEG, Mr 8 x 10(3)) in the concn range 0-2.5% (w/v). C1q with native immunoglobulin bindings sites rendered inactive by diethylpyrocarbonate treatment did not bind to immune complexes in the presence of PEG. The ionic strength dependence of the binding was independent of the presence of PEG. There was a linear relationship between the logarithm of the apparent affinity constant of the C1q:immune complex interaction and PEG concn.
Mol Immunol 1988 Mar
PMID:Enhancement of the binding of C1q to immune complexes by polyethylene glycol. 325 72

Hydrogenase (EC 1.12) from Desulfovibrio gigas is a dimeric enzyme (26 and 62 (X 10(3) Mr) that catalyzes the reversible oxidation of molecular hydrogen. Single crystals of hydrogenase have been produced using the hanging drop method, with either PEG (polyethylene glycol) 6000 or ammonium sulfate as precipitants at pH 6.5. X-ray examination of the crystals indicates that those obtained with ammonium sulfate are suitable for structure determination to at least 3.0 A resolution when synchrotron radiation Sources are used (1 A = 0.1 nm). The crystals are monoclinic, with space group C2, and cell dimensions a = 257.0 A, b = 184.7 A, c = 148.3 A and beta = 101.3 degrees, and contain between four and ten molecules per asymmetric unit. The enzyme can be reactivated within the crystals under reducing conditions without crystal damage.
J Mol Biol 1987 Jun 20
PMID:Crystallization, preliminary X-ray study and crystal activity of the hydrogenase from Desulfovibrio gigas. 330 47

The results demonstrate the presence in cod serum of beta 2-microglobulin (beta 2m)-binding molecules. Upon fractionation on Sephadex G-200, the bound beta 2m appears mainly in the void volume, but also as a minor peak with the apparent size of albumin. The complexes show affinity to Con A-Sepharose and Lens culinaris lectin-Sepharose, respectively, indicating that they contain glycoproteins. Because of the high molecular weight of the beta 2m-containing complexes they can be separated from unbound beta 2m by polyethyleneglycol (PEG-6000) precipitation, thus allowing rapid analysis. These beta 2m-binding molecules exhibited size- and charge-homogeneity when separated by gel filtration (Sepharose 4B) and by ion-exchange chromatography (DEAE-cellulose), respectively. The binding is temperature-dependent. At 37 degrees C, maximum binding is reached after about 2 hr. The dissociation is considerably slower, complete dissociation taking about 2 days. According to Scatchard analysis, the association constant is of the order 2 X 10(9)/M. The binding is sensitive to denaturating agents and high salt concentrations. Optimum binding is seen at neutral pH and the beta 2m-binding activity is heat-labile at 50 degrees C. The binding of heterologous beta 2m by cod serum can be used as a cross-reactive assay specific for the detection of beta 2m. Whereas unlabelled human, guinea-pig, and rat beta 2m all give similar inhibition, higher concentrations of rabbit beta 2m are needed for the same degree of inhibition.
Mol Immunol 1983 Aug
PMID:Binding of mammalian beta 2-microglobulin by glycoproteins in fish serum. 635 6

Human intraspecific hybrids were formed between tumor cells isolated from both primary and metastatic tumors and a tissue culture adapted cell line, D98OR, a HeLa derivative which is thioguanine and ouabain resistant. Five different tumor types in all were attempted: renal cell carcinoma, colon adenocarcinoma, melanoma, chrondrosarcoma, and hepatocarcinoma. The tumor tissue was either (1) immediately dissociated and fused, or (2) frozen and later thawed, dissociated, and fused. Two different PEG concentrations were used. The results reported here demonstrate that: (1) hybrid tumor cell lines can be made from several types of cancer, (2) unfrozen tumor tissue fused with D98OR by exposure to 50% PEG appears optimal, (3) chromosome loss, as determined by flow cytometry studies of hybrid DNA content, is minimal, and (4) hybrids have characteristics consistent with derivation from tumor cells rather than derivation from the nonmalignant cells of a tumor.
Somat Cell Mol Genet 1984 Mar
PMID:Somatic cell hybridization of human tumor samples. 658 90

A small-angle reflexion in X-rayograms and an intense band at A approximately 270 nm in the CD spectrum are assigned to compact particles that arise when mixing water-salt solutions of PEG with water-salt solutions of double-stranded RNA and those of poly(A) poly(U) and poly(I) poly(C). The discrepancy between the 35-40 A small-angle and the approximately 20 A small-angle reflexion typical for double-stranded poly-nucleotide crystals together with the presence of the intense band in the CD spectra point out to the fact that the double-stranded RNA molecules and the molecules of polyribonucleotides exist in a mesophase (liquid crystalline) state. The compact particles of double-stranded RNA and those of polyribonucleotides are shown to be able to have either a positive or a negative band of the CD spectrum depending on PEG concentration, ionic strength or temperature of the solution.
Mol Biol (Mosk)
PMID:[Mesophase state of double-stranded RNA and polyribonucleotides characterized by high optical activity]. 671 23


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