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Biomedical subjects

C Min

Publications and source records attributed to C Min.

12 recordsLinked to original sources
PubMed1996

Immobilized metal affinity chromatography of DNA.

Many of the most widely employed operations in molecular biology hinge upon the use of single-stranded DNA as a probe or template. Here we report a straightforward method by which to produce long single-stranded DNA molecules using the polymerase chain reaction (PCR) in combination with immobilized metal affinity chromatography (IMAC). We demonstrate that a tag consisting of six successive 6-histaminylpurine (H) residues (H6-tag) endows a DNA strand with selective retentivity onto a Ni2+-NTA-agarose chromatography matrix. The H6-tagged strand can then be eluted from the resin using 200 mM imidazole. Quantitative phosphorimaging analysis revealed that the PCR/IMAC procedure typically yields unmodified strands comprising >90% of the unbound DNA and H6-tagged strands comprising >95% of the bound fractions. DNA strands generated in this manner are shown to be excellent substrates for template-directed polymerization. The chemistry reported herein should facilitate a wide variety of operations in molecular biology, including automated DNA sequencing, hybridization screening of DNA libraries, assembly of gene cassettes, run-off transcription, site-directed mutagenesis and footprinting of protein-DNA complexes by template-directed interference footprinting.

Chromatography, Affinity↗
PubMed1995

Overexpression in Escherichia coli of 12 vitamin B12 biosynthetic enzymes.

The first 12 enzymes involved in the biosynthesis of vitamin B12 from its five-carbon precursor, aminolevulinic acid, have been overexpressed in recombinant strains of Escherichia coli. The activity of each enzyme has been demonstrated by the biosynthesis of hydrogenobyrinic acid from aminolevulinic acid.

Aminolevulinic Acid↗
PubMed1994

Biodegradable microspheres as controlled-release tetanus toxoid delivery systems.

Purified tetanus toxoid, a high-molecular-weight protein, was entrapped within poly(L-lactic acid) (PLA) and poly(D,L-lactic/glycolic acid) (PLGA) microspheres prepared by either a solvent extraction or a solvent evaporation method carried out in a multiple emulsion system (water-in-oil-in-water). The physical integrity and antigenicity of the protein treated under different processing conditions were investigated. A reduction of antigenicity that was related to the percentage of aggregated protein was noticed under some experimental conditions. This partial loss of antigenicity was associated with the lyophilization process and affected by the nature of the organic solvent. All types of microspheres prepared with different molecular weight PLA and PLGA displayed a high protein-loading efficiency (> 80%) but their size was strongly influenced by polymer molecular weight (3000 versus 100,000). Protein release pattern was influenced by both polymer molecular weight and composition (PLA versus PLGA). A constant release pattern after an induction period of 10 days was observed for microspheres composed of high-molecular-weight polymers (PLA and PLGA). The release rate was lower from PLA microspheres than from PLGA microspheres. In contrast, a continuously increasing release rate preceded by a burst was observed for low-molecular-weight (3000) PLGA microspheres. Microencapsulated tetanus toxoid was significantly more immunogenic in mice than fluid toxoid as determined by IgG anti-tetanus antibody levels and neutralizing antibodies. However, the magnitude and duration of the antibody response did not differ significantly from a similar dose of aluminium phosphate-adsorbed toxoid. We conclude that microencapsulated tetanus toxoid shows significant adjuvant activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Conducting states of a mammalian serotonin transporter.

We have studied permeation at a cloned rat 5-HT transporter expressed in Xenopus oocytes. [3H]5-HT uptake and [125I]RTI-55 binding yield a turnover rate of approximately 1/s that does not depend on membrane potential. However, in voltage-clamp experiments, three distinct currents results from 5-HT transporter expression. First, a steady-state, voltage-dependent transport-associated current is induced by 5-HT application. Second, a transient inward current is activated by voltage jumps to high negative potentials in the absence of 5-HT and is blocked by 5-HT itself. Third, a small leakage current is observed in the absence of 5-HT. All the observed currents are blocked by inhibitors of 5-HT uptake but are differentially affected by Na+, Li+, K+, Ba2+, Cs+, Cl-, and amiloride. The conducting states of the 5-HT transporter may reflect the existence of a permeation pathway similar to that of ionic channels.

Amiloride↗
PubMed1994

Genetically engineered synthesis of precorrin-6x and the complete corrinoid, hydrogenobyrinic acid, an advanced precursor of vitamin B12.

BACKGROUND: Genetically engineered synthesis, in which the gene products, cofactors, and substrates of a complete pathway are combined in vitro in a single flask to give the target, can be a viable alternative to conventional chemical construction of molecules of complex structure and stereochemistry. We chose to attempt to synthesize the metal-free corrinoid hydrogenobyrinic acid, an advanced precursor of vitamin B12. RESULTS: Cloning and overexpression of the genes necessary for the S-adenosyl methionine dependent conversion of 5-aminolevulinic acid (ALA) to precorrin-3 and those required for the synthesis of hydrogenobyrinic acid from precorrin-3 completed the repertoire of the 12 biosynthetic enzymes involved in corrin synthesis. Using these enzymes and the necessary cofactors, the multi-enzyme synthesis of hydrogenobyrinic acid from ALA can be achieved in 20% overall yield in a single reaction vessel, corresponding to an average of at least 90% conversion for each of the 17 steps involved. CONCLUSIONS: By replacing the cell wall with glass, and by mixing the soluble biosynthetic enzymes and necessary cofactors, the major segment of the physiological synthesis of vitamin B12 has been accomplished. Since only those enzymes necessary for the synthesis of hydrogenobyrinic acid from ALA are supplied, none of the intermediates is deflected from the direct pathway. This results in an efficiency which in fact surpasses that of nature.

Escherichia coli↗
PubMed1993

Biosynthesis of vitamin B12. Discovery of the enzymes for oxidative ring contraction and insertion of the fourth methyl group.

In the vitamin B12 biosynthetic pathway the enzymes responsible for the conversion of precorrin-3 to precorrin-4 have been identified as the gene products of cobG and cobJ from Pseudomonas denitrificans. CobG catalyzes the oxidation of precorrin-3 to precorrin-3x (a hydroxy lactone) whereas CobJ is a SAM-dependent C-17 methyl transferase and is necessary for ring contraction. A mechanism for ring contraction is proposed.

Bacterial Proteins↗
PubMed1993

Sequence of the Candida albicans erg7 gene.

The nucleotide sequence of the Candida albicans erg7 gene, which complements erg7 mutants of Saccharomyces cerevisiae and restores oxidosqualene cyclase activity, was determined. The gene encodes a 728-aa protein that displays homology with squalene-hopene cyclase, providing further evidence that erg7 is the gene encoding 2,3-oxidosqualene cyclase.

Amino Acid Sequence↗
PubMed1993

Receptors that couple to 2 classes of G proteins increase cAMP and activate CFTR expressed in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl- channel activated by phosphorylation, was expressed in Xenopus oocytes along with various combinations of several other components of the cAMP signalling pathway. Activation of the coexpressed beta 2 adrenergic receptor increased cAMP and led to CFTR activation. The activation of CFTR (1) requires only short (15 s) exposure to isoproterenol, (2) occurs for agonist concentrations 100-1000 fold lower than those that produce cAMP increases detectable by a radioimmunoassay, (3) requires injection of only 5 pg of receptor cRNA per oocyte, and (4) can be increased further by coexpression of cRNA for adenylyl cyclase type II or III or for Gs alpha. In addition, CFTR activation and cAMP increases by beta 2 activation were enhanced by activation of the coexpressed 5HT1A receptor, which is thought to couple to Gi. The additional activation by the 5HT1A receptor was enhanced by coexpression of adenylyl cyclase type II but not with type III and may proceed via the beta gamma subunits of a G protein. The sensitivity of the assay system is also demonstrated by responses to vasoactive intestinal peptide and to pituitary adenylate cyclase-activating polypeptide in oocytes injected with cerebral cortex mRNA.

Adenylyl Cyclases↗
PubMed1976

Vitamin D metabolite-binding proteins in human tissue.

Serum and post-microsomal supernatants of human lymphocyte, erythrocyte, skeletal muscle and parathyroid adenoma homogenates were examined for specific binding of 25-hydroxycholecalciferol (25-OHD3) and 1, 25-dihydroxycholecalciferol (1,25-(OH)2D3). Muscle, lymphocytes and parathyroid adenomata extracts contained a 6-S 25-OHD3-binding protein which was not found in erythrocyte extracts, and which was distinct from the smaller serum transport alpha-globulin. A cathodal, 1, 25-(OH)2D3-binding protein, which sedimented at 3-4 S was also detected in parathyroid tissue. These observations suggest the possibility of direct physiologic interaction between vitamin D metabolites and nucleated human tissues other than intestine and bone.

Adenoma↗
PubMed1976

Competition by 24,25-dihydroxycholecalciferol in the competitive protein binding radioassay of 25-hydroxycalciferol.

24,25-dihydroxycholecalciferol (24,25-(OH)2D3) is equipotent to 25-hydroxycholecalciferol in the displacement of 3H25-OHD3 from rat serum binding sites, and is extracted by the ethanol procedure recommended in non-chromatographic competitive protein binding radioassays for 25-OHD. Serum 24,25-(OH)2D content was measured following 24,25-(OH)2D3 isolation from lipid extracts by liquid-gel partition chromatography. Since normal serum 24,25-(OH)2D concentration is appreciable, non-chromatographic 25-OHD assays most probably overestimate serum 25-OHD levels as a result of their recognition of 24,25-(OH)2D3.

Animals↗
PubMedDate not supplied

Membrane-based, dry-reagent prothrombin time tests.

The authors describe a prototype membrane-based, dry-reagent prothrombin time assay for whole blood. This system uses an asymmetric polysulfone membrane to separate plasma from red blood cells, and works with samples as small as 10 microliters. The membrane contains calcium and thromboplastin, and permits the reactions of the complete extrinsic pathway to occur with minimal distortion from membrane surface interactions. Thrombin generation is monitored optically using a rhodamine-110-based fluorescent thrombin substrate. Fluorescence kinetics are analyzed to produce a prothrombin-time--equivalent parameter that can be converted to an international normalized ratio (INR) value. The system provides results that correlate well with conventional liquid phase prothrombin time assays (R2 = 0.96).

Anticoagulants↗