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Biomedical subjects

B R Rabin

Publications and source records attributed to B R Rabin.

At least 19 recordsLinked to original sources
PubMed1995

An amplified chemiluminescent assay for the detection of alkaline phosphatase.

An amplification assay for the measurement of alkaline phosphatase has been combined with a luminescent end point using the luminol-peroxidase system to produce the first enzyme-amplified chemiluminescent assay based on the principle of prosthetogenesis. This assay is both quantitative and extremely sensitive. When the assay was used to detect alkaline phosphatase in solution, the detection limit was 0.4 amol in a 5-min assay. The interassay variance ranged from 4 to 20% and 7 to 19% across the dynamic range of the assay for a chemiluminescent assay and an enhanced chemiluminescent assay, respectively, employing two different preparations of luminol.

Alkaline Phosphatase↗
PubMed1994

Purification and semienzymic synthesis of flavin adenine dinucleotide-3'-phosphate.

Nonradioactive immunoassays incorporating an element of amplification in their detection system require the use of components that are highly purified. Flavin adenine dinucleotide-3'-phosphate (FADP) is the primary substrate used in such an amplification assay. For incorporation into a simple, single-pot assay system, the concentration of contaminating flavin adenine dinucleotide (a prosthetic group for the enzyme D-aminoacid oxidase used in the amplification cascade assay) in this primary substrate must be minimized to achieve maximum sensitivity. Production of the substrate to a high degree of purity has been achieved using apo-glucose oxidase to specifically remove contaminating flavin adenine dinucleotide from solution and hydrolysis of a cyclic intermediate as a final production protocol by ribonuclease T2 to give the product in high yield. The use of continuous ultrafiltration reactors at each stage is described and compared to a final production step utilizing immobilized ribonuclease T2. These reactors allow large volumes of material to be handled and assist in the scale-up of these processes. The suitability of each protocol is assessed for the commercial production of FADP.

Apoproteins↗
PubMed1994

Benefits communication: its impact on employee benefits satisfaction under flexible programs.

Using field data from a private organization in upstate New York, a large statistically significant portion of benefits satisfaction was explained by benefits communication, employee benefits choice and change patterns, and demographics. Users of the organization's benefits communication materials and employees reporting fewer unmet benefits needs are more satisfied.

Adult↗
PubMed1993

A model for prosthetogenic enzyme amplification assays.

A mathematical model describing the behaviour of a new class of prosthetogenic enzyme amplification assays is described. The predictions of the model are favourably compared with an enzyme amplification assay for alkaline phosphatase. The model is used to kinetically characterise and optimise the enzyme amplification assay.

Alkaline Phosphatase↗
PubMed1993

Identification by site-directed mutagenesis of amino acids in the B2 subsite of bovine pancreatic ribonuclease A.

In addition to hydrolysing RNA, bovine pancreatic ribonuclease splits esters of pyrimidine nucleoside 3'-phosphates, including dinucleotides. For a series of 3':5'-linked dinucleotides of general structure CpN, where N is a 5' linked nucleoside, kcat for the release of N varies enormously with the precise structure of N. Structural studies have been interpreted to indicate that the group N interacts with a subsite, B2, on the enzyme that comprises Gln69, Asn71 and Glu111. We report studies by site-directed mutagenesis that indicate that Gln69 is not involved in productive interactions with any of the dinucleotide substrates and that Asn71 is an important component of subsite B2 for all dinucleotide substrates tested. Glu111 appears to be functionally involved in catalysis for dinucleotide substrates solely when N is guanosine.

Amino Acids↗
PubMed1992

Production of mature bovine pancreatic ribonuclease in Escherichia coli.

The coding sequence for the bovine pancreatic ribonuclease (RNase) precursor has been cloned and produced in Escherichia coli using the polymerase chain reaction (PCR) technique. A PCR amplification has been carried out utilizing as template the recombinant plasmid, pQR138, which contains the coding sequence for the RNase precursor, and primers that allow for the addition of new sequences at the 5' and 3' ends of the coding sequence. The resultant fragment contains two coding sequences, one for a hexapeptide and the other for pre-RNase. This fragment has been cloned into the expression vector, pKK223.3, under the control of the tac promoter, to form a two-cistron vector. Upon induction with IPTG, E. coli cells harboring this construct generate a bicistronic mRNA which upon translation produces a hexapeptide and pre-RNase. The RNase precursor is efficiently translocated into the periplasmic space of E. coli. Upon translocation, the signal sequence is removed generating mature RNase. Formation of the disulfide bridges in RNase is facilitated by the oxidative environment of the periplasm and a fully active protein is obtained. RNase produced in E. coli has been purified to homogeneity by cation-exchange chromatography, and the removal of the signal sequence has been verified by N-terminal sequencing. The total process from inoculation of media to obtaining pure and fully active recombinant RNase is achieved in 48 h.

Amino Acid Sequence↗
PubMed1992

Amplified assay of alkaline phosphatase using flavin-adenine dinucleotide phosphate as substrate.

A simple to use, robust, quantitative, and extremely sensitive colorimetric assay for alkaline phosphatase (EC 3.1.3.1), designed to be used as a detection system in diagnostic assays employing antibodies or gene probes, is described. This technology is based on the novel principle of prosthetogenesis, according to which a purpose-designed substrate (a prosthetogen) for a primary analyte-linked enzyme label is hydrolyzed to produce a prosthetic group for a detector enzyme system. The prosthetogen employed here is a derivative of FAD which is phosphorylated at the 3'-position of the ribose ring (FADP), the label enzyme is alkaline phosphatase, and the detector is a D-amino-acid oxidase/horseradish peroxidase-coupled system. Essentially each turnover of every molecule of alkaline phosphatase produces a molecule of D-amino-acid oxidase for detection. Thus enormous amplification of the initial signal is achieved in short time periods because of the relatively high turnover number of alkaline phosphatase for FADP. The system can be formatted as a stable, preformed, freeze-dried preparation containing all analytical components, which is reconstituted simply by addition of buffer solution. This methodology can quantitate less than 0.1 amol of alkaline phosphatase in 30 min at 25 degrees C using microtiter plates.

Alkaline Phosphatase↗
PubMed1991

Amplified colorimetric assay of alkaline phosphatase using riboflavin 4'-phosphate: a simple method for measuring riboflavin and riboflavin 5'-phosphate.

Alkaline phosphatase hydrolyzes riboflavin 4'-phosphate to produce riboflavin. This is converted to riboflavin 5'-phosphate, using riboflavin kinase, which reconstitutes apoglycolate oxidase to give hologlycolate oxidase. This enzyme catalyzes the oxidation of glycolate with simultaneous production of hydrogen peroxide which is detected via the formation of a colored product through the action of peroxidase. The system allows the detection of 4 amol after a 2-h incubation.

Alcohol Oxidoreductases↗
PubMed1991

Sensitive, colorimetric enzyme amplification cascade for determination of alkaline phosphatase and application of the method to an immunoassay of thyrotropin.

A highly sensitive flavin adenine dinucleotide-3'-phosphate (FADP)-based enzyme amplification cascade has been developed for determining alkaline phosphatase (ALP; EC 3.1.3.1). The cascade detects ALP via the dephosphorylation of the novel substrate FADP to produce the cofactor FAD, which binds stoichiometrically to inactive apo D-amino acid oxidase (D-AAO). The resulting active holo D-AAO oxidizes D-proline to produce hydrogen peroxide, which is quantified by the horseradish peroxidase-mediated conversion of 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine to a colored product. The FADP-based enzyme amplification cascade has been used in a novel releasable linker immunoassay (RELIA) to quantify thyrotropin (TSH). In the assay, TSH is first captured onto antibody-coated chromium dioxide particles. After formation of an antibody-TSH sandwich with a dethiobiotinylated second antibody, the complex is reacted with a streptavidin-ALP conjugate. Biotin is then used to release the conjugate into solution, and ALP is quantified in an automated version of the FADP-based amplification cascade on the aca discrete clinical analyzer (Du Pont). The sensitivity of the colorimetric RELIA assay for TSH (less than 0.1 milli-int. unit/L) is comparable with that of fluorometric assays. This technology provides a way to adapt to the aca high-sensitivity immunoassays for a wide range of analytes via colorimetric detection.

Clinical Enzyme Tests↗
PubMed1988

The effect of terpenoid compounds on cytochrome P-450 levels in rat liver.

We have investigated the ability of camphor, menthol, pinene, limonene and myrcene to induce in rats members of a cytochrome P-450 sub-family termed PB P-450. These proteins have recently been designated as members of the P450IIB sub-family. None of these naturally occurring terpenoids significantly changed the total content of cytochromes P-450 or cytochrome b5. Radioimmunoassay results showed that PB P-450 was induced 6-fold by camphor and to a lesser extent by menthol and pinene. The induction was confirmed by Western blotting. It was shown by nucleic acid hybridization that induction of PB P-450 by terpenoids was mediated by an increase in the amount of the corresponding mRNA. Analysis of the denaturation of mRNA-cDNA hybrids demonstrated that the mRNA induced by the terpenoids was encoded by a member of the P450IIB sub-family. None of the terpenoids had an effect on the amount of mRNA coding for P450IA2 (a cytochrome P-450 inducible by beta-naphthoflavone and isosafrole). The results indicate that cytochromes P-450 induced by a synthetic compound, phenobarbital, may have originally evolved in response to terpenoid compounds normally present in the environment.

Animals↗
PubMed1985

Induction of cytochrome P-450 by phenobarbital is mediated at the level of transcription.

We have previously shown that the 43-fold induction by phenobarbital of the major phenobarbital-inducible cytochrome P-450 of rat liver microsomal membranes (PB P-450) is mediated by a 20-fold increase in the amount of its mRNA in the cytoplasm. Here we demonstrate that the induction of the mRNA can be almost entirely accounted for by an increase in the rate of transcription of genes coding for PB P-450, and involves little or no change in the rates of processing, transport or degradation of the mRNA. Phenobarbital treatment resulted in no amplification or rearrangement of PB P-450 genes.

Animals↗
PubMed1985

Isolation and sequence of a human cytochrome P-450 cDNA clone.

A previously reported cDNA clone [pP450(1)] coding for a phenobarbital-inducible cytochrome P-450 variant of rat liver microsomal membranes, designated P-450e(U.C.), was used as a specific hybridization probe to screen a human liver cDNA library. Restriction mapping showed that two of the colonies isolated contained plasmids coding for overlapping regions of the same cDNA sequence. The clone [pHP450(1)] having the longer cDNA insert (1.25 kilobase pairs) was sequenced. The homology between the rat and human cDNAs is 62% in their coding regions but is only random (24%) in the 3'-noncoding nucleotides. The amino acid sequence deduced from the human cDNA is 50% identical to that of P-450e(U.C.). The homology increases to 72% if conservative changes in amino acid residues are permitted. The hydropathy profile of the polypeptide encoded by pHP450(1) is almost identical to that of P-450e(U.C.). Regions known to be highly conserved in cytochrome P-450 isozymes isolated from rat, rabbit, and mouse were found to be conserved in the amino acid sequence derived from pHP450(1). Analysis by Southern blotting indicated that the human cytochrome P-450 encoded by pHP450(1) is part of a multigene family.

Amino Acid Sequence↗
PubMed1983

Preparation and properties of a complex from rat liver of polyribosomes with components of the cytoskeleton.

Gel filtration with 1% agarose (Bio-Gel A-150m) separates polyribosomes bound to microsomal membranes from 'free' polyribosomes when these fractions are prepared by standard centrifugal techniques. However, when polyribosomes contained in an unfractionated postmitochondrial supernatant are run on an identical column, over 90% of the total polyribosomes are present as aggregates, designated 'membrane-cytomatrix', which are eluted in the column void volume. Polyribosomes are not released from these aggregates on removal of microsomal phospholipids by treatment of postmitochondrial supernatant with 1% Triton X-100, a neutral detergent. The aggregates are disrupted by the usual ultracentrifugation techniques used in subcellular fractionation. After treatment of membrane-cytomatrix with Triton X-100 to remove phospholipids and membrane proteins, 58% of the polyribosomes still remain associated with protein-containing complexes in the form of a cytomatrix and are not 'free'. Preparations of both membrane-cytomatrix and cytomatrix are capable of sustained protein synthesis. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that the cytoskeletal proteins actin and myosin are present in the cytomatrix. Incubation of cytomatrix preparations with the actin-depolymerizing agent deoxyribonuclease I caused release of the polyribosomes. Polyribosome release by deoxyribonuclease I was prevented by prior incubation with phalloidin, which is known to stabilize F-actin. Thus polyribosomes are associated with cytoskeletal elements in rat liver, and this association is dependent on polymeric forms of actin.

Animals↗
PubMed1983

Quantification of NADPH: cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique.

We have developed a specific radioimmunoassay to quantify NADPH: cytochrome P-450 reductase. The assay is based on the use of 125I-labelled NADPH: cytochrome P-450 reductase as the radiolabelled antigen and can detect quantities of this protein in amounts as low as 30 pg. The results of the radioimmunoassay demonstrates that the 2.7-fold increase in enzyme activity in rat liver microsomal membranes after phenobarbital treatment is due to increased amounts of the protein. beta-Naphthoflavone treatment, however, did not alter the activity or the quantity of this enzyme in microsomes. The quantification of NADPH: cytochrome P-450 reductase in the microsomes isolated from control and phenobarbital- and beta-naphthoflavone-treated animals permits the calculation of the ratio of this protein to that of total cytochromes P-450. A molar ratio of 15:1 (cytochromes P-450/NADPH: cytochrome P-450 reductase) was calculated for control and phenobarbital-treated animals. This ratio increased to 21:1 after beta-naphthoflavone treatment. Thus the molar ratio of these proteins in liver microsomes can vary with exposure of the animals to particular xenobiotics.

Animals↗
PubMed1983

Induction and repression of the major phenobarbital-induced cytochrome P-450 measured by radioimmunoassay.

Two independent radioimmunoassay techniques for the major phenobarbital-inducible cytochrome P-450 (PB P-450) of rat liver microsomal membranes are described. The first technique employs as the source of radiolabelled antigen the products of translation in vitro labelled with [35S]methionine. The second technique employs purified antigen labelled with 125I and is quicker, less expensive and more precise. Both assays are highly specific for PB P-450 and can detect quantities of this variant as small as 1 ng. This is several orders of magnitude more sensitive than any method described previously for the quantification of cytochromes P-450, and consequently the technique is particularly well suited for the quantification of so-called constitutive cytochrome P-450 variants that are present in very low amounts. The results of the radioimmunoassays demonstrate that the apparent 2.6-fold induction of total cytochromes P-450 after phenobarbital treatment is due to a 43-fold increase in Pb P-450. Although beta-naphthoflavone increases the total content of cytochrome P-450 of microsomal membranes 1.4-fold, it actually causes a 55% decrease in the amount of PB P-450. Thus different xenobiotics can have differential effects on the expression of the genes for specific cytochrome P-450 variants.

Animals↗
PubMed1983

A rapid one-step purification of NADPH-cytochrome c (P-450) reductase from rat liver microsomes.

NADPH-cytochrome c (P-450) reductase from liver microsomes of phenobarbital-treated rats has been purified in a single step by affinity chromatography on agarose-hexane-adenosine 2',5'-diphosphate. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme assay, and radioimmunoassay the protein obtained by this single step procedure is as pure as that isolated by multicolumn procedures.

Animals↗
PubMed1983

Cloning and sequence analysis of a rat liver cDNA coding for a phenobarbital-inducible microheterogenous cytochrome P-450 variant: regulation of its messenger level by xenobiotics.

A rat liver cDNA library was prepared from total polyribosomal poly(A)+ RNA extracted from phenobarbital-treated animals. A cDNA clone coding for a phenobarbital-inducible cytochrome P-450 (PB P-450) was identified by differential colony hybridization to cDNAs synthesized from liver poly(A)+RNAs isolated from phenobarbital-treated rats for positive selection and cDNAs from either untreated rats or beta-naphthoflavone-treated rats as negative controls, followed by hybrid-selected translation and analysis of the translation products by immunoprecipitation. As the cloning and screening strategies involve no prior enrichment for specific mRNAs, they also permit the identification of sequences coding for phenobarbital-induced proteins other than cytochromes P-450. This relatively straightforward approach is generally applicable to the molecular cloning of sequences coding for other inducible cytochromes P-450. Nucleic acid sequencing data indicated that the cloned PB P-450 cDNA codes for a cytochrome P-450 variant [designated P-450e(U.C.)] that is very similar, but not identical, to P-450e. Sequence analysis of the section of cDNA specifying the 3'-non-coding region of the mRNA revealed that it lacked the usual poly(A) addition site signal sequence but contained three inverted repeat structures. Solution hybridization analysis demonstrated that PB P-450 mRNA is increased 20-fold by phenobarbital treatment and decreased 3-fold by beta-naphthoflavone treatment.

Animals↗