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Biomedical subjects

J A Barnes

Publications and source records attributed to J A Barnes.

50 records · Page 3Linked to original sources
PubMed1987

Characterization of the phospholipid and fatty acid composition of Sendai virus.

The lipid composition of Sendai virus, propagated in chicken eggs, was analyzed by high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and gas-liquid chromatography (GLC). Phosphatidylcholine was found to be the dominant phospholipid (37.3%) with phosphatidylethanolamine (26.8%) and phosphatidylserine (12.0%) also present in significant amounts. Analysis of the fatty acid methyl esters revealed that the dominant fatty acids in total phospholipid were: C16:0 (17.6%), C18:0 (15.4%), C18:1 (n-9) (22.0%), and C24:0 (6.0%). Cardiolipin, phosphatidylserine, and sphingomyelin contained higher levels of saturated fatty acids relative to phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine.

Cardiolipins↗
PubMed1983

Immunochemical studies of the non-specific interactions of cyanogen bromide-activated macroporous agarose-based immunoadsorbents.

This paper reports studies of the origin of the undesirable non-specific adsorption in immunoadsorption chromatography. The non-specificity of cyanogen bromide (CNBr)-activated macroporous agarose (Sepharose 4B)-based immunoadsorbents has been assessed from a comparative study of the following parameters: (1) The non-specific adsorption of protein (IgG) on unsubstituted cyanogen bromide-activated Sepharose 4B. (2) The non-specific adsorption of proteins (human serum IgG and sheep IgG) on cyanogen bromide-activated Sepharose 4B-sheep IgG immunoadsorbent. (3) The non-specific adsorption of proteins, immunoglobulin G(IgG) and human serum albumin (HSA) on cyanogen bromide-activated Sepharose 4B-sheep anti-human IgG immunoadsorbent and (4) the non-specific adsorption of peptides and amino acids on cyanogen-bromide activated Sepharose 4B-sheep anti-human IgG immunoadsorbent.

Amino Acids↗
PubMed1983

The use of alginate ester films on solid supports in the preparation of water-insoluble immunoadsorbents for purification of antigens.

A novel method developed for the preparation of immunoadsorbents is based on the technique of coating potential column supports with a propylene glycol alginate ester film created by emulsion polymerisation. The cross-linking of the film with 1,2-diaminoethane in alkaline solution resulted in a water-permeable but water-insoluble film which was stable at high pH. Sheep anti-(human IgG) was subsequently coupled to the cyanogen-bromide-activated film and the resultant immunoadsorbents were used to purify IgG from whole human serum. The production of the film-coated supports, chemical coupling of antibody and the chromatographic characteristics of four alginate-film-coated supports are described. With the use of Sepharose--alginate-ester--sheep-anti-(human IgG) immunoadsorbent the overall yield for IgG from five serial adsorption-elution cycles was 94% of the adsorbed IgG. This immunoadsorbent proved to be of high binding capacity and allowed the elution of specific antigen in almost 100% yield and high purity with minimal non-specific interference from other serum proteins.

Alginates↗
PubMed1983

Isolation of thyroxine-binding globulin (TBG) by immunoadsorption chromatography: some physical and immunochemical characteristics of TBG.

Thyroxine-binding globulin (TBG) was isolated from pooled whole human serum by diethylaminoethyl (DEAE) Sephadex anion exchange chromatography followed by immunoadsorption chromatography on a cyanogen bromide-activated Sepharose 4B-sheep anti-human TBG immunoadsorbent. Sodium dodecyl sulphate (SDS)-poly-acrylamide gel electrophoresis (PAGE) of the purified TBG revealed a major protein band with a molecular mass of 65 000 and a weak band of molecular mass 54 000 in both reducing and non-reducing buffers. Sedimentation velocity analysis revealed an S20,w coefficient of 4.5S and a calculated molecular mass of 60 000. Immunochemical analysis confirmed the purity of the TBG preparation which gave a single precipitin peak on two-dimensional immunoelectrophoresis.

Amino Acids↗
PubMed1981

Use of F(ab')2 antibody fragments in the synthesis of immunoadsorbents for preparing monospecific antigen.

Class specific F(ab')2 antibody fragments were prepared by pepsin digestion of the labile Fc immunoglobulin fragments in antigen--antibody precipitates. The F(ab')2 fragments were covalently coupled to cyanogen bromide-activated Sepharose 4B and the resultant immunoadsorbent used to isolate IgG from human serum with a single chromatographic step, in high yield and purity and negligible non-specific interaction. This technique affords a simple method for preparing an enriched source of class specific affinity-purified immunoglobulin antibodies suitable for many immunochemical applications.

Antibody Specificity↗
PubMedDate not supplied

PEST sequences in calmodulin-binding proteins.

Many short-lived proteins which are devoid of proteolytic activity contain PEST sequences which are segments along the polypeptide chain that are rich in proline (P), glutamate (E), serine (S) and threonine (T). These designated PEST sequences are believed to be putative intramolecular signals for rapid proteolytic degradation. Calmodulin is a ubiquitous, 17 kDa, acidic Ca(2+)-binding protein which plays an important role in the regulation of many physiological processes through its interaction with a wide range of calmodulin-binding proteins. Several calmodulin-binding proteins are known to contain PEST sequences and are susceptible to proteolysis by endogenous neutral proteases such as calpain I and calpain II. In this report, we discuss the functions of PEST sequences in calmodulin-binding proteins and assess the correlation between calmodulin-binding proteins and PEST sequences.

Amino Acid Sequence↗
PubMedDate not supplied

High molecular weight calmodulin-binding protein is phosphorylated by calmodulin-dependent protein kinase VI from bovine cardiac muscle.

A high molecular weight calmodulin-binding protein (HMW CaMBP) from bovine heart cytosolic fraction was purified to apparent homogeneity. A novel CaM-dependent protein kinase was originally discovered when the total CaM-binding protein fraction from cardiac muscle was loaded on a gel filtration column. The CaM-dependent protein kinase was shown by gel filtration chromatography to have an apparent molecular mass of 36,000 daltons. The CaM-dependent protein kinase has been highly purified by sequential chromatography on DEAE-Sepharose Cl 6B (to remove calmodulin), CaM-Sepharose 4B, phosphocellulose, Sepharose 6B gel filtration and Mono S column chromatographies. The highly purified protein kinase stoichiometrically phosphorylated the HMW CaMBP in a Ca2+/CaM-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the HMW CaMBP. The distinct substrate specificity of this protein kinase indicates that it is not related to the known protein kinases (I, II, III, IV and V) that have been already characterized, therefore we would like to designate this novel kinase as a CaM-dependent protein kinase VI.

Animals↗
PubMedDate not supplied

Simultaneous phosphorylation of three human calpactins by kinase C.

We evaluated the concurrent phosphorylation of reconstituted mixtures of three purified human placental calpactins (or lipocortins) by purified bovine brain protein kinase C (PKC). Calpactin-I (p36 or lipocortin-II), calpactin-II (p38 or lipocortin-I), and a 70-kilodalton calpactin-related protein, calpactin-p70, when present together as substrates for PKC, all demonstrated comparable kinetic parameters (Vmax values = 0.3-0.5 nmol phosphate incorporated/min), with calpactin-II and calpactin-p70 exhibiting lower apparent Km values (40 and 30 nM, respectively) than did calpactin-I (Km, 200 nM). Because of the higher Vmax/Km ratios for calpactin-II and calpactin-70 (12.5 and 10.0, respectively) compared with the ratio for calpactin-I (2.0), our data suggest that, intracellularly, where all three calpactins might be co-localized, the higher molecular mass calpactins could be preferred substrates for PKC. Nonetheless, the requirement for relatively high calcium concentrations (greater than or equal to 0.5 mM) suggests that PKC-mediated phosphorylation of the calpactins may take place only in restricted intracellular compartments, wherein calcium concentrations might transiently reach levels much higher than those that are normally found intracellularly (less than or equal to 0.25 mM).

Annexins↗
PubMedDate not supplied

Insulin dosage reduction. Evaluation of an education-stabilization programme for patients with insulin-dependent diabetes.

A retrospective survey was conducted of 52 consecutive subjects with insulin-treated diabetes who were attending an education-stabilization programme. Eleven subjects (21%) achieved more than a 20% reduction in daily insulin dosage six months after completion of the programme. Compared with the remainder of the group, these subjects had a significantly earlier age of onset, higher initial insulin dosage (mean, 61 units/day, compared with 39 units/day), higher initial insulin carbohydrate and fat consumption, but similar initial glycosylated haemoglobin levels. These results suggest that overinsulinization is a relatively frequent occurrence and should be particularly considered in younger patients who are receiving relatively large doses of insulin.

Age Factors↗