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J A Higgins

Publications and source records attributed to J A Higgins.

At least 19 recordsLinked to original sources
PubMed1992

Determination of the intracellular distribution and pool sizes of apolipoprotein B in rabbit liver.

We have investigated the intracellular distribution of apolipoprotein B (apo B) in rabbit liver by immunoblotting, radioimmunoassay (r.i.a.) and enzyme-linked immunoassay (e.l.i.s.a.). Apo B100 was detected in total microsomes, rough microsomes, smooth microsomes, trans-enriched Golgi and cis-enriched Golgi and membrane and cisternal-content subfractions prepared from these fractions. There was also evidence of degradation of apo B100 in the Golgi membrane fractions. The amount of apo B in the subcellular fractions detected by competitive r.i.a. or e.l.i.s.a. ranged from 1.5 micrograms/mg of protein in the rough endoplasmic reticulum to 13 micrograms/mg of protein in the trans-Golgi fraction. Using internal standards (NADPH-cytochrome c reductase for the endoplasmic reticulum and galactosyltransferase for the Golgi membranes) it was calculated that all the apo B of liver is recovered within the secretory compartment, with 63% of the total apo B in the endoplasmic reticulum and the remainder in the Golgi. When the subcellular fractions were separated into membranes and cisternal contents, 60%, 50%, 60% and 30% of the total apo B was recovered in the membrane of the rough microsomes, smooth microsomes, cis-Golgi and trans-Golgi respectively. Using competitive e.l.i.s.a. we found that the membrane-bound form of the apo B was exposed at the cytosolic surface of the intact subcellular fractions. These observations are consistent with a model for assembly of very-low-density lipoproteins (VLDL) in which newly synthesized apo B is incorporated into a membrane-bound pool and a lumenal pool. The membrane-bound pool not used for VLDL assembly may be degraded, possibly in the Golgi region.

Animals↗
PubMed1992

Quantification of apolipoprotein B-48 and B-100 in rat liver endoplasmic reticulum and Golgi fractions.

We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound.

Animals↗
PubMed1992

Membrane-bound apolipoprotein B is exposed at the cytosolic surface of liver microsomes.

We have used a competitive enzyme-linked immunoassay with a panel of monoclonal antibodies to probe the topography of the membrane-bound form of apolipoprotein B (apo B) in rabbit microsomes. All epitopes investigated were found to be expressed at the cytosolic side of the microsomal membrane under conditions in which the vesicles remained sealed. These results indicate that the membrane-associated form of apolipoprotein B is either at the cytosolic side of the endoplasmic reticulum membrane or integrated into the membrane. From this site apo B may be translocated to the lumen for assembly into VLDL or may be degraded.

Animals↗
PubMed1992

Peptide-induced nonresponsiveness of HLA-DP restricted human T cells reactive with Dermatophagoides spp. (house dust mite).

The activation of CD4+ T lymphocytes, which play a central role in allergic inflammation, depends on the recognition of allergen-derived peptides in association with major histocompatibility complex class II gene products. In this report we demonstrate, at a clonal level, that a component of the T-cell repertoire reactive with Dermatophagoides spp. (house dust mite) in atopic individuals, is restricted by HLA-DP class II molecules. This supports the recent results emerging from genetic epidemiologic studies that indicate positive associations between the HLA-DP phenotype and immune responsiveness to a variety of common allergens. Our findings also reveal that the T cells restricted by HLA-DP recognize a species-specific epitope located in the group I allergen of Dermatophagoides pteronyssinus (residues 101-119). Furthermore, we report that the pretreatment of the T cells restricted by HLA-DP with the Der p I peptide renders them nonresponsive to an immunogenic challenge with house dust mite allergen, and the loss of antigen-dependent proliferation is associated with downregulation of membrane expression of the T-cell antigen receptor. The ability to functionally inactivate T cells restricted by HLA-DP, as well as those that recognize allergen in association with HLA-DR class II molecules, suggests that desensitization with allergen-derived peptides may have therapeutic potential in the management of allergic diseases irrespective of their HLA class II association.

Animals↗
PubMed1992

Effect of beta-agonists on expression of calpain and calpastatin activity in skeletal muscle.

Administration of beta-adrenergic agonists to domestic species can lead to skeletal muscle hypertrophy, probably by reducing the rate of myofibrillar protein breakdown. Myofibrillar breakdown is associated with the calcium-dependent proteinase system (calpains I,II and calpastatin) whose activity also changes during beta-agonist treatment. A number of growth trials using the agonists cimaterol and clenbuterol with cattle, sheep, chicken and rat are reported which suggest a general mechanism whereby beta-agonists reduce calpain I activity, but increase calpain II and calpastatin activity in skeletal muscle. Parallel changes in specific mRNAs indicate that changes in gene expression or stabilisation of mRNA could in part explain the changes in activity.

Adrenergic beta-Agonists↗
PubMed1991

Protective antibodies to hepatitis B virus in haemophiliacs.

Haemophilic patients are at increased risk from hepatitis B virus infection because of their need for blood product therapy. They are potentially poor responders to hepatitis B vaccine due to immunological abnormalities resulting from two causes: infection with the human immunodeficiency virus and treatment with clotting factor concentrates. The protective antibody response to hepatitis B virus in vaccinated haemophiliacs was investigated using a competitive enzyme-linked immunosorbent assay which employs a monoclonal antibody, RF-HBs-1, that recognises a virus-neutralising epitope on HBsAg. Serum samples from 55 haemophilic patients were studied at 7, 12, and 24 months after the first injection with HB vaccine. Twenty-four vaccinated normal subjects were used as controls. The level of neutralising antibody was found to correlate with the polyclonal anti-HBs response in the majority of subjects in both the control and patient groups. There was a small but statistically significant reduction in both antibody responses in the patients compared with the normal controls. Treatment with FVIII or FIX concentrate did not influence the antibody response in the patients. Eleven of the haemophilic patients were anti-HIV seropositive. This group had a significantly lower antibody response than anti-HIV negative patients, and this correlated with the duration of anti-HIV seropositivity, rather than with their T4 counts. We conclude that, following vaccination, the majority of haemophiliacs are able to mount a protective antibody response to hepatitis B virus. HIV infection was found to be the sole cause of immunological suppression of this response.

Adolescent↗
PubMed1989

Phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis as a probe for the distribution of phosphatidylinositol in hepatocyte membranes.

Phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Bacillus thuringiensis has been used as a probe for the distribution of phosphatidylinositol in hepatocyte membranes. Approx. 50% of this phospholipid was hydrolysed in microsomal vesicles (endoplasmic reticulum) with no significant hydrolysis of the remaining membrane phospholipids. Latency of mannose-6-phosphatase was retained during treatment indicating that the vesicles remained impermeable. Stripping of the ribosomes did not increase hydrolysis of phosphatidylinositol; however, when the vesicles were opened using dilute sodium carbonate, hydrolysis increased to greater than 90%. Hydrolysis of phosphatidylinositol of Golgi membranes was 35% and of plasma membranes was 50%. After treatment with PI-PLC, radiolabelled secretory proteins were retained in Golgi membranes and trapped lactate dehydrogenase was retained in plasma-membrane preparations indicating that the vesicles remained closed. Hydrolysis of phosphatidylinositol increased to greater than 90% when the membranes were opened by treatment with dilute sodium carbonate. These observations indicate that PI-PLC of Bacillus thuringiensis is a suitable probe for the distribution of phosphatidylinositol in membranes, and that in liver membranes this phospholipid occurs on each side of the bilayer, a topography consistent with its diverse roles.

Animals↗
PubMed1988

Evidence that during very low density lipoprotein assembly in rat hepatocytes most of the triacylglycerol and phospholipid are packaged with apolipoprotein B in the Golgi complex.

Rat liver lipids were labelled by an intraportal injection of [3H]palmitic acid followed by isolation of rough and smooth endoplasmic reticulum or 'cis' or 'trans'-enriched Golgi fractions. The preparations were separated into membrane and contents and the apolipoprotein B of the content fractions was immunoprecipitated. More than 90% of the labelled triacylglycerol and phospholipid secreted into the blood immunoprecipitated with apolipoprotein B. Under the same experimental conditions 8, 12, 27 and 59% of the lipids of the rough, smooth, 'cis-Golgi' and 'trans-Golgi' contents, respectively, were immunoprecipitated. Thus, the 'trans-Golgi' region appears to be the major intracellular site of assembly of apolipoprotein B with triacylglycerol and phospholipid.

Animals↗
PubMed1988

The relation between dietary restriction or clenbuterol (a selective beta 2 agonist) treatment on muscle growth and calpain proteinase (EC 3.4.22.17) and calpastatin activities in lambs.

1. Lamb growth trials were designed to modify growth and protein content of muscle by diet and also by beta-agonist treatment, and to correlate any changes to the activities of calpain proteinases (EC 3.4.22.17) and their inhibitor calpastatin. 2. Wether lambs in a control group were fed on a barley-based diet designed to give a growth rate of 350 g/d; a second group was fed on the same diet but the intake was restricted to give an expected gain of 44 g/d; a third group was fed on the same diet as the first group but the diet included 2 mg clenbuterol/kg. At the end of a 6-week trial, longissimus dorsi wet weights were 635 (n6), 377 (n4) and 788 g (n6) (standard error of difference 53.0) in the three groups respectively. 3. Minced L. dorsi was extracted in low-salt buffers and analysed by a fast protein liquid-chromatographic system for calpain I (low calcium-requiring), calpain II (high Ca2+-requiring) and calpastatin activities. No significant changes in the three activities were associated with reduced muscle weight in the restricted-intake group. The inclusion of clenbuterol in the diet, however, led to highly significant increases (P less than 0.001) in calpain II and calpastatin to approximately double the control values. 4. The results did not support a direct relation between these activities and muscle growth, except when protein accretion was stimulated by a beta-agonist, suggesting a role for this enzyme system in the mechanism by which these agents exert their effect.

Animals↗
PubMed1988

Factors influencing the ingestion of Onchocerca cervicalis microfilariae by Culicoides variipennis (Diptera: Ceratopogonidae).

Culicoides variipennis were fed under controlled conditions on two ponies that exhibited seasonal changes in Onchocerca cervicalis microfilarial (mf) skin density and skin distribution. The seasonal changes did not radically affect mf ingestion. Flies were fed on the umbilicus of infected ponies by two methods: individual feeding in consecutive order at the same site, or by mass feeding. Linear regression analysis indicated that ingestion of microfilaria was independent of feeding time and engorged weight. In the individual feeding data, there was a trend toward an increased ingestion of mf as the feeding time increased. Mass feeding trials suggest early feedings by C. variipennis may influence mf ingestion rates of flies that feed on subsequent days. Of the 1,104 flies feeding on both ponies over a 2-year period, 220 (20%) ingested mf and 99 (9% of all engorged flies) ingested only one mf.

Animals↗
PubMed1987

Reversible activation/inactivation of the deacylation/acylation cycle in rat liver microsomes.

Rat liver microsomes incorporate [14C]palmitoyl CoA into membrane phospholipids via the deacylation/acylation cycle. This activity is reversibly inactivated/activated by treatment of the microsomes with ATP, MgCl2, and 105,000 g supernatant or with 105,000 g supernatant alone. These observations suggest that the acylation cycle is controlled by a mechanism involving phosphorylation/dephosphorylation. As the pool of lysolecithin in the membranes is not altered by conditions increasing incorporation of palmitoyl CoA into phospholipid, it is probable that the site of regulation of deacylation/acylation is at the acyltransferase rather than the phospholipase.

Acylation↗
PubMed1987

Phosphatidylcholine synthesis for incorporation into membranes or for secretion as plasma lipoproteins by Golgi membranes of rat liver.

Phosphatidylcholine, the major phospholipid of very low density lipoproteins, is packaged with triglyceride in the Golgi cisternae. CTP-phosphocholine cytidyltransferase and CDP-choline phosphotransferase activities of Golgi subfractions were higher than those of rough or smooth microsomes measured under the same conditions, indicating that phosphatidylcholine synthesis can occur in Golgi membranes. Consistent with this, the specific activity of phosphatidylcholine of Golgi membranes rose more rapidly than that of rough and smooth microsomes after injection of [14C]choline in vivo. The specific activity of the Golgi content phosphatidylcholine (non-membrane fraction) remained low. The S-adenosylmethionine phosphatidylethanolamine methyltransferase activity of Golgi subfractions was also higher than that of rough or smooth microsomes. After injection of [3H]methyl-labeled methionine in vivo, the specific activity of phosphatidylcholine of the Golgi membranes rose in parallel with that of the rough and smooth microsomes. The specific activity of the Golgi content phosphatidylcholine rose above that of the Golgi membranes and exhibited a different pattern, suggesting that this pathway may selectively label phosphatidylcholine which is secreted as lipoproteins. These observations indicate that the Golgi membranes have the enzymes necessary for synthesis of phosphatidylcholine, and incorporation of lipid precursors indicates that synthesis of phosphatidylcholine by Golgi membranes occurs in vivo.

Animals↗
PubMed1986

Linear gastric erosion. A lesion associated with large diaphragmatic hernia and chronic blood loss anemia.

A prospective study was undertaken to identify mucosal lesions that might cause chronic blood loss anemia in patients with large diaphragmatic hernia. Patients with one-third or more of the stomach above the diaphragm on barium x-ray were examined by a gastroscopist who was given no clinical information. A total of 109 patients were included: 55 with anemia and 54 with a large hernia but no anemia. The incidence of esophagitis and peptic ulcer did not differ significantly in the anemic and nonanemic groups. Linear gastric erosions were found on the crests of mucosal folds at or near the level of the diaphragm in 23 anemic patients and 13 without anemia (p less than 0.05). Blood on the surface of a linear erosion was found in 14 anemic patients and 4 without anemia (p less than 0.05). We suggest that these erosions are due to trauma and can cause chronic blood loss anemia in hernia patients.

Aged↗
PubMed1985

Asymmetric synthesis and transmembrane movement of phosphatidylethanolamine synthesised by base-exchange in rat liver endoplasmic reticulum.

Using trinitrobenzenesulphonic acid (TNBS) as a probe we have observed that phosphatidylethanolamine (PE) formed by base-exchange is initially concentrated in the cytosolic leaflet of the membrane bilayer. At 2 min, the specific activity of the PE in this leaflet was 3-times that of the PE in the cisternal leaflet. After 30 min, the specific activities of the two pools of PE, determined with either phospholipase C or TNBS, were similar. Transbilayer movement of PE was slow at low temperature, prevented by EDTA and restored by the addition of calcium ions after EDTA treatment. Trypsin treatment of microsomes, under conditions in which the vesicles remained closed, inhibited the incorporation of ethanolamine into PE by 87%. The cytosolic location of the ethanolamine base-exchange enzyme is consistent with the initial concentration of newly synthesised PE at this site prior to its transmembrane movement to the cisternal leaflet.

Animals↗
PubMed1985

Microsomal membranes contain phosphatidylcholine that equilibrates across the bilayer, and phosphatidylcholine that does not.

Results of experiments using phosphatidylcholine transfer protein and phospholipase C as probes indicate that there are at least two pools of phosphatidylcholine in rat liver microsomes. One of these is preferentially labelled with [14C]choline and does not equilibrate across the bilayer. The second pool is labelled with [3H]glycerol and does equilibrate across the bilayer. Our observations also confirm that phosphatidylcholine exchange protein does not modify the distribution of phospholipids or cause randomization of the inner and outer leaflet pools of phosphatidylcholine when these are differentially labelled by [14C]choline.

Androgen-Binding Protein↗