Search PubMed⌕ Search

Biomedical subjects

J A Higgins

Publications and source records attributed to J A Higgins.

At least 37 records · Page 2Linked to original sources
PubMed1999

Dietary fish oils inhibit early events in the assembly of very low density lipoproteins and target apoB for degradation within the rough endoplasmic reticulum of hamster hepatocytes.

Dietary fish oils inhibited secretion and stimulated intracellular degradation of apolipoprotein (apo)B in hamster hepatocytes, while dietary sunflower oils stimulated secretion and had no effect on degradation of apoB. To investigate the intracellular site at which fish oils act, we have made use of our previous observations that inhibition of degradation by N-acetyl-leucyl-leucyl-norleucinal (ALLN) results in accumulation of apoB in the trans -Golgi membrane and does not stimulate secretion, while inhibition of degradation by o-phenanthroline results in accumulation of apoB in the rough endoplasmic reticulum membrane and stimulates secretion. Thus, ALLN protects apoB which has been diverted from secretion and o -phenanthroline protects apoB which is targetted for secretion. Addition of o -phenantholine to the incubation medium of hepatocytes from fish oil-fed hamsters inhibited degradation of apoB and stimulated its secretion in particles of the density of VLDL, while addition of ALLN had no effect. These observations suggest that dietary fish oils reversibly inhibit early steps in the assembly of very low density lipoprotein precursors and target apoB for degradation in the rough endoplasmic reticulum.

Animals↗
PubMed1999

Isolated rabbit enterocytes as a model cell system for investigations of chylomicron assembly and secretion.

A method is described for the isolation of viable enterocytes from rabbit small intestine. The procedure can also be used to isolate populations of epithelial cells from the crypt/villus gradient. The isolated enterocytes synthesized and secreted apoB-48 and triacylglycerol in particles of the density of chylomicrons. Secretion was stimulated by addition of bile salt/lipid micelles. Pulse-chase experiments demonstrated that newly synthesized apoB-48 is degraded intracellularly and that degradation is inhibited by provision of lipid micelles, suggesting that regulation of chylomicron assembly and secretion is broadly similar to that of very low density lipoprotein assembly in hepatocytes. This procedure for preparation of isolated enterocytes will provide a useful model system for investigation of the molecular details of chylomicron assembly.

Animals↗
PubMed1999

Increased dietary triacylglycerol markedly enhances the ability of isolated rabbit enterocytes to secrete chylomicrons: an effect related to dietary fatty acid composition.

Dietary fats are efficiently absorbed in the small intestine and transported into the blood via the lymph as chylomicrons, despite enormous variations in the amount and composition of the dietary lipid. The aim of the present study was to investigate how enterocytes respond to increased dietary fats of different composition. Rabbits were fed a low fat chow diet, and chow supplemented with sunflower oil (high n-6 polyunsaturated fatty acids), fish oil (high n-3 polyunsaturated fatty acids), or an oil mixture of a composition similar to that of the typical western diet. Feeding fat for 2 weeks markedly stimulated the ability of the isolated enterocytes to synthesize and secrete apolipoprotein B48, triacylglycerol, and cholesteryl ester (up to 18-, 50-, and 80-fold, respectively) in particles of chylomicron density. The magnitude of stimulation was sunflower oil > western diet lipid > fish oil. Single doses of lipid given 18 h prior to isolation of enterocytes stimulated chylomicron secretion by only 10% of that observed after 2 weeks of dietary supplementation. Enterocytes are replaced rapidly (half-life 1-2 days) by cells which move from the crypts to the tips of the villi, where absorption of nutrients takes place. Our observations suggest that dietary lipids modulate the function of enterocytes as they move from the crypts, so that the cells are 'turned-on' to lipid absorption. The results also show that diets of different fatty acid composition vary in their effects.

Animals↗
PubMed1998

Janus carcinogens and mutagens.

Janus carcinogens are carcinogenic agents that, under differing conditions of cell type or dose, can instead act as anticarcinogens. Studies by Haseman and Johnson [J.K. Haseman, F.M. Johnson, Analysis of rodent NTP bioassay data for anticarcinogenic effects, Mutat. Res. , 350 (1996) 131-142], have demonstrated that many chemicals that are carcinogenic for one tissue type can have anticarcinogenic action on another tissue type. As Magni et al. [G.E. Magni, R.C. von Borstel, S. Sora, Mutagenic action during meiosis and antimutagenic action during mitosis by 5-aminoacridine in yeast, Mutat. Res., 1 (1964) 227-230] have shown in 1964, this principle holds true for chemical mutagens as well, that is 9-aminoacridine is an antimutagen in the vegetative cell and a mutagen in the sporulating cell. The conclusion can be drawn that two established carcinogens, tobacco and ionizing radiation, are indeed Janus carcinogens. In their review of 'ambiguous carcinogens' (their name), Weinberg and Storer [A.M. Weinberg, J.B. Storer, Ambiguous carcinogens and their regulation, Risk Anal., 5 (1985) 151-156], pointed out that tobacco can be classified as an ambiguous carcinogen. The strong carcinogenicity and anticarcinogenicity of tobacco smoke and/or tobacco itself (i.e., chewing tobacco) may be due to components in the mixture, not that of a single carcinogenic chemical that also may be anticarcinogenic. Kondo [S. Kondo, Health Effects of Low-Level Radiation, Kinki Univ. Press, Osaka, Japan and Medical Physics Publishing, Madison, WI, 1995, 213 pp.] has compiled data that demonstrate that human populations who survive exposures to ionizing radiation generally live longer and have less cancer than unirradiated human populations, and this Janus phenomenon goes beyond the more trivial concept of increased sensitivity to radiation of rapidly dividing tumor cells. Thiabendazole is an interesting compound in that it is both aneugenic and antimutagenic, and yet it does not appear to be a carcinogen or a mutagen. It is discussed here because aneugenesis and antimutagenesis are at extremes of the mutagenic spectrum. In general, mutagenic or carcinogenic actions usually are at least partially understood at a molecular level, whereas antimutagenic and anticarcinogenic actions usually are not. It is possible there may be numerous specific mechanisms underlying the Janus activity of different chemicals.

Animals↗
PubMed1998

Topical reversion at the HIS1 locus of Saccharomyces cerevisiae. A tale of three mutants.

Mutants of the HIS1 locus of the yeast Saccharomyces cerevisiae are suitable reporters for spontaneous reversion events because most reversions are topical, that is, within the locus itself. Thirteen mutations of his1-1 now have been identified with respect to base sequence. Revertants of three mutants and their spontaneous reversion rates are presented: (1) a chain termination mutation (his1-208, née his1-1) that does not revert by mutations of tRNA loci and reverts only by intracodonic suppression; (2) a missense mutation (his1-798, née his1-7) that can revert by intragenic suppression by base substitutions of any sort, including a back mutation as well as one three-base deletion; and (3) a -1 frameshift mutation (his1-434, née his1-19) that only reverts topically by +1 back mutation, +1 intragenic suppression, or a -2 deletion. Often the +1 insertion is accompanied by base substitution events at one or both ends of a run of A's. Missense suppressors of his1-798 are either feeders or nonfeeders, and at four different locations within the locus, a single base substitution encoding an amino acid alteration will suffice to turn the nonfeeder phenotype into a feeder phenotype. Late-appearing revertants of his1-798 were found to be slowly growing leaky mutants rather than a manifestation of adaptive mutagenesis. Spontaneous revertants of his1-208 and his1-434 produced no late-arising colonies.

Adenine↗
PubMed1998

Molecular identification of Rickettsia typhi and R. felis in co-infected Ctenocephalides felis (Siphonaptera: Pulicidae).

Rickettsia typhi and R. felis, 2 closely related rickettsial species, often have been identified in cat fleas, Ctenocephalides felis (Bouché) from the same geographical location. However, no fleas have been found to be naturally infected concurrently with both rickettsial species. To examine whether one rickettsial species can develop simultaneously with another species in the same flea host, cat fleas, naturally infected with R. felis, were allowed to feed on blood containing R. typhi (Ethiopian strain, 10(7) PFU/ml). Experimental controls consisted of uninfected cat fleas, fleas infected with only R. typhi; and fleas naturally infected with only R. felis. After 9 d at 28 degrees C, the fleas were examined by PCR amplification and subsequent restriction digest analysis and dot blot hybridization of PCR products. Results from these studies demonstrated that R. felis and R. typhi are capable of co-existing in the same flea host. Subsequent isolation of R. typhi from dually infected fleas by tissue culture indicated that R. typhi was viable and capable of being maintained in fleas naturally infected with R. felis. As more studies confirm the presence of R. felis and R. typhi in domestic pets and peridomestic vertebrates in urban areas, the ability of the individual cat fleas, which live on these animals, to support both rickettsial species could be an epidemiologically important consideration.

Animals↗
PubMed1998

5' nuclease PCR assay to detect Yersinia pestis.

The 5' nuclease PCR assay uses a fluorescently labeled oligonucleotide probe (TaqMan) to rapidly detect and quantitate DNA templates in clinical samples. We developed a 5' nuclease PCR assay targeting the plasminogen activator gene (pla) of Yersinia pestis. The assay is species specific, with a detection threshold of 2.1 x 10(5) copies of the pla target or 1.6 pg of total cell DNA. The assay detected Y. pestis in experimentally infected Xenopsylla cheopis fleas and in experimentally infected monkey blood and oropharyngeal swabs. The TaqMan assay is simple to perform and rapid and shows promise as a future field-adaptable technique.

Animals↗
PubMed1998

Dietary fish oils modify the assembly of VLDL and expression of the LDL receptor in rabbit liver.

Supplementation of the diet of rabbits with fish oil or sunflower oil resulted in significant changes in the lipoproteins and lipids in serum. Compared with chow-fed rabbits, dietary fish oils decreased very low density lipoprotein (VLDL), increased low density lipoprotein (LDL), and shifted the peak of the LDL to denser fractions, whereas sunflower oil increased high density lipoprotein and shifted LDL to the lighter fractions. The amount of LDL receptors in fish oil-fed rabbit liver decreased by > 70% while there was only a small fall in these levels in sunflower oil-fed rabbit liver. The concentrations of apolipoprotein (apo) B in the subcellular organelles of the secretory compartment (rough and smooth endoplasmic reticula and Golgi fractions) were also changed by dietary lipids. In both sunflower oil- and fish oil-fed liver, apo B was increased in the lumen of the rough endoplasmic reticulum compared with fractions from chow-fed rabbit liver. The apo B in the trans-Golgi lumen from fish oil-fed livers was reduced and occurred in particles of d approximately 1.21 g/mL. In contrast, apo B in the trans-Golgi lumen from livers of sunflower oil-fed rabbits was increased and occurred in particles of d < 1.21 g/mL. These results suggests that feeding of fish oils causes an interruption in the intracellular transfer of apo B and hence assembly of VLDL. This leads to an enrichment of the rough endoplasmic reticulum membranes with cholesterol, thus downregulating the expression of the LDL receptor.

Animals↗
PubMed1997

Investigation of the role of lipids in the assembly of very low density lipoproteins in rabbit hepatocytes.

Our aims were i) to determine which lipids colocalize with newly synthesized apolipoprotein (apo) B in the lumen of the rough endoplasmic reticulum (RER), and thus may play a role in the stabilization and/or translocation of this protein; and ii) to determine the intracellular sites of assembly of lipids into very low density lipoprotein (VLDL). In order to do this, we have developed a new method for the separation of ER-derived microsomes on self-generated gradients of iodixanol. Rabbit liver microsomes were resolved into two broad peaks, the lighter peak contained smooth vesicles and the heavier peak contained rough vesicles. Each peak was collected in a number of subfractions. A single gradient thus separates the initial events in the secretion process (RER fractions), from later events (smooth endoplasmic reticulum (SER) fractions). The microsomal fractions were separated into membranes and lumenal contents, and the mass of apoB and VLDL lipids determined by ELISA or high performance thin-layer chromatography, respectively. The biosynthetic relationships of apoB and lipids were investigated, in timed or chase experiments, by incubation of isolated rabbit hepatocytes with radiolabeled precursors of apoB or lipids, followed by isolation and analysis of the microsomal fractions. The results indicate that very small amounts of triacylglycerol, cholesterol, and cholesteryl ester co-localize with apoB into the lumen of the RER. The bulk of the VLDL lipids were in the lumen of the SER. However, some newly synthesized triacyl-glycerol, phospholipid, cholesterol, and cholesteryl ester were also transferred to the lumen of the RER and were chased into the SER lumen. Double-labeling experiments showed that cholesteryl ester produced from newly synthesized cholesterol (labeled with [3H]mevalonate and [14C]oleate) was almost exclusively present in the RER, while cholesteryl ester in the SER was labeled only with [14C]oleate. Thus, distinct intracellular lipid-pools may be involved at different stages in the assembly of VLDL.

Acyltransferases↗
PubMed1996

Intracellular degradation in the regulation of secretion of apolipoprotein B-100 by rabbit hepatocytes.

Isolated rabbit hepatocytes were incubated with [35S]methionine to label intracellular pools of apolipoprotein B (apo-B). The cells were then reincubated with an excess of unlabelled methionine in the presence of oleate or protease inhibitors and the intracellular sites of accumulation of radiolabelled apo-B and the mass of apo-B were determined by isolation and analysis of subcellular fractions. Oleate or inhibitors of metalloproteases (o-phenanthroline), serine proteases (aprotinin), serine/cysteine proteases (leupeptin) or cysteins proteases (calpain inhibitor I; ALLN) but not aspartate proteases (pepstatin) resulted in inhibition of the cellular degradation of apo-B. The effect of o-phenanthroline was reversed by the addition of zinc ions. Oleate, o-phenanthroline and leupeptin also stimulated secretion of radiolabelled apo-B; the effects of the inhibitors and oleate were additive, suggesting that they could act via different mechanisms. o-Phenanthroline caused accumulation of apo-B in the rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) membranes; leupeptin caused accumulation of apo-B in the SER and cis-Golgi membranes, and ALLN and aprotinin caused accumulation of apo-B in the trans-Golgi membranes. These results suggest that intracellular degradation of apo-B occurs in the endoplasmic reticulum and in the trans-Golgi membranes and involves different proteases. Apo-B that accumulates in the ER membrane can be diverted into the lumen for secretion; however, apo-B that accumulates in the trans-Golgi membrane is irretrievably diverted from secretion.

Animals↗
PubMed1996

A novel method for the rapid separation of plasma lipoproteins using self-generating gradients of iodixanol.

We describe a new method for the rapid fractionation of plasma lipoproteins, which makes use of a new non-ionic, iodinated, density gradient medium, iodixanol, commercially available as Optiprep(TM). The method is simple: plasma or serum is mixed with iodixanol followed by centrifugation in a vertical or near vertical rotor. Separation of VLDL, LDL and HDL can be achieved in 3 h and the lipoprotein fractions are comparable in density and composition with those prepared using conventional salt based gradients. Each class of lipoprotein can be removed in a single fraction, or a profile of lipoprotein distribution can be obtained using a gradient fractionator. Because the medium is inert, fractions from the gradient can be analysed by agarose gel electrophoresis or assayed for lipid content or apolipoprotein composition by SDS-PAGE without removing the iodixanol. Small differences in electrophoretic mobility of HDL and LDL across several gradient fractions suggest that subfractionation of these classes may occur. The new method is simple, rapid and versatile with potential application for preparation of lipoproteins and for analysis of lipoprotein profiles in the research or clinical laboratory.

Apolipoproteins↗
PubMed1996

Incorporation of 2-deoxy-D-glucose into glycogen. Implications for measurement of tissue-specific glucose uptake and utilisation.

Estimation of glucose uptake in vivo using 2-deoxy-D-[2,6-3H]glucose (2DG) relies upon the assumption that the phosphorylated form, 2-deoxy-D-2,6-3H]glucose 6-phosphate (2DGP), cannot be further metabolised. We aimed to determine whether this assumption leads to underestimation of glucose uptake due to the incorporation of 2DGP into glycogen. Rats were infused with [U-14C]glucose and 2-[3H]DG, and the incorporation into glycogen was measured. These were compared to the accumulation of 2-[3H]DGP in heart, liver, muscle, white adipose tissue and brown adipose tissue. 2DG was incorporated into glycogen in an insulin-dependent manner (e.g. in soleus, at basal, physiological and supraphysiological insulin concentrations, glycogen synthesis rates from 2DG were 17.81 +/- 3.07, 64.47 +/- 7.47 and 203.23 +/- 44.52 nmol glycogen incorporated/g min-1, respectively). The rate of glycogen synthesis from 2-[3H]DG was identical to that for [U-14C]glucose in all tissues studied except for heart and brown adipose tissue (e.g. in soleus at physiological insulin concentration, 2-[3H]DG incorporation was 64.47 +/- 7.47 and [U-14C]glucose incorporation was 61.87 +/- 7.56 nmol glucose/g min-1). Furthermore, the proportion of 2DG incorporated into glycogen was significant with respect to total glucose uptake at all plasma insulin concentrations (10.7% +/- 0.9, 14.0 +/- 1.9 and 25.6% +/- 5.6 at basal, physiological and supraphysiological insulin concentrations, respectively). 2DG was metabolised to glycogen in all tissues studied causing an underestimation of the rate of glucose uptake by measurement of 2DGP accumulation alone. In addition, use of 2DG could provide a method for assessing the rate of direct glycogen synthesis in the rat.

Adipose Tissue↗
PubMed1996

Presence of calreticulin in vector fleas (Siphonaptera).

Calreticulin has been defined in the cat flea, Ctenophalides felis (Bouché), and oriental rat flea, Xenopsylla cheopis (Rothschild). Calreticulin, a major endoplasmic reticulum protein, was previously identified as a component of ixodid tick saliva. Using a riboprobe generated from tick calreticulin complementary DNA (cDNA), we distinguished 2 transcripts for calreticulin in cat fleas by Northern blot analysis. Increased expression of calreticulin was not evident in fed versus unfed adult fleas. We were able to amplify a calreticulin flea product from fed female messenger RNa (mRNA) using primers designed from the tick calreticulin gene. One of these products hybridized to the tick riboprobe. Localization of specific antibody to cat flea tissues showed calreticulin in the midgut with no detection in the salivary glands. We also observed specific labeling of calreticulin with antibody in the ovaries of fed females. Several cat flea polypeptides appear to crossreact with anticalreticulin antibody in Western blots. We did not detect a calreticulin using antibody to the tick-secreted protein in cat flea salivary glands. This antibody did recognize a protein in the rate flea salivary glands. Our results show that fleas have calreticulin and, possibly, several isoforms. It appears that the salivary glands of the cat and oriental rat flea differ in detectable levels of calreticulin. The specific antibody labeling of the ovaries is interesting and remains to be understood. Calreticulin's appearance in the midgut suggests a possible source of calreticulin as a flea secretion. Further studies are in progress to complete the sequencing of the flea polymerase chain reaction (PCR) product to compare to tick-secreted calreticulin. Comparisons to other blood-feeding arthropods at the protein and gene level are also being done. We hope to define further the expression of calreticulin in fleas, and in general, blood-feeding arthropods, with respect to its role in feeding and pathogen transmission.

Animals↗