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Biomedical subjects

W H Evans

Publications and source records attributed to W H Evans.

At least 19 recordsLinked to original sources
PubMed1992

Predictive relationships for sidestream smoke cigarette yields.

Relationships have been sought for smoking machine generated sidestream smoke yields of cigarettes for particulate matter (water and nicotine free) (PMWNF) and nicotine. Comparisons have been made with the corresponding mainstream smoke yields, other readily measurable factors in machine smoking and with the physical characteristics of cigarettes. The sidestream smoke yields have been found to be related to the puff count during machine smoking multiplied by the cross-sectional area of a cigarette or to the total weight of cigarette tobacco burnt during machine smoking. For the brands retailed in the U.K. these relationships permit calculation of the sidestream smoke contributions of PMWNF and nicotine to environmental tobacco smoke. From the data used to derive these relationships, and based on 31 cigarette brands representing 71% of cigarette sales in the U.K., the sales weighted sidestream PMWNF yield is 27.1 mg/cigarette and that for nicotine is 5.12 mg/cigarette.

Humans↗
PubMed1992

Differential expression of asialoglycoprotein receptor subunits in the endocytic compartment during liver regeneration.

Asialoglycoprotein receptors, responsible for the removal of circulating asialoglycoproteins by the liver, are located in at least two different membrane locations in hepatocytes. Receptors on the cell surface account only for a minor proportion (20-36%), for the majority of receptors in the liver are located intracellularly, mainly in the endocytic membrane networks. An understanding of the basis of receptor distribution and the underlying trafficking of receptors between the hepatocyte's polarised cell surface and the endocytic compartment would be aided if biochemical differences between the receptors in these pools were established. We now show, using three antibodies that recognise the receptor subunits in rat liver (RHL-1, RHL-2 and RHL-3), that the asialoglycoprotein receptors located in the plasma membrane domains and the endocytic compartment differ in oligomeric composition, sialic acid content, and solubility in Triton X-114 using two-phase systems. It is well established that the expression of the asialoglycoprotein receptor is down-regulated in livers regenerating after a partial hepatectomy. We demonstrate that the levels of the receptor subtype that is located mainly in the endocytic compartment (RHL-1, 42 kDa) was elevated in regenerating liver by agents that regulate cAMP production, whereas the levels of the other receptor subtypes remained unchanged. The asialoglycoprotein receptor subtypes that are present in different subcellular locations are thus regulated independently.

Animals↗
PubMed1992

Reorganization of the endocytic compartment in regenerating liver.

Antibodies raised to two membrane proteins present in rat liver endosomal fractions were used to study changes occurring in the endocytic compartment of hepatocytes during liver regeneration. Antibodies to the 42-kDa subunit (RHL-1) of the asialoglycoprotein receptor showed, by Western blotting of liver microsomes and endosomes, that there was a reduced expression of the receptor in liver 24 h following a partial hepatectomy. Immunocytochemical staining of thin sections of regenerating livers using these antibodies indicated that there was an intracellular relocation of endocytic structures in hepatocytes. The two main endocytic regions immunocytochemically stained in normal liver--one located beneath the sinusoidal plasma membrane and the other abutting the bile canaliculus--were replaced, in regenerating liver, by staining more closely associated with a region underlying the baso-lateral plasma membrane. A 140-kDa pI 4.3 calmodulin-binding protein located in endocytic and plasma membranes was also demonstrated, using a radio-iodinated calmodulin-binding assay, to be present at reduced levels in endosomes isolated from regenerating livers. Antibodies to this calmodulin-binding protein stained the hepatocyte's cytoplasm in a punctate manner. However, in regenerating liver, the staining was located in regions underlying the baso-lateral and apical plasma membrane of hepatocytes. Together, the results demonstrate that a reorganization of the endocytic compartment has occurred in hepatocytes 24 h following hepatectomy, with two endosomal proteins becoming relocated to a region below the baso-lateral-apical surface regions of hepatocytes.

Animals↗
PubMed1991

Topography of connexin32 in rat liver gap junctions. Evidence for an intramolecular disulphide linkage connecting the two extracellular peptide loops.

A range of anti-peptide antibodies directed towards selected amino acid sequences of connexin32 was prepared and characterised. The site-directed antibodies that identified connexin32 were used to study by immunolocalization and by proteolytic treatment of intact and split gap junctions the arrangement of the protein in the membrane. These studies reinforce models of connexin topography in which the polypeptide traverses the junctional membrane four times, with the amino and carboxyl termini cytoplasmically located. The four transmembrane domains were shown to be linked by two extracellular loops with a single intracellular loop connecting the second and third transmembrane domains. Evidence is presented to show that the two extracellular domains of connexin32, which are important for intercellular adhesion and the insulated bridging of the extracellular space by channels allowing cell-cell communication across the gap junction, are connected by disulphide bond(s). The studies lead to a more detailed two-dimensional model of connexin32 in the membrane, incorporating the favoured theoretical arrangement of disulphide bonds at the extracellular domain of connexin32.

Amino Acid Sequence↗
PubMed1990

A novel 100 kDa protein, localized to receptor-enriched endosomes, is immunologically related to the signal-transducing guanine-nucleotide-binding proteins Gt and Gi.

Antisera raised against the C-terminus decapeptide of the alpha-subunit of the retinal guanine-nucleotide-binding protein (G-protein) transducing (Gt) cross-reacted with the alpha-subunit of the inhibitory G-protein Gi. The same antisera also reacted with a 100 kDa protein (p100) found in rat liver homogenates. The immunoreactivity of both Gt and p100 was specifically inhibited by the immunizing peptide with similar dose-dependencies [concn. causing 50% inhibition (IC50) = 300 ng/ml]. This similarity in inhibition profiles implies that p100 contains within its structure the C-terminal sequence shared by both alpha t and alpha i. Tissue distribution studies demonstrated that p100 was particularly enriched in the liver and kidney, but was also present in other rat tissues, as well as in a number of cell lines tested. In the liver, p100 was found in both the soluble and membrane fractions. The membrane-associated form of p100 was specifically localized to an endosomal fraction (termed D-R), previously shown to be a ligand-free but receptor-enriched subfraction of liver endosomal vesicles. Two-dimensional gel electrophoresis revealed that both the cytosolic and membrane-bound forms of p100 occurred as a series of 100 kDa polypeptides with considerable charge heterogeneity (pI 6-7). Because the C-terminus domains of both alpha t and alpha i facilitate their association with their respective receptors, this region has been functionally assigned as the receptor binding site. Therefore the presence of an immunologically similar region within p100, together with its localization to the receptor-rich endocytic vesicles, suggests that p100 may be a receptor binding protein involved in receptor trafficking.

Animals↗
PubMed1990

A two-dimensional electrophoretic analysis of the proteins and glycoproteins of liver plasma membrane domains and endosomes. Implications for endocytosis and transcytosis.

1. Polypeptides of liver plasma membrane fractions enriched in three surface domains of hepatocytes, blood-sinusoidal, lateral and bile canalicular, were analysed by isoelectric focusing (IEF) and non-equilibrium pH gel electrophoresis (NEPHGE) across a wide pH range, followed by SDS/PAGE. The overall Coomassie Blue-stained polypeptide patterns in the fractions were different. lateral plasma membrane fractions contained a characteristically higher number of polypeptides focusing at the basic pH range, whereas few basic polypeptides were present in sinusoidal plasma membrane fractions. The glycoproteins in these plasma membrane fractions stained by a lectin overlay technique with radio-iodinated concanavalin A, wheat-germ agglutinin and a slug lectin, were also different. 2. The polypeptides and glycoproteins of 'early' and 'late' endosome fractions were also compared by two-dimensional electrophoresis. Their composition was shown by Coomassie Blue staining, lectin overlay staining and in membranes metabolically labelled with [35S]methionine to be generally similar. The glycoproteins of sinusoidal plasma membranes and early and late endosomes were generally similar, but major differences in polypeptides of molecular mass 20-50 kDa, pI 7.5-8.5, in plasma membranes and endosomes were demonstrated, with a specific population of basic (pI 8-9) low-molecular-mass polypeptides being present at highest levels in 'late' endosomal fractions (shown by Coomassie Blue staining). 3. Analysis of the distribution of three specific membrane glycoproteins identified by using immunoblotting techniques showed that the asialoglycoprotein and the divalent-cation-sensitive mannose 6-phosphate receptors were present in sinusoidal plasma membrane and in early and late endocytic fractions: they were not detected in canalicular plasma membrane fractions. In contrast, 5'-nucleotidase was detected in all fractions examined. The role of the endocytic compartment in regulating trafficking pathways between the plasma membrane domains of the hepatocyte is discussed.

Animals↗
PubMed1990

Distribution of polypeptides binding guanosine 5'-[gamma-[35S]thio]triphosphate and anti-(ras protein) antibodies in liver subcellular fractions. Evidence for endosome-specific components.

The subcellular distribution in rat liver of polypeptides binding guanosine 5'-[gamma-[35S]thio]triphosphate [( 35S]GTP[S]) and seven antibodies against ras oncoproteins was evaluated. Multiple low-Mr (21,000-28,000) GTP-binding proteins were detected, but their relative distribution among the membrane fractions varied. A more specific compartmentation of polypeptides which bind antibodies generated against ras proteins was evident, with an Mr-28,000 polypeptide and a probable Mr-56,000 dimer, identified by six of the antibodies tested, being confined mainly to endosomes. An Mr-23,000 polypeptide was detected by some of the antibodies in all of the membrane fractions, but especially in the plasma membranes.

Animals↗
PubMed1990

Highly purified bile-canalicular vesicles and lateral plasma membranes isolated from rat liver on Nycodenz gradients. Biochemical and immunolocalization studies.

1. A liver canalicular plasma-membrane fraction enriched 115-155-fold in five marker enzymes relative to the tissue homogenate was obtained by sonication of liver plasma membranes followed by fractionation in iso-osmotic Nycodenz gradients. 2. Two lateral-plasma membrane fractions were also collected by this procedure; the lighter-density fraction was still associated with canalicular membranes, as assessed by enzymic and polypeptide analysis. 3. The polypeptide composition of the domain-defined plasma-membrane fractions was evaluated. It was demonstrated by immunoblotting that the 41 kDa alpha-subunit of the inhibitory G-protein, associated in high relative amounts with canalicular plasma-membrane fractions, was partially lost in the last stage of purification; however, this subunit was retained by lateral plasma membranes. 4. Antibodies to the proteins of bile-canalicular vesicles were shown to localize to the hepatocyte surface in thin liver sections examined by immunofluorescent and immuno-gold electron microscopy. Two subsets of antigens were identified, one present on both sinusoidal and canalicular plasma-membrane domains and another, by using antisera pre-absorbed with sinusoidal plasma membranes, that was confined to the bile-canalicular domain.

Animals↗
PubMed1990

Priority targeting of glycosyl-phosphatidylinositol-anchored proteins to the bile-canalicular (apical) plasma membrane of hepatocytes. Involvement of 'late' endosomes.

1. Liver plasma membranes originating from the sinusoidal, lateral and canalicular surface domains of hepatocytes were covalently labelled with sulpho-N-hydroxysuccinamide-biotin. After solubilization in Triton X-114, treatment with a phosphatidylinositol-specific phospholipase C (PI-PLC), two-phase partitioning and 125I-streptavidin labelling of the proteins resolved by PAGE, six major polypeptides (molecular masses 110, 85, 70, 55, 38 and 35 kDa) were shown to be anchored in bile canalicular membrane vesicles by a glycosyl-phosphatidylinositol (G-PI) 'tail'. 2. Permeabilized 'early' and 'late' endocytic vesicles isolated from liver were also examined. Two polypeptides (110 and 35 kDa) were shown to be anchored by a G-PI tail in 'late' endocytic vesicles. 3. Analysis of marker enzymes in bile-canalicular vesicles treated with PI-PLC showed that 5'-nucleotidase and alkaline phosphatase, but not leucine aminopeptidase and ecto-Ca2(+)-ATPase activities were released from the membrane. A low release and recovery of alkaline phosphodiesterase activity was noted. The cleavage from the membrane of 5'-nucleotidase as a 70 kDa polypeptide was confirmed by Western blotting using an antibody to this enzyme. 4. Antibodies raised to proteins released from bile-canalicular vesicles by PI-PLC treatment, and purified by partitioning in aqueous and Triton X-114 phases, localized to the bile canaliculi in thin liver sections. Antibodies to proteins not hydrolysed by this treatment stained by immunofluorescence the sinusoidal and canalicular surface regions of hepatocytes. 5. Antibodies generated to proteins cleaved by PI-PLC treatment of canalicular vesicles were shown to identify, by Western blotting, a major 110 kDa polypeptide in these vesicles. Two polypeptides (55 and 38 kDa) were detected in MDCK and HepG-2 cultured cells. 6. Since two of the six G-PI-anchored proteins targeted to the bile-canalicular plasma membrane were also detected in 'late' endocytic vesicles, the results suggest that a junction where exocytic and endocytic traffic routes meet occurs in a 'late' endocytic compartment.

Animals↗
PubMed1990

[Biochemical features and functional activity of the endocytic compartment in the liver cell].

When many ligands, polypeptidic hormones, growth factors, metabolic carriers, plasma glycoproteins, etc., bind to cell-surface receptors, ligand-receptor complexes are internalized by a process called receptor-mediated endocytosis towards the endocytic compartment. The endocytic compartment is an extensive network of anastomosing vesiculo-tubular membranes that differs biochemical and functionally from other intracellular organelles. Endosome fractions were prepared and antibodies raised against endosome membrane proteins. In addition to a detailed biochemical study of proteins and glycoproteins the antibodies were used to immunolocalize the endocytic structures in the hepatic cell. These studies aided to demonstrate the involvement of endocytic compartment not only in the sorting of proteins to specific domains of the plasma membrane but in the identification of "resident" endosome components.

Animals↗
PubMed1989

Distribution of G-proteins in rat liver plasma-membrane domains and endocytic pathways.

1. The distribution of the alpha- and beta-subunits of nucleotide-binding G-proteins among rat liver sinusoidal, lateral and canalicular plasma membranes, endosomes, Golgi membranes and lysosomes was investigated. 2. Pertussis-toxin-catalysed ADP-ribosylation identified a 41 kDa inhibitory alpha-subunit in all liver plasma-membrane functional domains as well as in endosomes. An antibody to a synthetic peptide corresponding to a C-terminal sequence of the inhibitory alpha-subunit also identified the 41 kDa polypeptide in all plasma-membrane domains, in 'early' and 'late' endosomes and in Golgi membranes; this polypeptide was not detected in lysosomes. The antibody-binding studies showed that bile-canalicular plasma membranes had the highest content of the inhibitory alpha-subunit. 3. Immunofluorescent microscopy confirmed the presence of the inhibitory alpha-subunit in all regions of the hepatocyte's cell surface. 4. An antibody recognizing the beta-subunit showed that a 36 kDa polypeptide was present in all plasma membranes and in 'early' and 'late' endosomes; it was not detected in lysosomes. The relative distribution among the fractions of this polypeptide was similar to the distribution of the inhibitory alpha-subunit. 5. The presence of high levels of the G-protein inhibitory alpha-subunit in bile-canalicular plasma membranes was confirmed by demonstration of its co-fractionation with marker enzymes in Nycodenz gradients and by free-flow electrophoresis. The significance of this location is discussed.

Animals↗
PubMed1989

Evidence for a factor in normal human serum that induces human neutrophilic granulocyte end-stage maturation in vitro.

The end-stage maturation of neutrophilic granulocyte precursor cells isolated from normal human bone marrow by Ficoll density centrifugation was studied in a liquid culture assay system used previously to study the maturation of guinea pig granulocyte precursors. Dialyzed normal human serum induced end-stage morphological maturation of human granulocyte precursors and this induction was proportional to a serum level of up to 5.0% in the assay medium. At serum concentrations greater than 5.0% a pronounced inhibition of maturation was observed. Passage of serum through a DEAE-Fractogel 650S column equilibrated with 0.01 M phosphate buffer (pH 7.0) resulted in the binding of the end-stage granulocyte maturation factor to the column. The activity eluted from the column in a fraction containing 17% of the starting serum protein that was inhibitor-free and was also capable of inducing the appearance of granulocyte alkaline phosphatase, a specific biochemical marker for granulocyte end-stage maturation. GMF is most likely a protein since it was destroyed by protease digestion. The data also indicate that neither purified human transferrin nor human recombinant granulocyte colony-stimulating factor can substitute for human serum GMF as a granulocyte end-stage maturation factor in this assay system. It was observed, however, that purified human transferrin greatly potentiated the effect of GMF suggesting that transferrin plays a supporting role in the end-stage maturation of human granulocytes in vitro. To our knowledge the evidence presented here indicates for the first time the existence of a neutrophilic granulocyte end-stage maturation factor in normal human serum.

Alkaline Phosphatase↗
PubMed1989

Artifactual chemiluminescence in the determination of nitrogen monoxide in the vapour phase of mainstream cigarette smoke.

Evidence is presented for the artifactual chemiluminescence from olefins in the determination of nitrogen monoxide in the vapour phase of mainstream cigarette smoke. The interference may be quantified by measuring the residual chemiluminescence remaining in the vapour phase of mainstream cigarette smoke after 24 h. For the nitrogen monoxide analyser used, corrections to the yield of most cigarette brands retailed in the UK were found to be about 1.5 micrograms of nitrogen monoxide per puff, each machine puff being defined by international standards.

Luminescence↗
PubMed1989

Determination of formaldehyde and acetaldehyde in mainstream cigarette smoke by high-performance liquid chromatography.

A method is described for the determination of formaldehyde and acetaldehyde in mainstream cigarette smoke. This involved the collection and reaction of the aldehydes with 2,4-dinitrophenylhydrazine in aqueous acetonitrile. The high-performance liquid chromatographic separation and measurement of the various components directly in this reaction solution eliminated the need for a clean-up stage. Cigarette yields of greater than 5 micrograms of formaldehyde and 50 micrograms of acetaldehyde could be determined to estimated relative standard deviations of 0.07 and 0.05, respectively.

Acetaldehyde↗