Biomedical subjects

J C Brown

Publications and source records attributed to J C Brown.

At least 91 records · Page 5Linked to original sources

PubMed1994

Structure and stability of the triple-helical domains of human collagen XIV.

Two triple-helical domains, Col 1 and Col 2, were obtained from a pepsin digest of human placental collagen XIV and separated from each other under nondenaturing conditions. Edman degradation demonstrated 106 amino acids residues in the Col 1 and 149 residues in the Col 2 domain. All except one of the 37 prolines in the Yaa position of the Gly-Xaa-Yaa triplets were completely hydroxylated to 4-hydroxyproline, and there were three imperfections in the triplet repeat. Partial or complete hydroxylation and glycosylation were found for all seven lysines in the Yaa position. Domain Col 1 was joined by disulfide bonds into a trimer, while Col 2 appeared as a mixture of monomers and disulfide-linked dimers. Circular dichroic spectra were typical for the collagen triple helix and revealed relatively high melting temperatures for Col 1 (38 degrees C) and Col 2 (43 degrees C). An almost perfect refolding of the triple helix was observed for Col 1 but not for Col 2, emphasizing the importance of disulfide bonds for the folding kinetics and in part the stability of the triple helix. Circular dichroic spectra of the large nontriple helical domain, NC3, of collagen XIV indicated 11% alpha helix and 63% beta structure. Comparative melting profiles of NC3 and intact collagen XIV indicated that the triple helices in intact collagen XIV have a melting temperature of 44 degrees C.

Amino Acid Sequence↗

PubMed1994

Energy balance and expenditure while consuming guar gum at various fat intakes and ambient temperatures.

We devised a new model to evaluate whether dietary composition affects whole-body energy metabolism in rats. Dietary guar gum, but not corn starch, elevates both fecal energy excretion and energy expenditure. Neither ambient temperature (21 vs 28 degrees C) nor fat content of the basal diet (15% vs 33% metabolizable energy) has a marked effect on guar gum's thermogenic stimulus. Starch and guar gum each contribute 17.5 kJ/g to gross energy intake. This energy is fully available from the starch but guar gum contributes only 9-13 kJ/g to digestible energy intake on the low-fat diet (15% of energy), only 6 kJ/g to digestible energy on the higher-fat diet (33% of energy) (because of a fat-gum interaction), decreases urinary energy loss by 1.4-2.7 kJ/g gum, and elevates energy expenditure by 11.6-14.8 kJ/g. The thermogenic effect of the guar gum is highly reproducible. Several potential mechanisms to explain such elevated energy expenditure are considered. The model used to quantify the thermogenic stimulus is highly sensitive and could be applied to the trial of potentially thermogenic drugs as well as dietary ingredients.

Animals↗

PubMed1994

Polyunsaturated fatty acids of the n - 3 series influence intestinal crypt cell production in rats.

The effects of saturated and polyunsaturated dietary lipids on intestinal mucosal cell proliferation were investigated in rats. Animals were randomly allocated to three groups of 10, and fed fibre-free diets containing lipid in the form of lard, corn oil or fish oil at a level of 80 g/kg. Total energy intake was kept constant by pair-feeding. After 14 days the crypt cell production rates (CCPR) at two sites in the small intestine, one site in the caecum and two sites in the colon were determined by the metaphase arrest technique, together with circulating levels of enteroglucagon and gastrin and parameters of mucosal morphology. Consumption of fish oil led to lower CCPR compared to corn oil at all sampling positions except the caecum (Treatment approximately 50% of control values; P < 0.05). In animals fed lard the CCPR in the small bowel was not significantly different to that of animals fed corn oil but their rates were lower in the colon. Post-prandial enteroglucagon and gastrin levels were lower in animals fed fish oil compared to the other two groups. These results suggest that polyunsaturated fatty acids of the n - 3 series may support a relatively low rate of crypt cell proliferation in some regions of the alimentary tract and might thereby tend to suppress the progression of colorectal neoplasms.

Animals↗

PubMed1994

Rapid isolation and purification of lactococcal bacteriophage DNA without the use of caesium chloride gradients.

Intact bacteriophage of Lactococcus lactis were recovered from small volumes of lysate by centrifugation at 15,000 g without precipitation with polyethylene glycol and sodium chloride, or ultracentrifugation in a caesium chloride gradient. DNA was then extracted and purified by standard protocols. This DNA was readily digested with restriction endonucleases and used successfully in hybridization experiments.

Bacteriophages↗

PubMed1994

Induced extrusion of DNA from the capsid of herpes simplex virus type 1.

DNA-filled capsids (C capsids) of herpes simplex virus type 1 were treated in vitro with guanidine-HCl (GuHCl) and analyzed for DNA loss by sucrose density gradient ultracentrifugation and electron microscopy. DNA was found to be lost quantitatively from virtually all capsids treated with GuHCl at concentrations of 0.5 M or higher, while 0.1 M GuHCl had little or no effect. DNA removal from 0.5 M GuHCl-treated capsids was effected without significant change in the capsid protein composition, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or in its structure, as judged by electron microscopy. Electron microscopic examination of capsids in the process of emptying showed that DNA was extruded from multiple, discrete sites which appeared to coincide with capsid vertices. DNA exited the capsid in the form of thick strands or fibers that varied in diameter from approximately 4 to 13 nm with preferred diameters of 7 and 11 nm. The fibers most probably correspond to multiple, laterally aligned DNA segments, as their diameters are nearly all greater than that of a single DNA double helix. The results suggest that GuHCl treatment promotes an alteration in the capsid pentons which allows DNA to escape locally. Hexons must be more resistant to this change, since DNA loss appears to be restricted to the pentons. The ability of GuHCl to cause loss of DNA from C capsids with no accompanying change in capsid morphology or protein composition suggests that penton sites may open transiently to permit DNA exist and then return to their original state.

Capsid↗

PubMed1994

The protease of herpes simplex virus type 1 is essential for functional capsid formation and viral growth.

The herpes simplex virus type 1 protease and related proteins are involved in the assembly of viral capsids. The protease encoded by the UL26 gene can process itself and its substrate ICP35, encoded by the UL26.5 gene. To better understand the functions of the protease in infected cells, we have isolated a complementing cell line (BMS-MG22) and constructed and characterized a null UL26 mutant virus, m100. The mutant virus failed to grow on Vero cells and required a complementing cell line for its propagation, confirming that the UL26 gene product is essential for viral growth. Phenotypic analysis of m100 shows that (i) normal amounts of the c and d forms of ICP35 were produced, but they failed to be processed to the cleaved forms, e and f; (ii) viral DNA replication of the mutant proceeded at near wild-type levels, but DNA was not processed to unit length or encapsidated; (iii) capsid structures were observed in thin sections of m100-infected Vero cells by electron microscopy, but assembly of VP5 into hexons of the capsid structure was conformationally altered; and (iv) nuclear localizations of the protease and ICP35 are independent of each other, and the function(s) of Na, at least in part, is to direct the catalytic domain N(o) to the nucleus.

Animals↗

PubMed1994

Phenotype of the herpes simplex virus type 1 protease substrate ICP35 mutant virus.

The herpes simplex virus type 1 ICP35 assembly protein is involved in the formation of viral capsids. ICP35 is encoded by the UL26.5 gene and is specifically processed by the herpes simplex virus type 1 protease encoded by the UL26 gene. To better understand the functions of ICP35 in infected cells, we have isolated and characterized an ICP35 mutant virus, delta ICP35. The mutant virus was propagated in complementing 35J cells, which express wild-type ICP35. Phenotypic analysis of delta ICP35 shows that (i) mutant virus growth in Vero cells was severely restricted, although small amounts of progeny virus was produced; (ii) full-length ICP35 protein was not produced, although autoproteolysis of the protease still occurred in mutant-infected nonpermissive cells; (iii) viral DNA replication of the mutant proceeded at wild-type levels, but only a very small portion of the replicated DNA was processed to unit length and encapsidated; (iv) capsid structures were observed in delta ICP35-infected Vero cells by electron microscopy and by sucrose sedimentation analysis; (v) assembly of VP5 into hexons of the capsids was conformationally altered; and (vi) ICP35 has a novel function which is involved in the nuclear transport of VP5.

Animals↗

PubMed1994

Cell-free assembly of the herpes simplex virus capsid.

Herpes simplex virus type 1 (HSV-1) capsids were found to assemble spontaneously in a cell-free system consisting of extracts prepared from insect cells that had been infected with recombinant baculoviruses coding for HSV-1 capsid proteins. The capsids formed in this system resembled native HSV-1 capsids in morphology as judged by electron microscopy, in sedimentation rate on sucrose density gradients, in protein composition, and in their ability to react with antibodies specific for the HSV-1 major capsid protein, VP5. Optimal capsid assembly required the presence of extracts containing capsid proteins VP5, VP19, VP23, VP22a, and the maturational protease (product of the UL26 gene). Assembly was more efficient at 27 degrees C than at 4 degrees C. The availability of a cell-free assay for HSV-1 capsid formation will be of help in identifying the morphogenetic steps that occur during capsid assembly in vivo and in evaluating candidate antiherpes therapeutics directed at capsid assembly.

Capsid↗

PubMed1994

A radioimmunoassay for oncorhynchid growth hormone targeted to the physiological range.

This study describes the development of an oncorhynchid growth hormone (GH) radioimmunoassay using recombinant chum salmon GH (rsGH) and a rabbit antiserum (TJK-1) raised against this recombinant material. The assay was designed to measure the wide range of circulating immunoreactive GH (IRGH) levels in Pacific salmonids, resulting in a standard curve capable of accurately determining plasma levels of IRGH from 0.5 to 250 ng/mL without dilution. The assay ED50 and ED90 values averaged 13.1 and 0.5 ng/mL, respectively. This radioimmunoassay specifically recognizes oncorhynchid IRGH, showing no cross-reactivity with recombinant porcine and bovine GH, or natural chum salmon prolactin at concentrations up to 10 micrograms/mL. Curves approximately parallel to the standard curve were obtained with purified natural coho salmon GH and plasma from chinook salmon. Recovery of rsGH from plasma was complete over the full range of the standard curve. Intra- and inter-assay coefficients of variation were 6.0 and 12.9%, respectively. Plasma IRGH levels in fed coho salmon were 30.6 +/- 5.3 ng/mL, while those in fish starved for 2 weeks were 132.9 +/- 53.9 ng/mL. Starvation for an additional 4 weeks had no significant effect. Plasma IRGH levels in control rainbow trout injected with saline were significantly higher 45 min post-injection. In contrast, fish injected with recombinant porcine GH exhibited no elevation in IRGH. It is speculated that exogenous GH inhibits the production of endogenous GH.

Animals↗

PubMed1994

Gastric inhibitory polypeptide and glucagon-like peptide-1(7-36) amide exert similar effects on somatostatin secretion but opposite effects on gastrin secretion from the rat stomach.

Previous studies on the isolated perfused stomach have shown that gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1(7-36) amide (GLP-1(7-36) amide) stimulate release of somatostatin (somatostatin-like immunoreactivity, SLI). GIP produced a paradoxical increase in gastrin secretion, whereas GLP-1(7-36) was inhibitory. In the current study, the actions of synthetic (sp) and native (np) porcine and synthetic human (sh) GIP, GLP-1(7-36), and GLP-1(7-37) on SLI and gastrin secretion were compared using a gradient perfusion of peptide. All peptides increased SLI secretion at a threshold concentration of approximately 50 pmol/L (p < 0.05). The initial rate of increase in response to spGIP (119 +/- 39 pg/min) was greater than with other forms of GIP or GLP-1. Maximal increases obtained with the two porcine peptides did not differ. Gastrin secretion was increased by concentrations of spGIP and npGIP similar to those increasing SLI secretion, but the maximal response to shGIP was lower. In contrast to GIP-induced increases, both GLP-1(7-36) and GLP-1(7-37) suppressed gastrin secretion. It is concluded that human and porcine GIP, GLP-1(7-36), and GLP-1(7-37) all stimulate SLI secretion but with different maximal effects, and GIP stimulates gastrin secretion whereas both forms of GLP-1 inhibit gastrin secretion.

Animals↗

PubMed1994

Release of gastric inhibitory polypeptide from cultured canine endocrine cells.

Canine intestinal duodenal and jejunal epithelial cell preparations enriched for endocrine cells were obtained by sequential collagenase digestion and centrifugal elutriation and maintained in culture for a 40-h period. Adherent cells contained a total cell content (TCC) of 11.5 +/- 2.5 ng (mean +/- SE) immunoreactive gastric inhibitory peptide (IRGIP)/well and 1.4 +/- 0.2 ng immunoreactive somatostatin (IRS)/well. Release experiments were performed by incubation of the cells with various stimuli over a 2-h period. Basal release of IRGIP in 5 mM glucose-5 mM K+ was 2.7 +/- 0.4% TCC. Incubation with concentrations of K+ > 20 mM or glucose > 15 mM significantly increased IRGIP release, as did the addition of a somatostatin immunoneutralizing antibody to the basal media. The addition of the Ca2+ ionophore, A-23187 (10 microM), or the adenylate cyclase activator, forskolin (100 microM), resulted in an IRGIP output greater than four times basal. Porcine gastrin-releasing peptide (GRP), at 1-100 nM, significantly stimulated IRGIP release in a concentration-dependent fashion. IRS release was increased significantly by 55 mM K+, 20 mM glucose, 10 microM A-23187, 100 nM GRP, or 100 microM forskolin.

Animals↗

PubMed1994

Protein binding and cell adhesion properties of two laminin isoforms (AmB1eB2e, AmB1sB2e) from human placenta.

Two isoforms of laminin were extracted from human placenta by neutral buffer containing EDTA, copurified through several steps and finally separated by Mono Q anion exchange chromatography. One variant consisted of disulphide-linked 340, 230 and 190 kDa subunits, which were identified by immunoblotting as Am, B1e and B2e chains. In the other variant, the B1e chain was replaced by B1s of 180 kDa. After rotary shadowing, both variants showed a similar cross-shaped structure. The nidogen content of these laminins was substoichiometric and variable (3-70%), indicating loss by endogenous proteolysis. Yet both human isoforms were able to bind mouse nidogen with an affinity (Kd approximately 0.5 nM) comparable to that of AeB1eB2e laminin from a mouse tumour. Since the binding site is known to be contributed by a single EGF-like motif of the B2e chain, this demonstrates that activity of this site is independent of chain assembly. Binding activity of both isoforms to collagen IV and the heparan sulphate proteoglycan perlecan was correlated to the nidogen content and could be enhanced by adding nidogen. Binding to heparin was only partial and heparin did not inhibit perlecan binding. This indicated a crucial role for nidogen in mediating the integration of these laminin isoforms into basement membranes. Variant AmB1sB2e showed calcium-dependent binding to fibulin-1, while only a little activity was found for AmB1eB2e. Both isoforms promoted adhesion and spreading of several cell lines. Adhesion could be completely inhibited by antibodies to the integrin beta 1 subunit but not, or only weakly, by antibodies against beta 3, alpha 2, alpha 3, alpha 5 and alpha 6 subunits. No inhibition was observed with an Arg-Gly-Asp-containing peptide.

Animals↗

PubMed1994

Characterization of acetylcholine-induced increases in cytosolic free calcium concentration in individual rat pancreatic beta-cells.

The intracellular free Ca2+ ion concentration ([Ca2+]i) was measured using fura-2 microspectrofluorimetry in individual rat pancreatic beta-cells prepared by enzymatic digestion and fluorescence-activated cell sorting. The mean basal concentration of [Ca2+]i in beta-cells in the presence of 4.4 mM glucose and 1.8 mM Ca2+ was 112 +/- 1.6 nM (n = 207). The action of acetylcholine (ACh) was concentration-dependent, and raising the concentration resulted in [Ca2+]i spikes of increasing amplitude and duration in some, but not all of the beta-cells. In addition, the beta-cells demonstrated variable sensitivity to ACh. The increases in [Ca2+]i were rapid, transient and were blocked by atropine at 10(-6) M. A brief exposure to 50 mM K+ resulted in a transient increase in [Ca2+]i similar to that induced by ACh, but resistant to atropine. A high concentration of ACh (100 microL 10(-4) M or 10(-3) M) induced [Ca2+]i oscillations in 11 out of 57 beta-cells in the presence of 4.4 mM glucose. Using calcium channel blockers and Ca2+ free medium, the source of the increase in [Ca2+]i was deduced to be from extra-cellular spaces. Changing the temperature from 22 to 37 degrees C did not affect the action of ACh on [Ca2+]i. These data strongly suggest that ACh exerted a direct action on [Ca2+]i in normal rat pancreatic beta-cells and support a role for Ca2+ as a second messenger in the action of ACh.

Acetylcholine↗

PubMed1993

Insertion sequence analysis of protoplast fused strains of Lactococcus lactis ssp. cremoris.

The use of insertion sequence probes for the analysis of fusants obtained following protoplast fusion is described. Hybridization of both total and plasmid DNA from parent and fusant strains with probes to IS904 and ISS1 showed that of the four protoplast fusions examined, three appeared to involve a rearrangement of genetic material while in the fourth the fusant appeared similar to one of the parental strains. This method of analysis provides more information about the changes induced by protoplast fusion than that obtained by monitoring the acquisition or loss of individual characteristics.

Base Sequence↗

PubMed1993

Structure of the herpes simplex virus capsid. Molecular composition of the pentons and the triplexes.

The molecular anatomy of the herpes simplex virus (HSV-1) capsid has been examined by conventional electron microscopy, cryoelectron microscopy combined with three-dimensional image reconstruction, and scanning transmission electron microscopy (STEM). Studies were carried out with purified capsids before and after treatment with urea and guanidine hydrochloride (GuHCl) at concentrations that maintain the capsid's icosahedral geometry, but selectively extract certain of its protein components. Treatment with 6.0 M urea was found to remove the pentons quantitatively from the capsid vertices, but it caused no appreciable loss of hexons. Penton loss was correlated with solubilization of a small amount of VP5, the major HSV-1 capsid protein, and the amount solubilized (6.1%) was in good agreement with the amount expected (6.3%) if pentons are each composed of five copies of VP5. We conclude that the pentons, like the hexons, are composed of VP5, which exists as a pentamer at the capsid vertices (the pentons) and as a hexamer in all other capsomers (the hexons). Control capsids and capsids extracted with 2.0 M GuHCl (G2.0 capsids) were examined by cryoelectron microscopy and the resulting images were employed to compute three-dimensional reconstructions. Also, the masses of control and G2.0 capsids were determined by dark-field STEM and the results were used to calculate copy numbers for the proteins present. The three-dimensional reconstructions showed that control and G2.0 capsids are similar in structure, except that G2.0 capsids lack all 12 pentons and 120 of the 320 trigonal nodules or "triplexes" that connect HSV-1 capsomers in groups of three. The missing triplexes are the ten closest to each capsid vertex. Thus, the tightness with which triplexes are bound to the VP5 matrix varies according to position on the T = 16 icosahedral surface lattice, those closest to the pentons being most easily detached. Biochemical analyses revealed partial loss of the minor capsid proteins VP19 and VP23 in G2.0 compared to control capsids. Taking into account the STEM data on capsid protein stoichiometry, we propose that HSV-1 triplexes are heterotrimers composed of one copy of VP19 and two copies of VP23.

Capsid↗

PubMed1993

Structure and binding properties of collagen type XIV isolated from human placenta.

Collagen XIV was isolated from neutral salt extracts of human placenta and purified by several chromatographic steps including affinity binding to heparin. The same procedures also led to the purification of a tissue form of fibronectin. Collagen XIV was demonstrated by partial sequence analysis of its Col1 and Col2 domains and by electron microscopy to be a disulphide-linked molecule with a characteristic cross-shape. The individual chains had a size of approximately 210 kD, which was reduced to approximately 180 kD (domain NC3) after treatment with bacterial collagenase. Specific antibodies mainly to NC3 epitopes were obtained by affinity chromatography and used in tissue and cell analyses by immunoblotting and radioimmunoassays. Two sequences from NC3 were identified on fragments obtained after trypsin cleavage. They were identical to cDNA-derived sequences of undulin, a noncollagenous extracellular matrix protein. This suggests that collagen XIV and undulin may be different splice variants from the same gene. Heparin binding was confirmed in ligand assays with a large basement membrane heparan sulphate proteoglycan. This binding could be inhibited by heparin and heparan sulphate but not by chondroitin sulphate. In addition, collagen XIV bound to the triple helical domain of collagen VI. The interactions with heparin sulphate proteoglycan and collagen VI were not shared by the NC3 domain, or by reduced and alkylated collagen XIV. No or only low binding was observed for collagens I-V, pN-collagens I and III, and several noncollagenous matrix proteins, including laminin, recombinant nidogen, BM-40/osteonectin, plasma and tissue fibronectin, vitronectin, and von Willebrand factor. Insignificant activity was also shown in cell attachment assays with nine established cell lines.

Amino Acid Sequence↗

PubMed1993

Glucose-dependent insulinotropic polypeptide stimulated insulin release from a tumor-derived beta-cell line (beta TC3).

The beta TC3 tumor cell line was examined for the presence of functional glucose-dependent insulinotropic polypeptide (GIP) receptors. Increasing amounts of natural porcine GIP decreased the binding of HPLC-purified [125I]GIP to beta TC3 cells in a concentration-dependent manner. Displacement of GIP was significant at concentrations as low as 500 pM, and the radioligand was fully displaced at 100 nM. GIP(1-30) produced a displacement of [125I]GIP comparable with that produced by GIP(1-42), and glucagon yielded 20% displacement at a concentration of 1 microM but was without effect at 100 mM. Incubation of beta TC3 cells in the presence of glucose concentrations of 2-20 mM yielded a concentration-dependent stimulation of immunoreactive insulin (IRI) release. GIP and glucagon-like peptide-I(7-36) amide (tGLP-I) at concentrations of 1 nM or greater significantly stimulated IRI release in the presence of 2 mM glucose. The threshold glucose concentration for GIP-stimulated IRI release from beta TC3 cells was 0.5 mM, and maximal potentiation of IRI release by GIP occurred at 5 mM glucose. Somatostatin significantly inhibited GIP-stimulated IRI release in the presence of 5 mM glucose. It is concluded that beta TC3 cells have functional GIP receptors and may provide a useful model for the study of IRI secretion at the cellular level.

Animals↗

PubMed1993

Inhibitory actions of a somatostatin analog SMS 201-995 on endocrine secretion from the isolated perfused stomach and pancreas of the rat.

An analog of somatostatin, SMS 201-995 (SMS; Sandostatin), has been shown to have increased potency in the inhibition of hormone secretion in vivo. In this study, the effect of SMS on the secretion of immunoreactive insulin, gastrin and gastric somatostatin was examined in the rat using in situ vascularly perfused organ preparations. The analog was found to inhibit both basal somatostatin and gastrin secretion from the perfused stomach in a concentration-dependent manner. SMS (9.4 x 10(-8) mol/l) also inhibited somatostatin release stimulated by gastric inhibitory polypeptide (GIP), but did not suppress gastrin release under the same conditions; an opposite effect was obtained when l-isoproterenol was used to stimulate somatostatin release. In the perfused pancreas both SS-14 (6.1 x 10(-9) mol/l) and SMS (9.4 x 10(-9) mol/l) inhibited the GIP (2 x 10(-9) mol/l) or acetylcholine (1 x 10(-6) mol/l), but not 17.8 x 10(-9) mol/l glucose-stimulated insulin secretion. Insulin secretion stimulated by 17.8 x 10(-3) mol/l glucose was unaffected by either peptide at this concentration. These results show that SMS is a potent inhibitor of gastric and pancreatic endocrine secretion. However, the inhibitory effect of SMS on gastric endocrine secretion was uncoupled in the presence of different secretagogues, suggesting that the action of SMS was dependent on the activation of different mechanisms or pathways in the stomach. In the pancreas, SMS appears to be acting on the beta-cell via a similar mechanism as somatostatin.

Animals↗