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J C Brown

Publications and source records attributed to J C Brown.

At least 73 records · Page 4Linked to original sources
PubMed1995

Influence of the physical form of barley grain on the digestion of its starch in the human small intestine and implications for health.

It has been suggested that incomplete digestion of cereal starch explains the low energy values of certain cereals of large particle size. We used human subjects with ileostomies to investigate the digestion of barley and to determine whether the physical form of barley affects stomal excretion of starch, glucooligosaccharides, nitrogen, fat, and calculated energy. Only 2 +/- 1% of starch remained undigested after finely milled barley was eaten, but after flaked barley was eaten 17 +/- 1% resisted digestion, partly as oligosaccharides (G1-G10) but largely as intact unpitted starch granules bound by intact cell walls. The calculated energy excretion from the stoma was three times higher after flaked than after milled barley [51.5 decreasing to 15.3 kJ/g nonstarch polysaccharide (NSP, P < 0.001]. NSP, starch, and fat made almost equal contributions to the higher energy excretion. It is concluded that possibly the botanical source of cereals and certainly processing, other than retrogradation of the starch, are important determinants of starch digestibility and energy value. Possible clinical implications are introduced.

Adult↗
PubMed1995

Whole body metabolism is not restricted to D-sugars because energy metabolism of L-sugars fits a computational model in rats.

Can L-sugars contribute to whole body energy metabolism? Energy balance studies were undertaken in rats fed L-sugars at a rate of 10 g/100 g basal diet. Partial metabolizable energy values (MEVp) during the last 28 of 56 d while consuming the L-sugar diets showed that L-glucose contributed no energy (0.3 +/- 0.9 kJ/g, P > 0.5, mean +/- SEM), whereas L-fructose and L-glucose contributed 6.9 +/- 0.9 (P < 0.001) and 8.8 +/- 1.8 (P < 0.001) kJ/g, respectively. Over periods from 0 to 28 and 0 to 56 d of L-sugar treatment, measurements were made of energy intake, the average lean mass of animals, fat and lean mass deposition. Using these measurements and a computational model, estimates were made of each supplement's net metabolizable energy value for maintenance (NEVs). These estimates confirmed the supply of energy from L-fructose, with NEVs of 5.6 +/- 3.2 (P < 0.32) and 6.1 +/- 1.7 (P < 0.01) kJ/g over 28 and 56 d, respectively, and L-gulose with NEVs of 10.2 +/- 3.4 (P < 0.01) and 11.4 +/- 1.8 (P < 0.001) kJ/g over 28 and 56 d, respectively. A lack of energy supply from L-glucose was confirmed with NEVs of -2.6 +/- 3.2 (P > 0.5) and 0.0 +/- 1.6 (P > 0.5) kJ/g over 28 and 56 d, respectively. As reference substrates, sucrose and cellulose gave expected energy values in all determinations. Methods comparison analysis showed no differences between MEVp and NEVS. The assimilation of energy from L-fructose and L-gulose is probably via large bowl microorganisms. These observations show that L-gulose and L-fructose contribute to whole body energy metabolism while L-glucose does not.

Animals↗
PubMed1995

Detection of dairy Leuconostoc strains using the polymerase chain reaction.

This paper reports the design of a Leuconostoc-specific oligonucleotide based on 16S rRNA sequence data. When this oligonucleotide was used in a polymerase chain reaction (PCR) in conjunction with an oligonucleotide to a conserved region of the 16S rRNA sequence, a Leuconostoc-specific PCR product of approximately 470 bp was produced. The use of a second oligonucleotide to a conserved region allowed the production of an approximately 350 bp product in all PCRs, acting as a positive control. The PCR procedure described was particularly useful for detecting the presence of Leuconostoc in mixed mesophilic starter cultures. The Leuconostoc-specific oligonucleotide was used also as a specific hybridization probe.

Base Sequence↗
PubMed1995

Release of the catalytic domain N(o) from the herpes simplex virus type 1 protease is required for viral growth.

The herpes simplex virus type 1 (HSV-1) protease and its substrate, ICP35, are involved in the assembly of viral capsids and required for efficient viral growth. The full-length protease (Pra) consists of 635 amino acid (aa) residues and is autoproteolytically processed at the release (R) site and the maturation (M) site, releasing the catalytic domain No (VP24), Nb (VP21), and a 25-aa peptide. To understand the biological importance of cleavage at these sites, we constructed several mutations in the cloned protease gene. Transfection assays were performed to determine the functional properties of these mutant proteins by their abilities to complement the growth of the protease deletion mutant m100. Our results indicate that (i) expression of full-length protease is not required for viral replication, since a 514-aa protease molecule lacking the M site could support viral growth; and that (ii) elimination of the R site by changing the residue Ala-247 to Ser abolished viral replication. To better understand the functions that are mediated by proteolytic processing at the R site of the protease, we engineered an HSV-1 recombinant virus containing a mutation at this site. Analysis of the mutant A247S virus demonstrated that (i) the mutant protease retained the ability to cleave at the M site and to trans process ICP35 but failed to support viral growth on Vero cells, demonstrating that release of the catalytic domain No from Pra is required for viral replication; and that (ii) only empty capsid structures were observed by electron microscopy in thin sections of A247S-infected Vero cells, indicating that viral DNA was not encapsidated. Our results demonstrate that processing of ICP35 is not sufficient to support viral replication and provide genetic evidence that the HSV-1 protease has nuclear functions other than enzymatic activity.

Amino Acid Sequence↗
PubMed1995

Herpes simplex virus capsids assembled in insect cells infected with recombinant baculoviruses: structural authenticity and localization of VP26.

Recently, recombinant baculoviruses have been used to show that expression of six herpes simplex virus type 1 genes results in the formation of capsid-like particles. We have applied cryoelectron microscopy and three-dimensional image reconstruction to establish their structural authenticity to a resolution of approximately 2.7 nm. By comparing capsids assembled with and without the expression of gene UL35, we have confirmed the presence of six copies of its product, VP26 (12 kDa), around each hexon tip. However, VP26 is not present on pentons, indicating that the conformational differences between the hexon and penton states of the major capsid protein, VP5, extend to the VP26 binding site.

Animals↗
PubMed1995

Assembly of the herpes simplex virus capsid: requirement for the carboxyl-terminal twenty-five amino acids of the proteins encoded by the UL26 and UL26.5 genes.

Herpes simplex virus type 1 (HSV-1) intermediate capsids are composed of seven proteins, VP5, VP19C, VP21, VP22a, VP23, VP24, and VP26, and the genes that encode these proteins, UL19, UL38, UL26, UL26.5, UL18, UL26, and UL35, respectively. The UL26 gene encodes a protease that cleaves itself and the product of the UL26.5 gene at a site (M site) 25 amino acids from the C terminus of these two proteins. In addition, the protease cleaves itself at a second site (R site) between amino acids 247 and 248. Cleavage of the UL26 protein gives rise to the capsid proteins VP21 and VP24, and cleavage of the UL26.5 protein gives rise to the capsid protein VP22a. Previously we described the production of HSV-1 capsids in insect cells by infecting the cells with recombinant baculoviruses expressing the six capsid genes (D. R. Thomsen, L. L. Roof, and F. L. Homa, J. Virol. 68:2442-2457, 1994). Using this system, we demonstrated that the products of the UL26 and/or UL26.5 genes are required as scaffolds for assembly of HSV-1 capsids. To better understand the functions of the UL26 and UL26.5 proteins in capsid assembly, we constructed baculoviruses that expressed altered UL26 and UL26.5 proteins. The ability of the altered UL26 and UL26.5 proteins to support HSV-1 capsid assembly was then tested in insect cells. Among the specific mutations tested were (i) deletion of the C-terminal 25 amino acids from the proteins coded for by the UL26 and UL26.5 genes; (ii) mutation of His-61 of the UL26 protein, an amino acid required for protease activity; and (iii) mutation of the R cleavage site of the UL26 protein. Analysis of the capsids formed with wild-type and mutant proteins supports the following conclusions: (i) the C-terminal 25 amino acids of the UL26 and UL26.5 proteins are required for capsid assembly; (ii) the protease activity associated with the UL26 protein is not required for assembly of morphologically normal capsids; and (iii) the uncleaved forms of the UL26 and UL26.5 proteins are employed in assembly of 125-nm-diameter capsids; cleavage of these proteins occurs during or subsequent to capsid assembly. Finally, we carried out in vitro experiments in which the major capsid protein VP5 was mixed with wild-type or truncated UL26.5 protein and then precipitated with a VP5-specific monoclonal antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

The C-terminal 25 amino acids of the protease and its substrate ICP35 of herpes simplex virus type 1 are involved in the formation of sealed capsids.

The herpes simplex virus type 1 protease and its substrate, ICP35, are involved in the assembly of viral capsids. Both proteins are encoded by a single open reading frame from overlapping mRNAs. The protease is autoproteolytically processed at two sites. The protease cleaves itself at the C-terminal site (maturation site) and also cleaves ICP35 at an identical site, releasing a 25-amino-acid (aa) peptide from each protein. To determine whether these 25 aa play a role in capsid assembly, we constructed a mutant virus expressing only Prb, the protease without the C-terminal 25 aa. Phenotypic analysis of the Prb virus in the presence and absence of ICP35 shows the following: (i) Prb retains the functional activity of the wild-type protease which supports virus growth in the presence of ICP35; (ii) in contrast to the ICP35 null mutant delta ICP35 virus, the Prb virus fails to grow in the absence of ICP35; and (iii) trans-complementation experiments indicated that full-length ICP35 (ICP35 c,d), but not the cleaved form (ICP35 e,f), complements the growth of the Prb virus. The most striking phenotype of the Prb virus is that only unsealed aberrant capsid structures are observed by electron microscopy in mutant-infected Vero cells. Our results demonstrate that the growth of herpes simplex virus type 1 requires the C-terminal 25 aa of either the protease or its substrate, ICP35, and that the C-terminal 25 aa are involved in the formation of sealed capsids.

Animals↗
PubMed1995

Presence and actions of vasoactive intestinal peptide in the isolated rabbit heart.

The objectives of these experiments were to localize vasoactive intestinal peptide immunoreactivity (VIP-IR) in the intracardiac ganglia of the interatrial septum of the rabbit heart and to examine the effects of vasoactive intestinal peptide (VIP) on the isolated perfused rabbit heart. Cell bodies of neurones containing VIP-IR were located in the interatrial septa of rabbit hearts by using immunocytochemistry. In addition, the effects of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) on the isolated rabbit heart were compared with those of VIP. Bolus injections of SP, NKA, VIP, and NKB caused dose-dependent decreases in the perfusion pressure of hearts perfused at constant flow with ED50 values of 2.73 +/- 0.10 fmol (n = 6), 0.18 +/- 0.01 pmol (n = 8), 93.75 +/- 1.88 pmol (n = 6), and 75.00 +/- 3.06 pmol (n = 6), respectively. The slope of the dose-response curve of VIP was much greater than those of SP and NKA, suggesting the presence of one receptor subtype for VIP and multiple receptor subtypes for the neurokinins on rabbit coronary vessels. Differences in the slopes of the dose-response curves and potency may reflect differences in the mechanism of vasodilatation. The maximal values of vasodilatation of all of the peptides did not differ. None of the peptides produced significant changes in heart rate, left ventricular pressure, or contractility. These results suggest that VIP found in the intracardiac neurones of the rabbit heart can mediate coronary vasodilatation.

Animals↗
PubMed1995

Effects of glucose-dependent insulinotropic polypeptide and glucagon-like peptide-I-(7-36) on insulin secretion.

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide (GLP)-I-(7-36) are probably the most important "incretins," but there is controversy as to their relative insulinotropic activities. The effects of natural (np) and synthetic porcine (sp) GIP, synthetic human (sh) GIP, and GLP-I-(7-36) on insulin secretion from the perfused rat pancreas were compared using gradient perfusion. Insulin secretion was increased by both spGIP and GLP-I-(7-36) at concentrations of approximately 16 pM. Maximal responses to GLP-I-(7-36) in the presence of 16.7 mM glucose were slightly greater than with npGIP or spGIP, but with 10 mM glucose spGIP and GLP-I-(7-36) exerted equivalent effects. Responses to shGIP were greatly reduced compared with spGIP. In the presence of 50 pM spGIP or GLP-I-(7-36) the glucose threshold was 4.5 +/- 0.11 mM. The data indicate that GLP-I-(7-36) and porcine GIP are equally insulinotropic and share the same glucose threshold for activity, whereas shGIP is less active. At the concentrations found postprandially, however, GIP is likely to be the more important incretin.

Animals↗
PubMed1995

Gastric inhibitory polypeptide release from a tumor-derived cell line.

A cell line derived from intestinal tumors of transgenic mice (STC-1) was subcloned to produce a stable line with approximately 30% immunoreactive gastric inhibitory polypeptide (irGIP)-containing cells (STC 6-14). High-performance liquid chromatography (HPLC) of STC 6-14 extracts indicated that the tumor cell-derived irGIP had the same retention time as synthetic porcine GIP-(1-42) (pGIP). Approximately 30% of the cells also contained immunoreactive somatostatin (irSS), which eluted as a single peak on HPLC, corresponding with SS-(1-14). On average, each well of extracted cells (5.0 x 10(5) cultured 4 days) contained 33.3 +/- 1.4 ng irGIP and 18.4 +/- 1.5 ng irSS. Basal release of irGIP in the presence of 5 mM glucose was 733 +/- 58 pg.ml cells-1.2h-1 (2.20 +/- 0.17% of total cell content; TCC) and doubled at 20 mM glucose (4.20 +/- 0.42% TCC). The response to glucose was augmented by addition of a SS neutralizing antibody (SOMA-10) and suppressed by 10 nM SS. Basal release of irSS in 5 mM glucose was 377 +/- 35 pg.ml cells-1.2h-1 (2.05 +/- 0.19% TCC) and was increased by glucose (> or = 15 mM) and the addition of pGIP (> or = 1 nM). The STC 6-14 cell line represents a model to study the synthesis, storage, and release of GIP and SS in a controlled environment.

Animals↗
PubMed1995

The collagen superfamily.

The collagens represent a large family of molecular proteins which are located in the extracellular matrix. They all share a triple-helical segment of variable length (100-450 nm) but differ considerably in the size and nature of their globular domains. The various collagen types I-XIX known so far in vertebrates are adjusted to different functions including the formation of fibrils, microfibrils and networks. Their biological role is now increasingly studied by genetic approaches.

Animals↗
PubMed1995

Functional expression of the rat pancreatic islet glucose-dependent insulinotropic polypeptide receptor: ligand binding and intracellular signaling properties.

Incretins are endogenous peptides released from the gastrointestinal tract into the circulation during a meal that potentiate glucose-stimulated insulin secretion. At present, there are two established incretins: glucose-dependent insulinotropic polypeptide (GIP) and the truncated glucagon-like peptides (tGLPs), which are now being investigated for use in the treatment of diabetes mellitus. In the present study we cloned a rat islet GIP receptor complementary DNA (GIP-R1) to answer several important questions regarding the ligand-binding and intracellular signaling properties of the GP receptor. GIP-R1, when expressed transiently in monkey kidney (COS-7) or stably in Chinese hamster ovary (CHO-K1) cells, demonstrated comparable high affinity binding for either synthetic porcine (sp) GIP or synthetic human (sh) GIP. The IC50 values for displacement of [125I]spGIP in CHO-K1 cells were 2.6 +/- 0.8 and 3.1 +/- 0.9 nM for two different preparations of shGIP, and 3.7 +/- 1.5 and 3.6 +/- 0.4 nM for two preparations of spGIP. Saturation isotherms obtained with both intact cells and membranes gave monophasic binding curves with apparent Kd values of 204 +/- 17 and 334 +/- 94 pM, respectively. Cells expressed 12-15 x 10(3) receptors/cell. In COS-7 cells, spGIP and shGIP also exhibited similar IC50 values (7.6 +/- 1.2 and 8.9 +/- 1.8 nM, respectively). The receptor in CHO-K1 cells bound GIP-(1-30) with lower affinity (IC50 = 39 +/- 17 nM), whereas the fragments GIP-(19-30), GIP-(18-28), and GIP-(21-26) showed no apparent binding. The specificity of the receptor was further examined using several structurally related peptides. Surprisingly, exendin-(9-39) [Ex-(9-39)], a GLP-1 receptor antagonist, and Ex-4-(1-39), a GLP-1 receptor agonist, demonstrated some affinity for the GIP receptor, with 39% and 21% displacement of [125I]spGIP, respectively, at 1 microM. Other members of the secretin/vasoactive intestinal peptide family of peptides tested showed no interaction. GIP-R1 receptor binding correlated with activation of the adenylyl cyclase system, whereby spGIP and shGIP evoked concentration-dependent increases in cAMP accumulation with EC50 values of 8.7 +/- 1.5 x 10(-10)M and 8.1 +/- 1.6 x 10(-10)M for spGIP and shGIP, respectively. Increases in cAMP in the presence of 10 nM spGIP were not dependent on the ambient glucose concentration, with 22- and 18-fold increases in cAMP accumulation at 0.1 and 5.5 mM glucose, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Binding of purified collagen receptors (alpha 1 beta 1, alpha 2 beta 1) and RGD-dependent integrins to laminins and laminin fragments.

Integrins alpha 1 beta 1 and alpha 2 beta 1 when purified by collagen affinity chromatography, showed distinct binding to mouse tumor laminin-1, which has the chain composition alpha 1 beta 1 gamma 1. The binding was, however, about 10-fold lower than to collagen IV. Only little (alpha 1 beta 1) or no binding (alpha 2 beta 1) was observed to two different laminin isoforms (alpha 2 beta 1 gamma 1, alpha 2 beta 2 gamma 1) from human placenta. Binding to laminin-1 was abolished by EDTA and could be specifically inhibited by antibodies to the respective integrin alpha subunit. These antibodies also inhibited cell adhesion to collagens. The binding of soluble integrins was weaker than that of immobilized integrins but could be enhanced by an activating anti(beta 1 integrin). No enhancement was observed for immobilized integrins. Studies with laminin-1 fragments demonstrated lack of binding to the major cell-adhesive fragment E8 from the long arm, fragments E3 and E4, involved in heparin-binding and self-assembly, respectively, and fragment P1, corresponding to the inner segments of the short arms. A larger short-arm fragment (E1XNd), which lacks the N-terminal beta 1 chain domains V and VI, was as active as laminin. Together, these results, suggested the localization of the binding sites for alpha 1 beta 1 and alpha 2 beta 1 to the N-terminal region of the laminin alpha 1 chain. Fragment P1 but not intact laminin-1 bound to alpha V beta 3 integrin in an EDTA-sensitive and RGD-sensitive manner, underscoring previous data on the cryptic nature of the RGD site in laminin-1. Further analyses by surface plasmon resonance assays demonstrated a KD = 50 nM for alpha 2 beta 1/laminin-1 binding and a KD = 450 nM for alpha V beta 3/fragment P1 binding and confirmed the anti-beta 1-mediated increase in affinity for alpha 2 beta 1.

Amino Acid Sequence↗
PubMed1994

Mapping the auxin-binding site of auxin-binding protein 1.

Auxin-binding protein 1 (ABP1) is a putative receptor for the class of plant growth hormones designated auxins of which indole-3-acetic acid (IAA) is the predominant endogenous member. ABP1 is a homodimeric glycoprotein consisting of subunits of 163 amino acid residues. We have performed a structural study of ABP1 that has localized a region along its primary sequence that is involved in hormone binding. We have used the photoaffinity labeling agent, 5-[7-3H]azidoindole-3-acetic acid (5-[3H]N3IAA), an active auxin analog, to covalently label residues that are within, or near, the auxin-binding site. Photolabeled ABP1 was digested to completion with trypsin, and the resulting peptides were purified by reverse phase high performance liquid chromatography. When 5-[3H]N3IAA was used at a concentration of 0.5 microM (one order of magnitude below the Kd for 5-N3IAA) only one peptide was labeled at a high specific activity. Labeling was blocked by the presence of 50 microM IAA, indicating that the interaction is specific. Sequence analysis determined that this tryptic fragment was derived from Ile130 to Leu145 of ABP1. We suggest that residue Asp134 is the specific target of the photolabeling and is within 1.48 A of the postulated hydrophobic platform of the auxin-binding site. We propose that Trp136 may serve as this hydrophobic platform in the binding site for the aromatic rings of auxins.

Affinity Labels↗
PubMed1994

Finding a needle in a haystack: detection of a small protein (the 12-kDa VP26) in a large complex (the 200-MDa capsid of herpes simplex virus).

Macromolecular complexes that consist of homopolymeric protein frameworks with additional proteins attached at strategic sites for a variety of structural and functional purposes are widespread in subcellular biology. One such complex is the capsid of herpes simplex virus type 1 whose basic framework consists of 960 copies of the viral protein, VP5 (149 kDa), arranged in an icosahedrally symmetric shell. This shell also contains major amounts of three other proteins, including VP26 (12 kDa), a small protein that is approximately equimolar with VP5 and accounts for approximately 6% of the capsid mass. With a view to inferring the role of VP26 in capsid assembly, we have localized it by quantitative difference imaging based on three-dimensional reconstructions calculated from cryo-electron micrographs. Purified capsids from which VP26 had been removed in vitro by treatment with guanidine hydrochloride were compared with preparations of the same depleted capsids to which purified VP26 had been rebound and with native (undepleted) capsids. The resulting three-dimensional density maps indicate that six VP26 subunits are distributed symmetrically around the outer tip of each hexon protrusion on VP26-containing capsids. Because VP26 may be readily dissociated from and reattached to the capsid, it does not appear to contribute significantly to structural stabilization. Rather, its exposed location suggests that VP26 may be involved in linking the capsid to the surrounding tegument and envelope at a later stage of viral assembly.

Capsid↗
PubMed1994

Application of the ligase chain reaction to the detection of nisinA and nisinZ genes in Lactococcus lactis ssp. lactis.

This paper reports on the application of the ligase chain reaction (LCR) to the specific detection of variants of the nisin structural gene (nisinA and nisinZ) in nisin producing strains of Lactococcus lactis ssp lactis. The LCR assay was used to screen nisin producing strains to determine which form of the nisin structural gene they contained. This method of differentiating the nisin structural gene variants provides a useful alternative to the only other available genetic differentiation, that of sequencing the gene.

Base Sequence↗
PubMed1994

The laminins.

Laminins are extracellular matrix proteins which consist of alpha, beta and gamma chains with molecular masses of 140-400 kDa. Chain association occurs through a large triple alpha-helical coiled-coil domain towards the C-terminus of each chain. Eight genetically distinct laminin chains (alpha 1, alpha 2, alpha 3, beta 1, beta 2, beta 3, gamma 1, gamma 2) and seven different assembly forms (laminins-1 to -7) are known so far. The most extensively characterized laminin-1 (alpha 1 beta 1 gamma 1) shows calcium-dependent self assembly and heterotypic binding to perlecan, nidogen, fibulin-1 and other matrix components. This binding indicates a crucial role in the supramolecular organization of basement membranes. Laminins also possess binding sites for at least six different integrin receptors and are thus involved in many cell-matrix interactions. Such interactions have been shown to be important during embryonic development and for tissue homeostasis and remodelling.

Animals↗