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111.
Netrin binds discrete subdomains of DCC and UNC5 and mediates interactions between DCC and heparin 总被引:3,自引:0,他引:3
Geisbrecht BV Dowd KA Barfield RW Longo PA Leahy DJ 《The Journal of biological chemistry》2003,278(35):32561-32568
Netrins are secreted proteins that elicit both attractive and repulsive responses in migrating cells in the central and peripheral nervous systems. Netrins interact with members of two distinct families of transmembrane receptors, represented by DCC (deleted in colorectal cancer) and UNC5. A human netrin fragment (soluble netrin; sNetrin) was purified from an engineered Chinese hamster ovary cell line and used in a pull-down assay to map the interactions between netrin and its receptors. We find that sNetrin binds exclusively to the fifth fibronectin type III repeat of DCC and to each immunoglobulin repeat of UNC5. Both DCC and UNC5 bind to sNetrin with 1:1 stoichiometry in solution, and the minimal receptor fragments behave similarly to larger fragments in cross-linking experiments with purified sNetrin. We find no evidence for formation of a ternary complex between sNetrin and soluble forms of DCC and UNC5. We also find no evidence for an interaction between DCC and heparin and instead demonstrate that a loop on the fifth fibronectin type III repeat of DCC previously implicated in mediating interactions with heparin is important for sNetrin binding. Since netrin binds heparin, our results suggest that interactions between DCC and heparin are probably mediated by netrin. 相似文献
112.
The high-molecular-weight glutenin subunits (HMW-GSs) are considered to be one of the most important components of wheat gluten, contributing to the unique viscoelastic properties of wheat dough. The HMW-GSs are highly homologous in sequence and structure and a mixture of subunits is usually present in wheat flours. Consequently, it is difficult to purify these proteins separately in appreciable amounts. Expression in heterologous systems provides a clear opportunity to produce large amounts of single HMW-GS proteins, amounts (up to 100 mg) which are required for in vitro analysis of these proteins. However, since the first expression studies of HMW-GSs, over 10 years ago, this technology has not been widely utilized. Previous studies have been analytical or small scale (5-100 ml) and in most cases only partial purity was obtained. In the present paper, we describe in detail the expression of the HMW-GSs Glu1-Dx2, Dx5, Dy10, and Dy12 for the first time on a large scale, producing up to 100 mg of target protein from a 2-liter bacterial culture, using a Biostat fermenter. Our results include optimization of expression conditions to increase yield and stability of proteins. Results also include localization, differences between x- and y-type expression and small-scale versus large-scale expression. We also developed a large-scale purification procedure. The bacterially expressed proteins have the same molecular weight on SDS-PAGE and the same retention times on RP-HPLC as their native counterparts extracted from flour. Functionality tests, on the bacterially produced proteins, have shown a clear correlation with the equivalent native proteins from flour. These results provide a clear opportunity to produce protein in amounts necessary for more detailed studies of the structure and function of the HMW-GSs and glutenin polymers on dough development and quality. 相似文献
113.
Can labels suggestively influence sensory perceptions and taste? Using a “ Phantom Ingredient” taste test, we show that the presence or absence of a labeled ingredient (soy) and the presence or absence of a health claim negatively bias taste perceptions toward a food erroneously thought to contain soy. We found a label highlighting soy content made health claims believable but negatively influenced perceptions of taste for certain segments of consumers. Our results and discussion provide better direction for researchers who work with ingredient labeling as well as for those who work with soybean products. 相似文献
114.
Rick PD; Hubbard GL; Kitaoka M; Nagaki H; Kinoshita T; Dowd S; Simplaceanu V; Ho C 《Glycobiology》1998,8(6):557-567
The polysaccharide chains of enterobacterial common antigen (ECA) consist
of linear trisaccharide repeat units with the structure -->3)-
alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1-->
4)-alpha-d-GlcNAc-(1-->, where Fuc4NAc is 4-acetamido-4,
6-dideoxy-d-galactose, ManNAcA is N - acetyl-d- mannosaminuronic acid, and
GlcNAc is N -acetyl-d-glucosamine. The major form of ECA (ECAPG) consists
of polysaccharide chains that are believed to be covalently linked to
diacylglycerol through phosphodiester linkage; the phospholipid moiety
functions to anchor molecules in the outer membrane. The ECA trisaccharide
repeat unit is assembled as a polyisoprenyl-linked intermediate which has
been tentatively identified as Fuc4NAc-ManNAcA-GlcNAc-
pyrophosphorylundecaprenol (lipid III). Subsequent chain-elongation
presumably occurs by a block-polymerization mechanism. However, the
identity of the polyisoprenoid carrier-lipid has not been established.
Accordingly, the current studies were conducted in an effort to
structurally characterize the polyisoprenyl lipid-carrier involved in ECA
synthesis. Isolation and characterization of the lipid carrier was
facilitated by the accumulation of a ManNAcA-GlcNAc-
pyrophosphorylpolyisoprenyl lipid (lipid II) in mutants of Salmonella
typhimurium defective in the synthesis of TDP-Fuc4NAc, the donor of Fuc4NAc
residues for ECA synthesis. Analyses of lipid II preparations by fast atom
bombardment tandem mass spectroscopy (FAB-MS/MS) resulted in the
identification of the lipid-carrier as the 55-carbon polyisoprenyl alcohol,
undecaprenol. These analyses also resulted in the identification of a novel
glycolipid which copurified with lipid II. FAB-MS/MS analyses of this
glycolipid revealed its structure to be 1,2-diacyl- sn
-glycero-3-pryophosphoryl-GlcNAc-ManNAcA (DGP- disaccharide). An
examination of purified ECAPGby phosphorus-31 nuclear magnetic resonance
spectroscopy confirmed that the polysaccharide chains are linked to
diacylglycerol through phosphodiester linkage. Thus, DGP-disaccharide does
not appear to be an intermediate in ECAPGsynthesis. Nevertheless, although
the available evidence clearly indicate that lipid II is a precursor of
DGP-disaccharide, the function of this novel glycolipid is not yet known,
and it may be an intermediate in the biosynthesis of a molecule other than
ECAPG.
相似文献
115.
The transport of free polymannose-type oligosaccharides from the lumen of
the endoplasmic reticulum into the cytosol has been recently demonstrated
(Moore,S.E.H., et al., 1995, EMBO J., 14, 6034-6042), but at present little
is known of the characteristics of this process. Here, it is shown that
inhibition of the transport of endogenously synthesized metabolically
radiolabeled free oligosaccharides out of the endoplasmic reticulum into
the cytosol of permeabilized HepG2 cells occurs when assays are conducted
in the presence of mannose (IC50, 4.9 mM), or its derivatives modified at
the first carbon (C1) of the sugar ring; alpha-methyl mannoside (IC50, 2.0
mM), mannoheptulose (IC50, 1.6 mM), and alpha-benzyl mannoside (IC50, 0.8
mM), whereas other monosaccharides (50 mM), differing from mannose at
position; C2 (glucose), C3 (altrose), C4 (talose), C5 (l-rhamnose), and C6
(mannoheptose), have little effect. N-Acetylglucosamine does not inhibit
oligosaccharide transport and, furthermore, although mannobioses and a
mannotriose inhibit free oligosaccharide transport, di-N-acetylchitobiose
is without effect. It is also shown that if the transport assay buffer is
either depleted of calcium ions, or supplemented with the Ca2+/Mg2+ATPase
inhibitor, thapsigargin, or with calcium ionophores, free oligosaccharide
transport out of the endoplasmic reticulum is inhibited. These results
demonstrate that the terminal nonreducing mannosyl residues of free
polymannose-type oligosaccharides and not their
N-acetylglucosamine-containing reducing termini, play an important role in
the interaction of the free oligosaccharide with the transport machinery,
and that this transport process requires the presence of calcium
sequestered in the lumen of the endoplasmic reticulum.
相似文献
116.
Thus far the life cycle of Trypanosoma (Megatrypanum) theileri has not been studied. We collected tabanids during the mass hatching, when only few tabanids are infected with trypanosomes. Tabanids were caught immediately after attacking a bait cow to serve as controls or after they had been allowed to engorge on the Trypanosoma (M.) theileri-infected cow. Tabanids were kept in the laboratory and used to study the developmental cycle of T. (M.) theileri in the tabanid gut. From day 1 to day 10 the presumably unfed controls and the engorged tabanids were dissected and cytological smears made from the mid- and hindgut. In total 2.6% (1/38) of the controls and 39% (23/59) of the engorged tabanids were positive for trypanosomes in the 1991 season. From day 1 to day 4 after engorgement trypanosomes were found in the midgut. Epimastigotes with a length of 29 μm on day 1 after infection multiplied by inequal division to form smaller epimastigotes of 26 μm on day 3. On day 4 morphologically indistinguishable trypanosomes of 21 μm total length were found in both mid- and hindgut. From day 5 to day 10 trypanosomes were found only in the hindgut in which the transformation to metacyclics was demonstrated, i.e. epimastigotes transformed to amastigote stages of 5 μm in total length. 相似文献
117.
118.
Organization of K88ac-encoded polypeptides in the Escherichia coli cell envelope: use of minicells and outer membrane protein mutants for studying assembly of pili 总被引:14,自引:7,他引:7
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Escherichia coli K-12 minicells, harboring recombinant plasmids encoding polypeptides involved in the expression of K88ac adhesion pili on the bacterial cell surface, were labeled with [35S]methionine and fractionated by a variety of techniques. A 70,000-dalton polypeptide, the product of the K88ac adhesion cistron adhA, was primarily located in the outer membrane of minicells, although it was less clearly associated with this membrane than the classical outer membrane proteins OmpA and matrix protein. Two polypeptides of molecular weights 26,000 and 17,000 (the products of adhB and adhC, respectively) were located in significant amounts in the periplasmic space. The 29,000-dalton polypeptide was shown to be processed in E. coli minicells. The 23.500-dalton K88ac pilus subunit (the product of adhD) was detected in both inner and outer membrane fractions. E. coli mutants defective in the synthesis of murein lipoprotein or the major outer membrane polypeptide OmpA were found to express normal amounts of K88ac antigen on the cell surface, whereas expression of the K88ac antigen was greatly reduced in perA mutants. The possible functions of the adh cistron products are discussed. 相似文献
119.
We have shown previously that the β-adrenergic agonist isoproterenol (2μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP. 相似文献
120.
Acetone and ether: acetone extracts of the mushrooms Lactarius fuliginosus (Fr. ex Fr.) Fr., L. fumosus fumosus Peck and L. fumosus. fumosoides (Smith and Hesler) Smith and Hesler were toxic to the corn earworm, Heliothis zea (L.), while water extracts were inactive. Ether: acetone extracts of L. fuliginosus and L. fumosus fumosus were toxic to the large milkweed bug, Oncopeltus fasciatus (L.), and in some cases caused precocious development. Profiles of compounds separated chromatographically and visualized with chromene reagents, literature reports of chromenes from L. fuliginosus, and known insecticidal/antihormone effects of chromenes suggest that chromenes may be responsible for the activity of some of the extracts.
Résumé L'activité insecticide d'extraits solubles du champignon européen L. fuliginosus Fr. et des espèces américaines étroitement apparentées L. fumosus var. fumosus Peck et L. fumosus var. fumosoides Smith & Hesler a été examinée. Les extraits à l'acétone et à l'éther: acétone des 3 champignons ont été toxiques pour Heliothis zea L., mais non les extraits aqueux. Les extraits éther: acétone de L. fuliginosus et de L. fumosus var. Fumosus ont été toxiques pour Oncopeltus fasciatus F. et ont induit dans quelques cas une métamorphose précoce. Les métabolites secondaires étaient presque aussi semblables entre L. fumosus var. fumosus et L. fumosus var. fumosoides qu'entre ces 2 variétés et L. fuliginosus. Les chromènes peuvent être responsables d'une partie de l'activité biologique contre les insectes.相似文献