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21.
Joey Lai Oliver K. Bernhard Stuart G. Turville Andrew N. Harman John Wilkinson Anthony L. Cunningham 《The Journal of biological chemistry》2009,284(17):11027-11038
C-type lectin receptors expressed on the surface of dendritic cells and
macrophages are able to bind glycoproteins of microbial pathogens via mannose,
fucose, and N-acetylglucosamine. Langerin on Langerhans cells,
dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin
on dendritic cells, and mannose receptor (MR) on dendritic cells and
macrophages bind the human immunodeficiency virus (HIV) envelope protein gp120
principally via high mannose oligosaccharides. These C-type lectin receptors
can also oligomerize to facilitate enhanced ligand binding. This study
examined the effect of oligomerization of MR on its ability to bind to mannan,
monomeric gp120, native trimeric gp140, and HIV type 1 BaL. Mass spectrometry
analysis of cross-linked MR showed homodimerization on the surface of primary
monocyte-derived dendritic cells and macrophages. Both monomeric and dimeric
MR were precipitated by mannan, but only the dimeric form was
co-immunoprecipitated by gp120. These results were confirmed independently by
flow cytometry analysis of soluble monomeric and trimeric HIV envelope and a
cellular HIV virion capture assay. As expected, mannan bound to the
carbohydrate recognition domains of MR dimers mostly in a calcium-dependent
fashion. Unexpectedly, gp120-mediated binding of HIV to dimers on
MR-transfected Rat-6 cells and macrophages was not calcium-dependent, was only
partially blocked by mannan, and was also partially inhibited by
N-acetylgalactosamine 4-sulfate. Thus gp120-mediated HIV binding
occurs via the calcium-dependent, non-calcium-dependent carbohydrate
recognition domains and the cysteine-rich domain at the C terminus of MR
dimers, presenting a much broader target for potential inhibitors of gp120-MR
binding.The mannose receptor
(MR)2 is a C-type
lectin receptor that is expressed on the surface of a variety of cells,
including immature monocyte-derived dendritic cells (MDDC), dermal dendritic
cells, macrophages, and hepatic endothelial cells. It is a multifunctional
protein, involved in antigen recognition and internalization during the early
stages of the innate immune response
(1) as well as physiological
clearance of the endogenous pituitary hormones lutropin and thyrotropin
(2,
3). Recognition of foreign
antigens occurs via mannose, fucose, and GlcNAc residues
(4,
5), which are generally not
found as terminal residues on mammalian glycoproteins but are highly abundant
on surface proteins of pathogens such as the HIV-1 envelope gp120
(6,
7). Once bound, pathogens can
be internalized by endocytosis or phagocytosis, where they are targeted to
lysosomes for proteolytic degradation and presentation on major
histocompatibility complex class II
(8). In immature DCs, soluble
recombinant HIV envelope proteins are processed by this pathway, initially
binding to both dendritic cell-specific intracellular adhesion molecule 3
grabbing non-integrin (DC-SIGN) and MR and ultimately co-localizing with MR
but not DC-SIGN in lysosomes
(9). Furthermore, in immature
DCs and to a greater extent mature DCs, a proportion of intact HIV-1 enters a
unique vesicular compartment that co-localizes with tetraspanin proteins such
as CD81 (10,
11). Recently, this
compartment has been shown to be continuous with the plasma membrane
(11) and does not represent a
continuation of the endolysosomal network. Interestingly, this compartment can
translocate virus from DCs to CD4 T cells, upon the formation of a virological
synapse
(10–12).
Although viral uptake can occur in DCs independent of HIV env
(2), the efficiency of HIV
binding and uptake is greatly enhanced by the presence of C-type lectin-env
interactions. At least initial binding to DC-SIGN (and most likely also MR) is
required for T cell trans-infection
(13).Structurally, the extracellular domain of MR consists of an N-terminal
cysteine-rich domain (Cys-RD), followed by a fibronectin type II domain and
eight carbohydrate recognition domains (CRD) on a single polypeptide backbone
(1). Of the eight CRDs, CRD
4–8 have been shown to be required for high affinity binding of ligands
containing terminal mannose/fucose/GlcNAc residues, with CRD 4 having
demonstrable monosaccharide binding in isolation
(14). Binding and release of
ligand within the low pH environment of the endolysosomal compartment are also
Ca2+-dependent. Acid-induced removal of Ca2+ binding in
CRD 4 and 5 was shown to cause a conformational rearrangement of the domain,
resulting in a loss of carbohydrate binding activity
(15). In contrast, binding of
sulfated carbohydrates to the Cys-RD appears to be Ca2+-independent
as no Ca2+-binding sites were observed in its crystal structure
(2,
16).Oligomerization of CLRs such as DC-SIGN
(17), Langerin
(18), and mannose-binding
protein (19) has been reported
to be essential for binding of oligosaccharide-bearing ligands. Early studies
on MR suggested that it exists solely as a monomeric molecule and that
clustering of multiple CRDs within the single polypeptide backbone was
necessary for high affinity binding of oligosaccharide moieties
(20). However, more recent
studies have shown that dimerization is possible in the presence of
Ca2+ (21) and that
an equilibrium may exist between monomeric and dimeric forms on the cell
surface (22). It is currently
unclear what effect dimerization has on ligand binding to the CRDs; however,
there is evidence that dimerization of MR is required for high affinity
binding of ligands bearing terminal N-acetylgalactosamine 4-sulfate
(GalNAc-4-SO4) such as lutropin and thyrotropin
(22) to the Cys-RD.To date, studies on the oligomerization and ligand binding activity of MR
have used solubilized protein from cell lysates
(20) or purified recombinant
fragments (21). Because the
membrane microenvironment can influence protein associations, soluble forms of
MR may not necessarily be a true model of the quaternary structure and
function of the native protein. Here, we used a well established method of
cross-linking (23) on MDDCs,
monocyte-derived macrophages (MDMs), and MR-transfected Rat-6 cells to
preserve lateral protein-protein interactions between MR on the cell surface
prior to solubilization. Mass spectrometry analysis of affinity-purified
complexes showed they were homo-oligomers, and further resolution of the
complex on a low percentage polyacrylamide gel by SDS-PAGE strongly indicates
that they are dimers. Dimerization of MR was also found to be essential for
binding mannan, monomeric gp120, native trimeric gp140, and HIV-1 viral
particles. Persistence of monomeric gp120 and trimeric gp140 binding to
dimeric MR in the presence of EGTA and various CRD and other inhibitors,
however, suggested that gp120-mediated HIV-1 binding is not
Ca2+-dependent and that at least binding probably occurs to both
Ca2+-dependent and -independent CRDs and also the Cys-RD. 相似文献
22.
A suitable medium was developed from modified Richard's medium plus V8 juice (RM8) to produce high levels of desiccation-tolerant conidia ofTrichoderma harzianumstrain 1295-22. The addition of 9% (v/v) glycerol to RM8 improved both biomass production and desiccation tolerance of the conidia ofT. harzianum.This medium was then used in a laboratory scale fermenter (1.5 liter) to determine optimal operating conditions. The optimal temperature for conidial production and desiccation tolerance improvement in the fermenter was 32°C when dissolved oxygen was maintained at 50% saturation of air, and the stirring rate was 1000 revolutions per minute. The initial water potential of the medium (with 9% glycerol) was −3.7 MPa, the pH was 6, and neither was controlled during fermentation. Changes in medium pH and dissolved oxygen were associated with the stages of morphological development and conidiation. The pH of the medium decreased concurrently with germ-tube elongation and mycelium development and then increased to 6.0–6.2 at phialide formation. Intensive conidiation occurred at pH 6.3–6.5 and reached its maximal level at 6.9–7.1. Changes in pH values could be used as indicators to monitor the morphological development and conidiation ofT. harzianumduring fermentation. The use of a 48-h-old culture inoculum, rather than conidial inoculum, to start fermentation reduced the time required to complete the shift from vegetative growth to phialide formation. Intensive conidiation occurred immediately after the addition of culture inoculum and reached maximum levels within 68 h of fermentation. Dry weight of biomass increased with the duration of fermentation and was greatest at 96 h. However, no improvements in conidia/gram and CFU/gram were achieved after 72 h of fermentation. The desiccation tolerance of conidia harvested at 72 or 96 h was significantly (P = 0.05) greater than that of conidia harvested at 48 h of fermentation. Results obtained from this study could be used for further scale-up of the fermentation process. 相似文献
23.
J. Davenport S. McCullough R. W. Thomas L. Harman R. McAllen 《Marine and Freshwater Behaviour and Physiology》2016,49(5):301-315
Observations of the behavioural responses of near-shore marine scavengers to fish carrion were made at two depths (1–2 m, 16–18 m). Gobies and juvenile whelks were the most numerous scavengers, but appeared to consume little biomass. The first scavengers to appear at carrion (seconds/minutes) were swimming forms, later (minutes) joined by fast-moving, crawling portunid crabs. Large scavengers (crabs/starfish/catsharks) arrived after tens of minutes/hours. Scavengers were ‘direct feeders’ on the bait (crabs and some fish) or ‘indirect feeders’ (gobies and whelks) on scraps generated by direct feeders. Scavengers spent little time in aggression. While fish spent relatively low proportions of their time feeding (e.g. Lipophrys pholis: 2.2–15.8%), crabs fed almost continuously (e.g. Carcinus maenas: 97.8–99.3%) before leaving baits. Crab presence depressed fish feeding. Crabs were wasteful feeders that macerated the baits, generating scraps for indirect feeders and attracting more scavengers. Large scavengers consumed most bait. 相似文献
24.
Brownridge P Holman SW Gaskell SJ Grant CM Harman VM Hubbard SJ Lanthaler K Lawless C O'Cualain R Sims P Watkins R Beynon RJ 《Proteomics》2011,11(15):2957-2970
In this paper, we discuss the challenge of large-scale quantification of a proteome, referring to our programme that aims to define the absolute quantity, in copies per cell, of at least 4000 proteins in the yeast Saccharomyces cerevisiae. We have based our strategy on the well-established method of stable isotope dilution, generating isotopically labelled peptides using QconCAT technology, in which artificial genes, encoding concatenations of tryptic fragments as surrogate quantification standards, are designed, synthesised de novo and expressed in bacteria using stable isotopically enriched media. A known quantity of QconCAT is then co-digested with analyte proteins and the heavy:light isotopologues are analysed by mass spectrometry to yield absolute quantification. This workflow brings issues of optimal selection of quantotypic peptides, their assembly into QconCATs, expression, purification and deployment. 相似文献
25.
N. Bishop Harman 《BMJ (Clinical research ed.)》1924,1(3292):203-204
26.
27.
Pedro LC Pinheiro João CR Cardoso Ana S Gomes Juan Fuentes Deborah M Power Adelino VM Canário 《BMC evolutionary biology》2010,10(1):373
Background
Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken. 相似文献28.
Role for Plasmacytoid Dendritic Cells in the Immune Control of Recurrent Human Herpes Simplex Virus Infection 总被引:1,自引:0,他引:1
Heather Donaghy Lidija Bosnjak Andrew N. Harman Valerie Marsden Stephen K. Tyring Tze-Chiang Meng Anthony L. Cunningham 《Journal of virology》2009,83(4):1952-1961
Plasmacytoid dendritic cells (pDC) are an important component of the innate immune response, producing large amounts of alpha interferon in response to viral stimulation in vitro. Under noninflammatory conditions, pDC are not found in the skin and are restricted in location to the blood and lymph nodes. Therefore, their role in mucosal and cutaneous herpes simplex virus (HSV) infection has not been well-defined. In this study we show a role for human pDC in the immune response to HSV infection. First, by confocal microscopy we showed that pDC infiltrate the dermis of recurrent genital herpes simplex lesions at early and late phases, often at the dermo-epidermal junction. We then showed that pDC in vitro are resistant to HSV infection despite expressing the entry receptors CD111, CD112, and HVE-A. Within the lesions, pDC were found closely associated with CD3+ lymphocytes and NK cells, especially those which were activated (CD69+). Furthermore, these HSV-exposed pDC were able to stimulate virus-specific autologous T-lymphocyte proliferation. We conclude from this work that pDC may contribute to the immune control of recurrent herpes virus infection in vivo. 相似文献
29.
Aphelenchoides resinosi n. sp. and Ektaphelenchus joyceae n. sp. are described and illustrated from red pines of the Allegheny plateau of Maryland, USA. The new species were found in trees infested with Bursaphelenchus xylophilus. Primary diagnostic characters of A. resinosi females are constriction of the head, basal stylet knobs, tong postuterine sac, two incisures in the lateral field, and conical tail four to five anal body widths long with a simple terminal mucro. Diagnostic characters of the males are two pairs of subventral caudal papillae and spicule shape: Primary diagnostic characters of E. joyceae females are a slight constriction of the head, six similar lips, conical tail, and short postuterine sac. Diagnostic characters of the males are spicule size and shape, a single row of spermatocytes, and one pair of caudal papillae. Within-tree distributions of A. resinosi and E. joyceae are presented. A total of 70% of both red-needled and chlorotic-needled trees in the study were positive for A. resinosi and E. joyceae. Branch hierarchy was related to the percentage of samples positive for A. resinosi. 相似文献
30.