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1.
2.
Barrie J. Anthony Kylie R. James Geoffrey N. Gobert Grant A. Ramm Donald P. McManus 《PloS one》2013,8(6)
Hepatic fibrosis induced by egg deposition is the most serious pathology associated with chronic schistosomiasis, in which the hepatic stellate cell (HSC) plays a central role. While the effect of Schistosoma mansoni eggs on the fibrogenic phenotype of HSCs has been investigated, studies determining the effect of eggs of
S
. japonicum
on HSCs are lacking. Disease caused by
S
. japonicum
is much more severe than that resulting from S. mansoni infection so it is important to compare the pathologies caused by these two parasites, to determine whether this phenotype is due to the species interacting differently with the mammalian host. Accordingly, we investigated the effect of
S
. japonicum
eggs on the human HSC cell line, LX-2, with and without TGF-β (Transforming Growth Factor beta) co-treatment, so as to determine the impact on genes associated with fibrogenesis, inflammation and matrix re-organisation. Activation status of HSCs was assessed by αSMA (Alpha Smooth Muscle Actin) immunofluorescence, accumulation of Oil Red O-stained lipid droplets and the relative expression of selected genes associated with activation. The fibrogenic phenotype of HSCs was inhibited by the presence of eggs both with or without TGF-β treatment, as evidenced by a lack of αSMA staining and reduced gene expression of αSMA and Col1A1 (Collagen 1A1). Unlike S. mansoni-treated cells, however, expression of the quiescent HSC marker PPAR-γ (Peroxisome Proliferator-Activated Receptor gamma) was not increased, nor was there accumulation of lipid droplets. In contrast,
S
. japonicum
eggs induced the mRNA expression of MMP-9 (Matrix Metalloproteinase 9), CCL2 (Chemokine (C-C motif) Ligand 2) and IL-6 (Interleukin 6) in HSCs indicating that rather than inducing complete HSC quiescence, the eggs induced a proinflammatory phenotype. These results suggest HSCs in close proximity to
S
. japonicum
eggs in the liver may play a role in the proinflammatory regulation of hepatic granuloma formation. 相似文献
3.
Metabolism of
d-Glycero-d-Manno-Heptitol,
Volemitol, in Polyanthus. Discovery of a Novel Ketose
Reductase
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Volemitol
(d-glycero-d-manno-heptitol,
α-sedoheptitol) is an unusual seven-carbon sugar alcohol that
fulfills several important physiological functions in certain species
of the genus Primula. Using the horticultural hybrid
polyanthus (Primula × polyantha) as
our model plant, we found that volemitol is the major nonstructural
carbohydrate in leaves of all stages of development, with
concentrations of up to 50 mg/g fresh weight in source leaves (about
25% of the dry weight), followed by sedoheptulose
(d-altro-2-heptulose, 36 mg/g fresh weight),
and sucrose (4 mg/g fresh weight). Volemitol was shown by the
ethylenediaminetetraacetate-exudation technique to be a prominent
phloem-mobile carbohydrate. It accounted for about 24% (mol/mol) of
the phloem sap carbohydrates, surpassed only by sucrose (63%).
Preliminary 14CO2 pulse-chase radiolabeling
experiments showed that volemitol was a major photosynthetic product,
preceded by the structurally related ketose sedoheptulose. Finally, we
present evidence for a novel NADPH-dependent ketose reductase,
tentatively called sedoheptulose reductase, in volemitol-containing
Primula species, and propose it as responsible for the
biosynthesis of volemitol in planta. Using enzyme extracts from
polyanthus leaves, we determined that sedoheptulose reductase has a pH
optimum between 7.0 and 8.0, a very high substrate specificity, and
displays saturable concentration dependence for both sedoheptulose
(apparent Km = 21 mm) and NADPH
(apparent Km = 0.4 mm). Our
results suggest that volemitol is important in certain
Primula species as a photosynthetic product, phloem
translocate, and storage carbohydrate.Alditols (sugar alcohols or acyclic polyols) may be chemically
described as reduction products of aldose or ketose sugars. The most
prevalent plant alditols are the hexitols sorbitol, mannitol, and
galactitol. However, as many as 17 different alditols occur naturally
in higher plants (for review, see Bieleski, 1982; Lewis, 1984; Loescher
and Everard, 1996). The lesser-known alditols are often restricted in
their occurrence but still fulfill important functions in those plants
where they do occur. Volemitol (Fig. (Fig.1)
1)
is a good example of a less common but important alditol. This
seven-carbon sugar alcohol seems to be confined to certain sections of
the genus Primula, so much so that it has been suggested as
a useful chemotaxonomical marker (Kremer, 1978). Very little is known
about the physiology and metabolism of volemitol in primulas, except
that it was an early photosynthetic product in cowslip (Primula
veris) and oxslip (Primula elatior) (Kremer, 1978).
Figure 1Fischer projections of volemitol and its four
structurally related seven-carbon sugars. Nomenclature follows that of
Collins (1987); trivial names are underlined.The physiological roles of alditols are manifold and largely resemble
those of disaccharides and oligosaccharides. They include
photosynthetic assimilation, translocation and storage of carbon, and
reducing power, as well as protection against different types of
stresses (for review, see Bieleski, 1982; Lewis, 1984; Loescher and
Everard, 1996; Stoop et al., 1996). The biosynthetic pathways of the
hexitols sorbitol (glucitol), mannitol, galactitol (dulcitol), and the
pentitol ribitol have been established in higher plants. They generally
use NADPH as a hydrogen donor and aldose phosphate as a hydrogen
acceptor, in concert with the corresponding phosphatases. One exception
might be galactitol, which was suggested to be formed directly from
unphosphorylated Gal (and NADPH) (Negm, 1986). Although all foliar
alditols are thought to be phloem-mobile (Lewis, 1984), this has only
been demonstrated for sorbitol, mannitol, and galactitol (Zimmermann
and Ziegler, 1975; Davis and Loescher, 1990; Moing et al., 1992; Flora
and Madore, 1993).To expand our knowledge of alditol metabolism in higher plants beyond
that of hexitols, we studied the carbohydrate metabolism of polyanthus
(Primula × polyantha). This popular
horticultural hybrid of primrose (Primula
vulgaris), oxlip, and cowslip (Mabberley, 1997) was
chosen because preliminary experiments showed that its volemitol
content is very high, similar to that of the wild-type species, and
because it may be easily grown both outdoors and indoors.We give a general overview on volemitol metabolism in polyanthus with
special emphasis on the role of volemitol in plant development and
phloem transport. We also report on a novel enzyme, a NADPH-dependent
ketose reductase, which forms volemitol by the reduction
of sedoheptulose. 相似文献
4.
Joao P. C. Pinto Araz Zeyniyev Harma Karsens Hein Trip Juke S. Lolkema Oscar P. Kuipers Jan Kok 《Applied and environmental microbiology》2011,77(18):6687-6690
Plasmid pSEUDO and derivatives were used to show that llmg_pseudo_10 in Lactococcus lactis MG1363 and its homologous locus in L. lactis IL1403 are suitable for chromosomal integrations. L. lactis MG1363 and IL1403 nisin-induced controlled expression (NICE) system derivatives (JP9000 and IL9000) and two general stress reporter strains (NZ9000::PhrcA-GFP and NZ9000::PgroES-GFP) enabling in vivo noninvasive monitoring of cellular fitness were constructed. 相似文献
5.
6.
7.
Yuusuke Maruyama Toshihiko Ogura Kazuhiro Mio Kenta Kato Takeshi Kaneko Shigeki Kiyonaka Yasuo Mori Chikara Sato 《The Journal of biological chemistry》2009,284(20):13676-13685
The Ca2+ release-activated Ca2+ channel is a
principal regulator of intracellular Ca2+ rise, which conducts
various biological functions, including immune responses. This channel,
involved in store-operated Ca2+ influx, is believed to be composed
of at least two major components. Orai1 has a putative channel pore and
locates in the plasma membrane, and STIM1 is a sensor for luminal
Ca2+ store depletion in the endoplasmic reticulum membrane. Here we
have purified the FLAG-fused Orai1 protein, determined its tetrameric
stoichiometry, and reconstructed its three-dimensional structure at 21-Å
resolution from 3681 automatically selected particle images, taken with an
electron microscope. This first structural depiction of a member of the Orai
family shows an elongated teardrop-shape 150Å in height and 95Å in
width. Antibody decoration and volume estimation from the amino acid sequence
indicate that the widest transmembrane domain is located between the round
extracellular domain and the tapered cytoplasmic domain. The cytoplasmic
length of 100Å is sufficient for direct association with STIM1. Orifices
close to the extracellular and intracellular membrane surfaces of Orai1 seem
to connect outside the molecule to large internal cavities.Ca2+ is an intracellular second messenger that plays important
roles in various physiological functions such as immune response, muscle
contraction, neurotransmitter release, and cell proliferation. Intracellular
Ca2+ is mainly stored in the endoplasmic reticulum
(ER).2 This ER system
is distributed through the cytoplasm from around the nucleus to the cell
periphery close to the plasma membrane. In non-excitable cells, the ER
releases Ca2+ through the inositol 1,4,5-trisphosphate
(IP3) receptor channel in response to various signals, and the
Ca2+ store is depleted. Depletion of Ca2+ then induces
Ca2+ influx from outside the cell to help in refilling the
Ca2+ stores and to continue Ca2+ rise for several
minutes in the cytoplasm (1,
2). This Ca2+ influx
was first proposed by Putney
(3) and was named
store-operated Ca2+ influx. In the immune system, store-operated
Ca2+ influx is mainly mediated by the Ca2+
release-activated Ca2+ (CRAC) current, which is a highly
Ca2+-selective inwardly rectified current with low conductance
(4,
5). Pathologically, the loss of
CRAC current in T cells causes severe combined immunodeficiency
(6) where many Ca2+
signal-dependent gene expressions, including cytokines, are interrupted
(7). Therefore, CRAC current is
necessary for T cell functions.Recently, Orai1 (also called CRACM1) and STIM1 have been physiologically
characterized as essential components of the CRAC channel
(8–12).
They are separately located in the plasma membrane and in the ER membrane;
co-expression of these proteins presents heterologous CRAC-like currents in
various types of cells (10,
13–15).
Both of them are shown to be expressed ubiquitously in various tissues
(16–18).
STIM1 senses Ca2+ depletion in the ER through its EF hand motif
(19) and transmits a signal to
Orai1 in the plasma membrane. Although Orai1 is proposed as a regulatory
component for some transient receptor potential canonical channels
(20,
21), it is believed from the
mutation analyses to be the pore-forming subunit of the CRAC channel
(8,
22–24).
In the steady state, both Orai1 and STIM1 molecules are dispersed in each
membrane. When store depletion occurs, STIM1 proteins gather into clusters to
form puncta in the ER membrane near the plasma membrane
(11,
19). These clusters then
trigger the clustering of Orai1 in the plasma membrane sites opposite the
puncta (25,
26), and CRAC channels are
activated (27).Orai1 has two homologous genes, Orai2 and Orai3
(8). They form the Orai family
and have in common the four transmembrane (TM) segments with relatively large
N and C termini. These termini are demonstrated to be in the cytoplasm,
because both N- and C-terminally introduced tags are immunologically detected
only in the membrane-permeabilized cells
(8,
9). The subunit stoichiometry
of Orai1 is as yet controversial: it is believed to be an oligomer, presumably
a dimer or tetramer even in the steady state
(16,
28–30).Despite the accumulation of biochemical and electrophysiological data,
structural information about Orai1 is limited due to difficulties in
purification and crystallization. In this study, we have purified Orai1 in its
tetrameric form and have reconstructed the three-dimensional structure from
negatively stained electron microscopic (EM) images. 相似文献
8.
Natalia Cadaxo Rochael Luize Gon?alves Lima Sandra Maria Pereira de Oliveira Marcello André Barcinski Elvira Maria Saraiva Robson Queiroz Monteiro Lucia Helena Pinto-da-Silva 《Memórias do Instituto Oswaldo Cruz》2013,108(6):679-685
Leishmania parasites expose phosphatidylserine (PS) on their
surface, a process that has been associated with regulation of host''s immune
responses. In this study we demonstrate that PS exposure by metacyclic
promastigotes of Leishmania amazonensis favours blood
coagulation. L. amazonensis accelerates in vitro coagulation of
human plasma. In addition, L. amazonensis supports the assembly
of the prothrombinase complex, thus promoting thrombin formation. This process
was reversed by annexin V which blocks PS binding sites. During blood meal,
Lutzomyia longipalpis sandfly inject saliva in the bite
site, which has a series of pharmacologically active compounds that inhibit
blood coagulation. Since saliva and parasites are co-injected in the host during
natural transmission, we evaluated the anticoagulant properties of sandfly
saliva in counteracting the procoagulant activity of L.
amazonensis . Lu. longipalpis saliva reverses
plasma clotting promoted by promastigotes. It also inhibits thrombin formation
by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS
vesicles or in L. amazonensis . Sandfly saliva inhibits factor
X activation by the intrinsic tenase complex assembled on PC/PS vesicles and
blocks factor Xa catalytic activity. Altogether our results show that metacyclic
promastigotes of L. amazonensis are procoagulant due to PS
exposure. Notably, this effect is efficiently counteracted by sandfly
saliva. 相似文献
9.
Maria C Anholeti Rodrigo C Duprat Maria R Figueiredo Maria AC Kaplan Marcelo Guerra Santos Marcelo S Gonzalez Norman A Ratcliffe Denise Feder Selma R Paiva Cicero B Mello 《Memórias do Instituto Oswaldo Cruz》2015,110(5):629-635
Studies evaluated the effects of hexanic extracts from the fruits and flowers
ofClusia fluminensis and the main component of the flower
extract, a purified benzophenone (clusianone), against Aedes
aegypti. The treatment of larvae with the crude fruit or flower extracts
from C. fluminensis did not affect the survival ofAe.
aegypti (50 mg/L), however, the flower extracts significantly delayed
development of Ae. aegypti. In contrast, the clusianone (50 mg/L) isolate from the
flower extract, representing 54.85% of this sample composition, showed a highly
significant inhibition of survival, killing 93.3% of the larvae and completely
blocking development of Ae. aegypti. The results showed, for the first time, high
activity of clusianone against Ae. aegypti that both killed and inhibited mosquito
development. Therefore, clusianone has potential for development as a biopesticide
for controlling insect vectors of tropical diseases. Future work will elucidate the
mode of action of clusianone isolated from C. fluminensis. 相似文献
10.
11.
The kinetic properties of main and subconductance states of a mutant mouse N-methyl-d-aspartate
(NMDA) receptor channel were examined. Recombinant receptors made of ζ-ε2 (NR1-NR2B) subunits having asparagine-to-glutamine mutations in the M2 segment (ζN598Q /ε2N589Q) were expressed in Xenopus oocytes. Single channel currents recorded from outside-out patches were analyzed using hidden Markov model techniques. In
Ca2+-free solutions, an open receptor channel occupies a main conductance (93 pS) and a subconductance (62
pS) with about equal probability. There are both brief and long-lived subconductance states, but only a single
main level state. At −80 mV, the lifetime of the main and the longer-lived sub level are both ∼3.3 ms. The gating
of the pore and the transition between conductance levels are essentially independent processes. Surprisingly, hyperpolarization speeds both the sub-to-main and main-to-sub transition rate constants (∼120 mV/e-fold change),
but does not alter the equilibrium occupancies. Extracellular Ca2+ does not influence the transition rate constants. We conclude that the subconductance levels arise from fluctuations in the energetics of ion permeation
through a single pore, and that the voltage dependence of these fluctuations reflects the modulation by the membrane potential of the barrier between the main and subconductance conformations of the pore. 相似文献
12.
13.
Toru Sugiyama Bruce D. Levy Thomas Michel 《The Journal of biological chemistry》2009,284(19):12691-12700
Tetrahydrobiopterin (BH4) is a key redox-active cofactor in endothelial
isoform of NO synthase (eNOS) catalysis and is an important determinant of
NO-dependent signaling pathways. BH4 oxidation is observed in vascular cells
in the setting of the oxidative stress associated with diabetes. However, the
relative roles of de novo BH4 synthesis and BH4 redox recycling in
the regulation of eNOS bioactivity remain incompletely defined. We used small
interference RNA (siRNA)-mediated “knockdown” GTP cyclohydrolase-1
(GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, and dihydrofolate
reductase (DHFR), an enzyme-recycling oxidized BH4 (7,8-dihydrobiopterin
(BH2)), and studied the effects on eNOS regulation and biopterin metabolism in
cultured aortic endothelial cells. Knockdown of either DHFR or GTPCH1
attenuated vascular endothelial growth factor (VEGF)-induced eNOS activity and
NO production; these effects were recovered by supplementation with BH4. In
contrast, supplementation with BH2 abolished VEGF-induced NO production. DHFR
but not GTPCH1 knockdown increased reactive oxygen species (ROS) production.
The increase in ROS production seen with siRNA-mediated DHFR knockdown was
abolished either by simultaneous siRNA-mediated knockdown of eNOS or by
supplementing with BH4. In contrast, addition of BH2 increased ROS production;
this effect of BH2 was blocked by BH4 supplementation. DHFR but not GTPCH1
knockdown inhibited VEGF-induced dephosphorylation of eNOS at the inhibitory
site serine 116; these effects were recovered by supplementation with BH4.
These studies demonstrate a striking contrast in the pattern of eNOS
regulation seen by the selective modulation of BH4 salvage/reduction
versus de novo BH4 synthetic pathways. Our findings suggest that the
depletion of BH4 is not sufficient to perturb NO signaling, but rather that
concentration of intracellular BH2, as well as the relative concentrations of
BH4 and BH2, together play a determining role in the redox regulation of
eNOS-modulated endothelial responses.Regulation of endothelial nitric oxide
(NO)2 production
represents a critical mechanism for the modulation of vascular homeostasis. NO
is released by endothelial cells in response to diverse humoral, neural, and
mechanical stimuli
(1–4).
Endothelial cell-derived NO activates guanylate cyclase in vascular smooth
muscle cells, leading to increased levels of cGMP and to smooth muscle
relaxation. Blood platelets represent another key target for the actions of
endothelium-derived NO (5):
platelet aggregation is inhibited by NO-induced guanylate cyclase activation.
Many other effects of NO have been identified in cultured vascular cells and
in vascular tissues, including the regulation of apoptosis, cell adhesion,
angiogenesis, thrombosis, vascular smooth muscle proliferation, and
atherogenesis, among other cellular responses and (patho)physiological
processes.The endothelial isoform of NO synthase (eNOS) is a membrane-associated
homodimeric 135-kDa protein that is robustly expressed in endothelial cells
(2,
4,
6,
7). Similar to all the
mammalian NOS isoforms, eNOS functions as an obligate homodimer that includes
a cysteine-complex Zn2+ (zinc-tetrathiolate) at the dimer interface
(8–10).
eNOS is a Ca2+/calmodulin-dependent enzyme that is activated in
response to the stimulation of a variety of Ca2+-mobilizing cell
surface receptors in vascular endothelium and in cardiac myocytes. The
activity of eNOS is also regulated by phosphorylation at multiple sites
(11) that are differentially
modulated following the activation of cell surface receptors by agonists such
as insulin and vascular endothelial growth factor (VEGF)
(12). The phosphorylation of
eNOS at Ser-1179 activates eNOS, but phosphorylation at Thr-497 or Ser-116 is
associated with inhibition of eNOS activity
(13–17).
eNOS is reversibly targeted to plasmalemmal caveolae as a consequence of the
protein''s N-myristoylation and thiopalmitoylation. The generation of
NO by eNOS requires several redox-active cofactors, including nicotinamide
adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD),
flavin mononucleotide (FMN), calmodulin, and tetrahydrobiopterin (BH4), which
have key roles in the electron flow required for eNOS catalysis. If the flow
of electrons within eNOS is disrupted, the enzyme is uncoupled from NO
production and other redox-active products are generated, including hydrogen
peroxide and superoxide anion radical
(18,
19).In vascular disease states such as diabetes, endothelial dysfunction is
characterized by a decrease in NO bioactivity and by a concomitant increase in
superoxide formation, while eNOS mRNA and protein levels are maintained or
even increased. “Uncoupled” eNOS generates reactive oxygen species
(ROS), shifting the nitroso-redox balance and having adverse consequences in
the vascular wall (20).
Several enzymes expressed in vascular tissues contribute to the production and
efficient degradation of ROS, and an enhanced activity of oxidant enzymes
and/or reduced activity of antioxidant enzymes may cause oxidative stress.
Various agonists, pathological conditions, and therapeutic interventions lead
to modulated expression and function of oxidant and antioxidant enzymes.
However, the intimate relationship between intracellular redox state, eNOS
regulation, and NO bioavailability remains incompletely characterized.BH4 is a key redox-active cofactor for activity of all NOS enzymes
(21). The exact role of BH4 in
NOS catalysis is not yet completely defined, but this cofactor appears to
facilitate electron transfer from the eNOS reductase domain and maintains the
heme prosthetic group of the enzyme in its redox-active form
(18,
22,
23). Moreover, BH4 promotes
formation of active NOS homodimers
(24) and inhibits the
formation of hydrogen peroxide or superoxide by uncoupled eNOS
(18,
19). It has been reported that
the endothelial dysfunction associated with diabetes is accompanied a decrease
in the abundance of bioactive BH4. Supplementation with BH4 has been shown to
improve endothelial function in the models of diabetes and hypertension
(25,
26,
27). Moreover, BH4 oxidation
is seen in vascular cells in the setting of oxidative stress associated with
diabetes (28) and hypertension
(29).BH4 can be formed either by a de novo biosynthetic pathway or by a
salvage pathway. Guanosine triphosphate cyclohydrolase-1 (GTPCH1) catalyzes
the conversion of GTP to dihydroneopterin triphosphate. BH4 is generated by
further steps catalyzed by 6-pyruvoyltetrahydropterin synthase and sepiapterin
reductase (30). GTPCH1 appears
to be the rate-limiting enzyme in BH4 biosynthesis; overexpression of GTPCH1
is sufficient to augment BH4 levels in cultured endothelial cells
(31). On the other hand,
dihydrofolate reductase (DHFR) catalyzes the regeneration of BH4 from its
oxidized form, 7,8-dihydrobiopterin (BH2), in several cell types
(30,
32). DHFR is mainly involved
in folate metabolism and converts inactive BH2 back to BH4 and plays an
important role in the metabolism of exogenously administered BH4. However, the
relative contributions of endothelial GTPCH1 and DHFR to the modulation of
eNOS-dependent pathways are incompletely understood.In these studies, we have used siRNA-mediated “knockdown” of
GTPCH1 and DHFR to explore the relative roles of BH4 synthesis and recycling
in the modulation of eNOS bioactivity, as well as in the regulation of
NO-dependent signaling pathways in endothelial cells. 相似文献
14.
15.
Laura M. Stefan Elena Gómez-Díaz Eric Elguero Heather C. Proctor Karen D. McCoy Jacob González-Solís 《PloS one》2015,10(12)
According to classic niche theory, species can coexist in heterogeneous environments
by reducing interspecific competition via niche partitioning, e.g. trophic or spatial
partitioning. However, support for the role of competition on niche partitioning
remains controversial. Here, we tested for spatial and trophic partitioning in
feather mites, a diverse and abundant group of arthropods. We focused on the two
dominant mite species, Microspalax brevipes and Zachvatkinia
ovata, inhabiting flight feathers of the Cory’s shearwater,
Calonectris borealis. We performed mite counts across and within
primary and tail feathers on free-living shearwaters breeding on an oceanic island
(Gran Canaria, Canary Islands). We then investigated trophic relationships between
the two mite species and the host using stable isotope analyses of carbon and
nitrogen on mite tissues and potential host food sources. The distribution of the two
mite species showed clear spatial segregation among feathers; M.
brevipes showed high preference for the central wing primary
feathers, whereas Z. ovata was restricted to the
two outermost primaries. Morphological differences between M.
brevipes and Z. ovata support
an adaptive basis for the spatial segregation of the two mite species. However, the
two mites overlap in some central primaries and statistical modeling showed that
Z. ovata tends to outcompete M.
brevipes. Isotopic analyses indicated similar isotopic values for
the two mite species and a strong correlation in carbon signatures between mites
inhabiting the same individual host suggesting that diet is mainly based on shared
host-associated resources. Among the four candidate tissues examined (blood, feather
remains, skin remains and preen gland oil), we conclude that the diet is most likely
dominated by preen gland oil, while the contribution of exogenous material to mite
diets is less marked. Our results indicate that ongoing competition for space and
resources plays a central role in structuring feather mite communities. They also
illustrate that symbiotic infracommunities are excellent model systems to study
trophic ecology, and can improve our understanding of mechanisms of niche
differentiation and species coexistence. 相似文献
16.
17.
John M. Harrington Sawyer Howell Stephen L. Hajduk 《The Journal of biological chemistry》2009,284(20):13505-13512
Trypanosome lytic factor (TLF) is a subclass of human high density
lipoprotein (HDL) that mediates an innate immune killing of certain mammalian
trypanosomes, most notably Trypanosoma brucei brucei, the causative
agent of a wasting disease in cattle. Mechanistically, killing is initiated in
the lysosome of the target trypanosome where the acidic pH facilitates a
membrane-disrupting activity by TLF. Here we utilize a model liposome system
to characterize the membrane binding and permeabilizing activity of TLF and
its protein constituents, haptoglobin-related protein (Hpr), apolipoprotein
L-1 (apoL-1), and apolipoprotein A-1 (apoA-1). We show that TLF efficiently
binds and permeabilizes unilamellar liposomes at lysosomal pH, whereas
non-lytic human HDL exhibits inefficient permeabilizing activity. Purified,
delipidated Hpr and apoL-1 both efficiently permeabilize lipid bilayers at low
pH. Trypanosome lytic factor, apoL-1, and apoA-1 exhibit specificity for
anionic membranes, whereas Hpr permeabilizes both anionic and zwitterionic
membranes. Analysis of the relative particle sizes of susceptible liposomes
reveals distinctly different membrane-active behavior for native TLF and the
delipidated protein components. We propose that lysosomal membrane damage in
TLF-susceptible trypanosomes is initiated by the stable association of the TLF
particle with the lysosomal membrane and that this is a property unique to
this subclass of human HDL.High density lipoproteins
(HDL)2 are complex yet
ordered macromolecules consisting of characteristic proteins embedded in a
phospholipid monolayer that surrounds a hydrophobic core of esterified
cholesterol and triglycerides. A subclass of HDL is responsible for an innate
immune killing of the African blood stream parasite Trypanosoma brucei
brucei
(1–3),
and very recently, has been shown to be cytotoxic to intracellular
Leishmania promastigotes
(4). The trypanolytic HDL
particle, termed trypanosome lytic factor (TLF), is characterized by the
presence of two proteins, apolipoprotein L-1 (apoL-1) and haptoglobin-related
protein (Hpr), as well as the HDL ubiquitous apolipoprotein A-1 (apoA-1)
(1,
5–7).
Killing of the susceptible parasite involves high affinity binding to a
cell-surface receptor, endocytosis, and trafficking of the TLF particle to the
lysosome
(8–12).
The acidic lysosomal environment facilitates a membrane-disrupting activity by
the TLF particle and subsequent cell death
(9,
13). It has been shown that
purified, delipidated apoL-1 or Hpr are sufficient for trypanosome killing.
When these proteins are incorporated into the same lipoprotein particle, a
several hundredfold increase in killing activity is exhibited
(5). In addition,
Molina-Portela et al.
(14) show that maximal
protection against T. b. brucei in a transgenic mouse model requires
the expression of human Hpr, apoL-1, and apoA-1, supporting a synergistic mode
of action.Haptoglobin-related protein evolved during primate evolution and is
restricted to apes, old world monkeys, and humans
(15). Haptoglobin-related
protein is highly similar (92%) to the acute phase serum protein haptoglobin
(Hp) (16). All mammals use Hp
as a scavenger of hemoglobin (Hb) released during hemolysis associated with
infection or trauma. Haptoglobin binds cell-free Hb with high affinity and
facilitates its removal from the circulation through a receptor-mediated
process in the liver (17).
Like Hp, Hpr binds free Hb, yet this Hpr·Hb complex is not recognized
by the requisite receptors in mammals and is thus not removed from the
circulation (18). TLF uptake
by susceptible trypanosomes requires specific binding to an Hpr·Hb
complex that facilitates trafficking of the TLF particle to the lysosome
(10). It has been proposed
that once inside the lysosomal compartment, Hpr·Hb contributes directly
to membrane disruption through the generation of oxygen radicals with the
bound Hb providing the iron necessary for Fenton chemistry
(7,
10,
19).Apolipoprotein L-1 is a unique member of the apolipoprotein L protein
family in that, unlike the remaining apoL proteins, it possesses an N-terminal
signal sequence and is thus secreted from cells. As is the case for Hpr,
apoL-1 appeared during primate evolution
(20–22).
Within the circulation of primates, apoL-1 is exclusively associated with HDL,
and the majority of the protein is in the TLF subclass
(5). The apoL family members
are all predicted to adopt amphipathic α-helical conformations,
suggesting that their physiological role involves membrane interaction
(20). Apolipoprotein L-1
shares limited homology with channel-forming colicins and, consistent with
this observation, has been shown to function as an ion channel when
incorporated into lipid bilayers
(23).The ultimate fate of TLF-targeted lysosomal membranes is not firmly
established. Several studies employing both in vivo cellular analysis
and artificial membrane systems address this point with conflicting results.
Electron microscopy studies with gold-conjugated TLF revealed accumulation of
TLF in intracellular vesicles and subsequent vesicle membrane breakdown and
appearance of gold particles in the cytoplasm
(9). Widener et al.
(10) observed efflux of
lysosomally localized large molecular mass dextrans (500 kDa) in TLF-treated
T. b. brucei. These data suggest that the lysosomal membrane
experiences large scale disruption. In contrast, Perez-Morga et al.
(23) and Vanhollebeke et
al. (24) report
uncontrollable lysosomal swelling in susceptible trypanosomes treated with
normal human serum, suggesting stability of the lamellar structure of the
lysosomal membrane after TLF attack. Swelling is attributed to apoL-1-mediated
influx of Cl– ions and concomitant osmotic flow of water into
the lysosome. However, Molina-Portela et al.
(25) observed the formation of
cation-selective pores in TLF-treated planar lipid bilayers composed of
trypanosome lipids. The diversity of activities reported for TLF and normal
human serum may reflect the packaging of multiple toxins within the same
complex that can act synergistically to provide optimal killing activity
(5,
14).Here we utilize model liposomes to monitor the membrane activity of TLF and
its protein constituents. We describe the effects of TLF, delipidated Hpr,
apoL-1, and apoA-1 on the permeability of unilamellar liposomes. Additionally,
we show that TLF, apoL-1, and apoA-1 exhibit lipid specificity and that Hpr,
apoL-1, and apoA-1 induce large scale changes in the geometry of liposomes.
These results provide a molecular basis for the recognition of lysosomal
membranes by this toxic HDL and support a multicomponent mechanism for
trypanosome killing. 相似文献
18.
19.
Erin L. Westman David J. McNally Armen Charchoglyan Dyanne Brewer Robert A. Field Joseph S. Lam 《The Journal of biological chemistry》2009,284(18):11854-11862
The lipopolysaccharide of Pseudomonas aeruginosa PAO1 contains an
unusual sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid
(d-ManNAc3NAcA). wbpB, wbpE, and wbpD
are thought to encode oxidase, transaminase, and N-acetyltransferase
enzymes. To characterize their functions, recombinant proteins were
overexpressed and purified from heterologous hosts. Activities of
His6-WbpB and His6-WbpE were detected only when both
proteins were combined in the same reaction. Using a direct MALDI-TOF mass
spectrometry approach, we identified ions that corresponded to the predicted
products of WbpB (UDP-3-keto-d-GlcNAcA) and WbpE
(UDP-d-GlcNAc3NA) in the coupled enzyme-substrate reaction.
Additionally, in reactions involving WbpB, WbpE, and WbpD, an ion consistent
with the expected product of WbpD (UDP-d-GlcNAc3NAcA) was
identified. Preparative quantities of UDP-d-GlcNAc3NA and
UDP-d-GlcNAc3NAcA were enzymatically synthesized. These compounds
were purified by high-performance liquid chromatography, and their structures
were elucidated by NMR spectroscopy. This is the first report of the
functional characterization of these proteins, and the enzymatic synthesis of
UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA.Gram-negative organisms such as Pseudomonas aeruginosa produce
lipopolysaccharide
(LPS)4 as an essential
component of the outer leaflet of the outer membrane. LPS can be conceptually
divided into three parts: lipid A, which anchors LPS into the membrane; core
oligosaccharide, which contributes to membrane stability; and the O-antigen,
which is a polysaccharide that extends away from the cell surface. In P.
aeruginosa, two types of O-antigen are observed: A-band O-antigen, which
is common to most strains, and B-band O-antigen, which is variable and
therefore used as the basis of the International Antigenic Typing Scheme
(1). P. aeruginosa
serotypes O2, O5, O16, O18, and O20 collectively belong to serogroup O2,
because they all share common backbone sugar structures in their O-antigen
repeat units consisting of two di-N-acetylated uronic acids and one
2-acetamido-2,6-dideoxy-d-galactose
(N-acetyl-d-fucosamine). The minor structural variations
in the O-antigen repeat units that differentiate this serogroup into five
serotypes are: the type of glycosidic linkage between O-units (alpha
versus beta) that is formed by the O-antigen polymerase (Wzy),
isomers present (d-mannuronic or l-guluronic acid), and
acetyl group substituents
(2–4).
The B-band O-antigen of P. aeruginosa PAO1 (serotype O5) contains a
repeating trisaccharide of
2-acetamido-3-acetamidino-2,3-dideoxy-d-mannuronic acid
(d-ManNAc3NAmA),
2,3-diacetamido-2,3-dideoxy-d-mannuronic acid
(d-ManNAc3NAcA), and 2-acetamido-2,6-dideoxy-d-galactose
(3).The biosynthesis of the two mannuronic acid derivatives has yet to be fully
understood and has been the subject of investigation by our group. To produce
UDP-d-ManNAc3NAcA, a five-step pathway has been proposed
(Fig. 1) that requires the
products of five genes localized to the B-band O-antigen biosynthesis cluster
(5). The O-antigen biosynthesis
cluster was shown to be identical for all serotypes within serogroup O2, which
further underscores the high similarity between these serotypes
(5). The five genes, including
wbpA, wbpB, wbpE, wbpD, and wbpI, have been shown to be
essential for B-band LPS biosynthesis, because knockout mutants of each of
these genes are deficient in B-band O-antigen
(6–8).
Homologs of all five of the proteins required for the
UDP-d-ManNAc3NAcA biosynthesis pathway are conserved in other
bacterial pathogens, including Bordetella pertussis, Bordetella
parapertussis, and Bordetella bronchiseptica.
Cross-complementation of P. aeruginosa knockout mutants lacking
wbpA, wbpB, wbpE, wbpD, or wbpI with the homologues from
B. pertussis could fully restore LPS production in the P.
aeruginosa LPS mutants, suggesting that the genes from B.
pertussis are functional homologs of the wbp genes
(7). Homologs of these genes
could be identified in diverse bacterial species, demonstrating the importance
of UDP-d-ManNAc3NAcA biosynthesis beyond its role in P.
aeruginosa (7).Open in a separate windowFIGURE 1.Proposed pathway for the biosynthesis of UDP-d-ManNAc3NAcA in
P. aeruginosa PAO1. The full names of the sugars are as follows:
GlcNAc, 2-acetamido-2-deoxy-d-glucose; GlcNAcA,
2-acetamido-2-deoxy-d-glucuronic acid; 3-keto-d-GlcNAcA,
2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid; GlcNAc3NA,
2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid; GlcNAc3NAcA,
2,3-diacetamido-2,3-dideoxy-d-glucuronic acid; ManNAc3NAcA,
2,3-diacetamido-2,3-dideoxy-d-mannuronic acid. Adapted from Ref.
8.The first enzyme of the UDP-d-ManNAc3NAcA biosynthesis pathway,
WbpA, is a 6-dehydrogenase that converts
UDP-2-acetamido-2-deoxy-d-glucose
(N-acetyl-d-glucosamine; UDP-d-GlcNAc) to
UDP-2-acetamido-2-deoxy-d-glucuronic acid
(N-acetyl-d-glucosaminuronic acid,
UDP-d-GlcNAcA) using NAD+ as a coenzyme
(9)
(Fig. 1). Following this, the
second step in UDP-d-ManNAc3NAcA biosynthesis is proposed to be an
oxidation reaction catalyzed by WbpB, forming
UDP-2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid
(3-keto-d-GlcNAcA), which in turn is used as the substrate for
transamination by WbpE, creating
UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid
(d-GlcNAc3NA).This residue is thought to be the substrate for WbpD, a putative
N-acetyltransferase of the hexapeptide acyltransferase superfamily
(10) that requires acetyl-CoA
as a co-substrate (8). WbpD has
been proposed to synthesize
UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid
(UDP-d-GlcNAc-3NAcA), which is utilized in the B-band O-antigen of
P. aeruginosa serotype O1. In P. aeruginosa serogroup O2,
the UDP-d-GlcNAc3NAcA is then epimerized by WbpI to create the
UDP-d-ManNAc3NAcA required for incorporation into B-band LPS
(11). A derivative of
UDP-d-ManNAc3NAcA is also used in the synthesis of B-band O-antigen
of P. aeruginosa serogroup O2. UDP-d-ManNAc3NAmA is
thought to be produced through additional modification of
UDP-d-ManNAc3NAcA via the action of WbpG, an amidotransferase,
which has also been demonstrated to be essential for the production of B-band
O-antigen (12,
13).In the current study, our aim was to define the function of WbpB, WbpE, and
WbpD, because only genetic evidence has previously been given for the
involvement of wbpB and wbpE
(7), and the reaction catalyzed
by WbpD could not be demonstrated due to the unavailability of its presumed
substrate, UDP-d-GlcNAc3NA
(8). The functional
characterization of these proteins is also important for understanding LPS
biosynthesis in B. pertussis, because the genes in the LPS locus of
this species, wlbA, wlbC, and wlbB, could cross-complement
knockouts of wbpB, wbpE, and wbpD, respectively, when
expressed in P. aeruginosa PAO1
(7). Furthermore, these three
proteins form a cassette for the generation of C-3 N-acetylated
hexoses and may be important for the biosynthesis of a variety of other
sugars. Capillary electrophoresis and MALDI-TOF mass spectrometry were used to
analyze reaction mixtures of WbpB and WbpE and showed that the expected
products were produced only when both enzymes were present together. Achieving
the enzymatic synthesis of the product of both enzymes, which was demonstrated
to be UDP-d-GlcNAc3NA by 1H NMR spectroscopy, was a key
breakthrough, because this rare sugar has never before been produced by any
means. UDP-d-GlcNAc3NA was also essential for use as the substrate
of WbpD, which not only allowed us to determine the enzymatic activity of this
protein but also allowed the enzymatic synthesis of
UDP-d-GlcNAc3NAcA to be achieved as well. Although this sugar had
previously been produced through a 17-step chemical synthesis
(11,
14), the 4-step concurrent
enzymatic reaction demonstrates the advantage of linking chemistry with
biology and represents a significant saving of both time and reagents as
compared with chemical synthesis. Finally, our data also showed the success in
reconstituting in vitro the 5-step pathway for the biosynthesis of
UDP-d-ManNAc3NAcA in P. aeruginosa. 相似文献
20.
Maria De Angelis Francesca Bottacini Bruno Fosso Philip Kelleher Maria Calasso Raffaella Di Cagno Marco Ventura Ernesto Picardi Douwe van Sinderen Marco Gobbetti 《PloS one》2014,9(9)
Lactobacillus rossiae is an obligately hetero-fermentative lactic acid bacterium, which can be isolated from a broad range of environments including sourdoughs, vegetables, fermented meat and flour, as well as the gastrointestinal tract of both humans and animals. In order to unravel distinctive genomic features of this particular species and investigate the phylogenetic positioning within the genus Lactobacillus, comparative genomics and phylogenomic approaches, followed by functional analyses were performed on L. rossiae DSM 15814T, showing how this type strain not only occupies an independent phylogenetic branch, but also possesses genomic features underscoring its biotechnological potential. This strain in fact represents one of a small number of bacteria known to encode a complete de novo biosynthetic pathway of vitamin B12 (in addition to other B vitamins such as folate and riboflavin). In addition, it possesses the capacity to utilize an extensive set of carbon sources, a characteristic that may contribute to environmental adaptation, perhaps enabling the strain''s ability to populate different niches. 相似文献