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Borate-Rhamnogalacturonan II Bonding Reinforced by
Ca2+ Retains Pectic Polysaccharides in Higher-Plant Cell
Walls
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The extent of in vitro formation of
the borate-dimeric-rhamnogalacturonan II (RG-II) complex was stimulated
by Ca2+. The complex formed in the presence of
Ca2+ was more stable than that without Ca2+. A
naturally occurring boron (B)-RG-II complex isolated from radish
(Raphanus sativus L. cv Aokubi-daikon) root contained
equimolar amounts of Ca2+ and B. Removal of the
Ca2+ by
trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic
acid induced cleavage of the complex into monomeric RG-II. These data
suggest that Ca2+ is a normal component of the B-RG-II
complex. Washing the crude cell walls of radish roots with a 1.5%
(w/v) sodium dodecyl sulfate solution, pH 6.5, released 98% of the
tissue Ca2+ but only 13% of the B and 22% of the pectic
polysaccharides. The remaining Ca2+ was associated with
RG-II. Extraction of the sodium dodecyl sulfate-washed cell walls with
50 mm
trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic
acid, pH 6.5, removed the remaining Ca2+, 78%
of B, and 49% of pectic polysaccharides. These results suggest that
not only Ca2+ but also borate and Ca2+
cross-linking in the RG-II region retain so-called chelator-soluble
pectic polysaccharides in cell walls.Boron (B) is an essential element for higher plant growth,
although its primary function is not known (Loomis and Durst, 1992).
Determining the sites of B in cells is required to identify its
function. In cultured tobacco cells more than 80% of cellular B is in
the cell wall (Matoh et al., 1993), whereas the membrane fraction
(Kobayashi et al., 1997) and protoplasts (Matoh et al., 1992) do not
contain a significant amount of B. In radish (Raphanus
sativus L. cv Aokubi-daikon) root cell walls, B cross-links two
RG-II regions of pectic polysaccharides through a borate-diol ester
(Kobayashi et al., 1995, 1996). The association of B with RG-II has
been confirmed in sugar beet (Ishii and Matsunaga, 1996), bamboo
(Kaneko et al., 1997), sycamore and pea (O''Neill et al., 1996), and
red wine (Pellerin et al., 1996). In cultured tobacco cells the B
associated with RG-II accounts for about 80% of the cell wall B
(Kobayashi et al., 1997) and RG-II may be the exclusive carrier of B in
higher plant cell walls (Matoh et al., 1996). Germanic acid, which
partly substitutes for B in the growth of the B-deprived plants (Skok,
1957), also cross-links two RG-II chains (Kobayashi et al., 1997).
These results suggest that the physiological role of B is to cross-link
cell wall pectic polysaccharides in the RG-II region and thereby form a
pectic network.It is believed that in the cell wall pectic polysaccharides are
cross-linked with Ca2+, which binds to carboxyl
groups of the polygalacturonic acid regions (Jarvis, 1984). Thus, the
ability of B and Ca2+ to cross-link cell wall
pectic polysaccharides needs to be evaluated. In this report we
describe the B-RG-II complex of radish root and the role of B-RG-II and
Ca2+ in the formation of a pectic
network. 相似文献
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Bertram T. Ober Artur Summerfield Christina Mattlinger Karl-Heinz Wiesmüller Günther Jung Eberhard Pfaff Armin Saalmüller Hanns-Joachim Rziha 《Journal of virology》1998,72(6):4866-4873
Pseudorabies virus (PRV; suid herpesvirus 1) infection causes heavy economic losses in the pig industry. Therefore, vaccination with live attenuated viruses is practiced in many countries. This vaccination was demonstrated to induce extrathymic virus-specific memory CD4+CD8+ T lymphocytes. Due to their major histocompatibility complex (MHC) class II-restricted proliferation, it is generally believed that these T lymphocytes function as memory T-helper cells. To directly prove this hypothesis, 15-amino-acid, overlapping peptides of the viral glycoprotein gC were used for screening in proliferation assays with peripheral blood mononuclear cells of vaccinated d/d haplotype inbred pigs. In these experiments, two naturally processed T-cell epitopes (T1 and T2) which are MHC class II restricted were identified. It was shown that extrathymic CD4+CD8+ T cells are the T-lymphocyte subpopulation that responds to epitope T2. In addition, we were able to show that cytokine secretion can be induced in these T cells through recall with inactivated PRV and demonstrated that activated PRV-primed CD4+CD8+ T cells are able to induce PRV-specific immunoglobulin synthesis by PRV-primed, resting B cells. Taken together, these results demonstrate that the glycoprotein gC takes part in the priming of humoral anti-PRV memory responses. The experiments identified the first T-cell epitopes so far known to induce the generation of virus-specific CD4+CD8+ memory T lymphocytes and showed that CD4+CD8+ T cells are memory T-helper cells. Therefore, this study describes the generation of virus-specific CD4+CD8+ T cells, which is observed during vaccination, as a part of the potent humoral anti-PRV memory response induced by the vaccine.Pseudorabies virus (PRV), a member of the Alphaherpesvirinae, is the causative agent of Aujeszky’s disease. This disease is lethal to young pigs and causes important economic losses (52). Therefore, vaccination of pigs is practiced in many countries.Several humoral immune system effector mechanisms are involved in the protection of pigs from PRV infection. Virus-neutralizing antibodies, antibodies mediating antibody-dependent cell-mediated cytotoxicity, and antibodies mediating complement-mediated lysis of PRV-infected target cells have been demonstrated (22, 23, 53, 54). The main targets of this humoral immune response were shown to be the viral glycoproteins (3, 45), and passive immunization with monoclonal antibodies (MAbs) against gB, gC, and gD protects pigs from a lethal challenge (20, 49).The protection conferred through cell-mediated immunity is poorly understood. An increase in major histocompatibility complex (MHC)-unrestricted cell-mediated cytotoxicity against uninfected and PRV-infected cells has been detected after infection or vaccination of pigs with PRV (16, 53, 54), and specific cellular immune responses to PRV infections could be demonstrated by stimulation of proliferation and lymphokine secretion of porcine PRV-immune lymphocytes (10, 17, 42, 43, 51) as well as by the detection of PRV-specific cytotoxic lymphocytes (21, 56).There are some difficulties in defining more precisely the impact of cell-mediated immune effector mechanisms to protection from PRV-infection and their interplay with the observed humoral immune response. Considerably fewer porcine than human or mouse differentiation markers are available (34). In addition, the immune system of swine differs considerably from that of humans and mice. The pig has a substantial number of CD4−CD8− T lymphocytes in the peripheral blood (4, 6, 12, 36, 39). In young animals, this subpopulation of T lymphocytes comprises up to 60% of the T lymphocytes and contains mainly γδ T lymphocytes. The pig is also the only species so far known to contain a substantial number of resting extrathymic CD4+CD8+ T lymphocytes (28, 36, 39). This T-lymphocyte population shows morphologically the phenotype of mature T lymphocytes (40) and increases with age to up to 60% of peripheral T lymphocytes (29, 35, 39, 55). Further, it was demonstrated that CD4+CD8+ T lymphocytes comprise memory T cells which proliferate upon stimulation with recall antigen (43, 55). Since the observed proliferative response was shown to be MHC class II-restricted, it was speculated that the porcine CD4+CD8+ T-cell subset contains memory T-helper lymphocytes (43). However, the ability of these T lymphocytes to secrete cytokines or to provide help to B cells has so far not been demonstrated.To gain a better understanding of immune effector mechanisms conferring protection from PRV infection, the function of these unusual extrathymic T-lymphocyte subsets has to be elucidated. In the present study, we identified two T-cell epitopes on glycoprotein gC which are primed during vaccination of d/d haplotype inbred pigs (41) against PRV and demonstrated that MHC class II-restricted, peripheral CD4+CD8+ memory T lymphocytes are the responding T lymphocytes. We were further able to show that PRV-specific, extrathymic CD4+CD8+ T lymphocytes are able to secrete cytokines and have the capacity to stimulate the secretion of PRV-specific immunoglobulins (Ig) by PRV-primed B cells. These results demonstrate that porcine CD4+CD8+ T lymphocytes can function as memory T-helper cells and can direct humoral anti-PRV memory responses. 相似文献
7.
An Important Role for Major Histocompatibility Complex Class I-Restricted T Cells,and a Limited Role for Gamma Interferon,in Protection of Mice against Lethal Herpes Simplex Virus Infection
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Ai-Xuan Holterman Kathleen Rogers Kurt Edelmann David M. Koelle Lawrence Corey Christopher B. Wilson 《Journal of virology》1999,73(3):2058
Herpes simplex virus (HSV) inhibits major histocompatibility complex (MHC) class I expression in infected cells and does so much more efficiently in human cells than in murine cells. Given this difference, if MHC class I-restricted T cells do not play an important role in protection of mice from HSV, an important role for these cells in humans would be unlikely. However, the contribution of MHC class I-restricted T cells to the control of HSV infection in mice remains unclear. Further, the mechanisms by which these cells may act to control infection, particularly in the nervous system, are not well understood, though a role for gamma interferon (IFN-γ) has been proposed. To address the roles of MHC class I and of IFN-γ, C57BL/6 mice deficient in MHC class I expression (β2 microglobulin knockout [β2KO] mice), in IFN-γ expression (IFN-γKO mice), or in both (IFN-γKO/β2KO mice) were infected with HSV by footpad inoculation. β2KO mice were markedly compromised in their ability to control infection, as indicated by increased lethality and higher concentrations of virus in the feet and spinal ganglia. In contrast, IFN-γ appeared to play at most a limited role in viral clearance. The results suggest that MHC class I-restricted T cells play an important role in protection of mice against neuroinvasive HSV infection and do so largely by mechanisms other than the production of IFN-γ.Two gene products of herpes simplex virus (HSV) block presentation of viral proteins by class I major histocompatibility complex (MHC) molecules: the viral host shutoff protein (vhs), which is present in the viral particle, and the immediate-early protein ICP47 (1, 14, 41, 42). Through the sequential action of these proteins, antigen presentation by MHC class I is inhibited early in the viral replication cycle. ICP47 binds to human transporter associated with antigen-processing proteins (TAP), thereby inhibiting peptide loading on MHC class I and recognition by HSV-specific, MHC class I-restricted, CD8+ T cells (1, 14, 42, 43). This effect is greatest in nonhematopoietic cells in which the abundance of MHC class I and TAP are lower than in antigen-presenting cells (41). As a consequence, HSV is more likely to impair recognition of infected target cells in the tissues than to block the generation of antigen-specific CD8+ T cells. Consistent with this, recent studies indicate that HSV antigen-specific CD8+ cytotoxic-T-lymphocyte (CTL) precursors can be readily detected in the blood and cutaneous lesions of HSV-infected individuals (16, 31, 32). However, NK cells and HSV antigen-specific CD4+ T cells are detected earlier than antigen-specific CD8+ T cells in lesions of humans with recurrent HSV-2 disease (16). This finding has led to the proposal that gamma interferon (IFN-γ) produced by infiltrating NK and CD4+ T cells overrides the inhibitory effects of HSV on TAP function and MHC class I expression (22, 41), thereby allowing the eradication of virus by CD8+ T cells, whose numbers increase in lesions around the time of viral clearance (16, 31). In patients with AIDS, a lower frequency in the blood of HSV antigen-specific CD8+ CTL precursors is associated with more frequent and severe recurrences of genital disease (32). These correlative data suggest that CD8+ T cells may play an important role in the clearance of HSV in humans, at least from mucocutaneous lesions.ICP47 inhibits murine TAP poorly (1, 42), which may explain the greater ease with which anti-HSV CD8+ CTLs have been detected in mice than in humans (3, 8, 28, 34, 35). Despite the weak interaction of ICP47 with murine TAP, results of a recent study (12) suggested that ICP47 impairs CD8+ T-cell-dependent viral clearance from the nervous system: CD8+ T cells protected susceptible BALB/c or A/J mice from lethal, nervous system infection with an HSV mutant lacking ICP47 but did not appear to protect against infection with wild-type HSV or to contribute to clearance of either virus from the eye. These findings are consistent with data suggesting that CD8+ T cells limit persistence of HSV in the spinal ganglia and decrease spread to the central nervous system (35, 36). However, other studies have concluded that CD4+ T cells but not CD8+ T cells play the critical role in viral clearance and protection from lethal primary infection with wild-type HSV (20, 23, 24) or that either CD4+ or CD8+ T cells are sufficient for protection (26, 37). Since the effects of ICP47 are likely to be greater in humans than in mice, if MHC class I-restricted CD8+ T cells do not play an important role in protection of mice from lethal, neuroinvasive infection due to wild-type HSV, an important role in humans would be unlikely.The mechanisms by which T cells may limit the spread of infection in the nervous system are not clearly understood. Studies by Simmons and colleagues suggested that CD8+ T cells may lyse infected Schwann cells or satellite cells but that they probably do not lyse infected neurons (31, 32). They and others have proposed that CD8+ T cells protect neurons through the production of cytokines, in particular IFN-γ (35, 36). IFN-γ contributes to the clearance of HSV from mucocutaneous sites (4, 24, 25, 37, 44). However, the role of IFN-γ in protection from lethal, neuroinvasive infection is uncertain and may vary with the strain of mice, method used to inhibit IFN-γ function, and route of inoculation (4, 5, 24, 37, 44). IFN-γ is produced in the ganglia of mice with acute or latent HSV infection (5, 13, 19). Both CD4+ and CD8+ T cells (and NK cells) produce IFN-γ, but CD4+ T cells appear to be the predominant source of IFN-γ following intravaginal infection with HSV (24, 25). Thus, it is possible that the disparity in results regarding the relative importance of CD4+ and CD8+ T cells in protection from lethal, neuroinvasive HSV infection reflects their redundant roles in production of this cytokine or that IFN-γ and CD8+ T cells contribute independently to control of infection in the nervous system.To address in parallel the contributions of MHC class I-restricted T cells and of IFN-γ to protection of mice from HSV, MHC class I and CD8+ T-cell-deficient β2 microglobulin knockout (β2KO) mice, IFN-γ knockout (IFN-γKO) mice, and mice deficient in both MHC class I and IFN-γ expression (IFN-γKO/β2KO) were studied. The results indicated that loss of MHC class I expression in β2KO mice substantially increased their susceptibility to HSV, whereas the loss of IFN-γ expression had a much more limited effect. These findings indicate that MHC class I-restricted T cells play an important role in protection against neuroinvasive HSV infection in mice and that they do so largely by mechanisms other than the production of IFN-γ. Though MHC class I expression is more severely impaired in β2KO mice than in human cells infected with wild-type HSV, these findings support the notion that inhibition of MHC class I expression is an important factor in the virulence of this virus. 相似文献
8.
Utilization of an NF-ATp Binding Promoter Element for EGR3 Expression in T Cells but Not Fibroblasts Provides a Molecular Model for the Lymphoid Cell-Specific Effect of Cyclosporin A 总被引:2,自引:1,他引:1
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Hans W. Mages Rima Baag Birgit Steiner Richard A. Kroczek 《Molecular and cellular biology》1998,18(12):7157-7165
9.
The effect of copper on
photoinhibition of photosystem II in vivo was studied in
bean (Phaseolus vulgaris L. cv Dufrix). The plants were
grown hydroponically in the presence of various concentrations of
Cu2+ ranging from the optimum 0.3 μm
(control) to 15 μm. The copper concentration of leaves
varied according to the nutrient medium from a control value of 13 mg
kg−1 dry weight to 76 mg kg−1 dry weight.
Leaf samples were illuminated in the presence and absence of lincomycin
at different light intensities (500–1500 μmol photons
m−2 s−1). Lincomycin prevents the concurrent
repair of photoinhibitory damage by blocking chloroplast protein
synthesis. The photoinhibitory decrease in the light-saturated rate of
O2 evolution measured from thylakoids isolated from treated
leaves correlated well with the decrease in the ratio of variable to
maximum fluorescence measured from the leaf discs; therefore, the
fluorescence ratio was used as a routine measurement of photoinhibition
in vivo. Excess copper was found to affect the
equilibrium between photoinhibition and repair, resulting in a decrease
in the steady-state concentration of active photosystem II centers of
illuminated leaves. This shift in equilibrium apparently resulted from
an increase in the quantum yield of photoinhibition (ΦPI)
induced by excess copper. The kinetic pattern of photoinhibition and
the independence of ΦPI on photon flux density were not
affected by excess copper. An increase in ΦPI may
contribute substantially to Cu2+ toxicity in certain plant
species.Cu2+ is an essential micronutrient but in
excess is toxic for plants. It is a redox-active metal that functions
as an enzyme activator and is an important part of prosthetic groups of
many enzymes (for review, see Sandmann and Böger, 1983). Copper
concentrations in healthy plant tissues range from 5 to 20 mg
kg−1 dry weight. In
Cu2+-rich environments, accumulation of
Cu2+ in plant tissues depends on the species and
cultivar. Cu2+ seems to have several sites of
action, which vary among plant species. Toxic concentrations of
Cu2+ inhibit metabolic activity, which leads to
suppressed growth and slow development. Most Cu2+
ions are immobilized to the cell walls of roots or of mycorrhizal fungi
(Kahle, 1993).When the tolerance mechanisms in the root zone become overloaded,
Cu2+ is translocated by both the xylem and phloem
up to the leaves. Excess Cu2+ may replace other
metals in metalloproteins or may interact directly with SH groups of
proteins (Uribe and Stark, 1982). Cu2+-induced
free-radical formation may also cause protein damage (for review, see
Fernandes and Henriques, 1991; Weckx and Clijsters, 1996). High
concentrations of Cu2+ may catalyze the formation
of the hydroxyl radical from O2 and
H2O2. This
Cu2+-catalyzed Fenton-type reaction takes place
mainly in chloroplasts (Sandmann and Böger, 1980). The hydroxyl
radical may start the peroxidation of unsaturated membrane lipids and
chlorophyll (Sandmann and Böger, 1980), and these inhibitory
mechanisms might contribute to the observed inhibition of
photosynthetic electron transport by excess Cu2+
(Clijsters and Van Assche, 1985).The role of Cu2+ as an inhibitor of
photosynthetic electron transport has been studied in vitro. Both the
donor (Cedeno-Maldonado and Swader, 1972; Samuelsson and Öquist,
1980; Schröder et al., 1994) and acceptor (Mohanty et al., 1989;
Yruela et al., 1992, 1993, 1996a, 1996b; Jegerschöld et al.,
1995) sides of PSII have been proposed to be the most sensitive site
for Cu2+ action. On the donor side,
Cu2+ is thought to inhibit electron transport to
P680, the primary donor of PSII (Schröder et al., 1994). On the
acceptor side, Cu2+ interactions with the
pheophytin-QA-Fe2+-domain
or Cu2+-induced modifications in the amino acid
or lipid structure close to the QA- and
QB-binding sites have been suggested to cause the
inhibition of electron transport (Jegerschöld et al., 1995;
Yruela et al., 1996a, 1996b).Celeno-Maldonado and Swader (1972) noticed that preincubation of
chloroplasts in the light enhanced the
Cu2+-induced inhibition of electron transport,
and that PSII was more susceptible to this kind of inhibition than was
PSI. The hypothetical acceptor- and donor-side mechanisms of the
light-induced inhibition of electron transport, photoinhibition,
involve the same domains of attack as Cu2+. Both
acceptor- and donor-side photoinhibition trigger the D1 polypeptide of
the PSII reaction center for degradation (for review, see Aro et al.,
1993). The damaged D1 protein is degraded, and the recovery of PSII
activity needs de novo synthesis of D1 protein. Photoinhibition occurs
at all light intensities (Tyystjärvi and Aro, 1996); therefore,
the cycle of PSII photoinhibition, which is followed by degradation,
and, finally, resynthesis of the D1 protein, runs constantly in plant
cells in the light. If the photoinhibition-repair cycle is allowed to
run for some time at a constant light intensity, equilibrium is
reached. At equilibrium (steady state), all three reaction rates
(photoinhibition, D1 degradation, and D1 synthesis) are equal. Healthy
plants are often able to maintain a high steady-state concentration of
active PSII under widely varying light intensities. Even if the
concentration of active PSII is lowered by high light, the
concentration of D1 protein tends to stay fairly constant (Cleland et
al., 1990; Kettunen et al., 1991). In the bean (Phaseolus
vulgaris L.) plants used in this study the steady-state D1 protein
content remained almost constant even in the presence of excess
Cu2+.The effect of Cu2+ on photoinhibition in
vivo has been studied very little. Vavilin et al. (1995) suggest that
excess Cu2+ may slow the PSII repair cycle in the
green alga Chlorella pyrenoidosa, and Ouzounidou et al.
(1997) suggest that Cu2+ inhibits adaptation to
light in maize. In the current study we show that excess
Cu2+ induces a large increase in the rate
constant of photoinhibition in vivo in a higher plant. 相似文献
10.
Cellular compartmentation of Zn in the leaves of the
hyperaccumulator Thlaspi caerulescens was investigated
using energy-dispersive x-ray microanalysis and single-cell sap
extraction. Energy-dispersive x-ray microanalysis of frozen, hydrated
leaf tissues showed greatly enhanced Zn accumulation in the epidermis
compared with the mesophyll cells. The relative Zn concentration in the
epidermal cells correlated linearly with cell length in both young and
mature leaves, suggesting that vacuolation of epidermal cells may
promote the preferential Zn accumulation. The results from single-cell
sap sampling showed that the Zn concentrations in the epidermal
vacuolar sap were 5 to 6.5 times higher than those in the
mesophyll sap and reached an average of 385 mm in plants
with 20,000 μg Zn g−1 dry weight of shoots. Even when
the growth medium contained no elevated Zn, preferential Zn
accumulation in the epidermal vacuoles was still evident. The
concentrations of K, Cl, P, and Ca in the epidermal sap generally
decreased with increasing Zn. There was no evidence of association of
Zn with either P or S. The present study demonstrates that Zn is
sequestered in a soluble form predominantly in the epidermal vacuoles
in T. caerulescens leaves and that mesophyll cells are
able to tolerate up to at least 60 mm Zn in their sap.Different mechanisms have been proposed to explain the tolerance
of plants to toxic heavy metals (Baker and Walker, 1990; Verkleij and
Schat, 1990). Some tolerant plant species, the so-called
“excluders,” use exclusion mechanisms by which uptake and/or
root-to-shoot transport of heavy metals are restricted. Other tolerant
plant species are able to cope with elevated concentrations of toxic
metals inside of their tissues through production of metal-binding
compounds, cellular and subcellular compartmentation, or alterations of
metabolism.An extreme strategy for metal tolerance that is in sharp contrast to
metal exclusion is “hyperaccumulation,” a term that was originally
used by Brooks et al. (1977) to describe plants that can accumulate
more than 1,000 μg Ni g−1 dry weight in their
aerial parts. Approximately 400 taxa of terrestrial plants have been
identified as hyperaccumulators of various heavy metals, with about 300
being Ni hyperaccumulators (Baker and Brooks 1989; Brooks, 1998). Only
16 species of Zn hyperaccumulators, which are defined as being able to
accumulate more than 10,000 μg Zn g−1 in the
aboveground parts on a dry weight basis in their natural habitat
(Brooks, 1998), have been reported. Thlaspi caerulescens J.
& C. Presl (Brassicaceae) is the best-known example of a Zn/Cd
hyperaccumulator. Under hydroponic culture conditions T.
caerulescens can accumulate up to 25,000 to 30,000 μg Zn
g−1 dry weight in the shoots without showing any
toxicity symptoms or reduction in growth (Brown et al., 1996a; Shen et
al., 1997). Recently, there has been a surge of interest in the
phenomenon of heavy-metal hyperaccumulation because this property may
be exploited in the remediation of heavy-metal-polluted soils through
phytoextraction and phytomining (McGrath et al., 1993; Brown et al.,
1995b; Robinson et al., 1997).The mechanisms for metal hyperaccumulation are not fully understood,
and this is particularly true in the case of the Zn/Cd
hyperaccumulators. To cope with the consequence of hyperaccumulation,
plants must also be hypertolerant to the heavy metals that accumulate.
Recent studies comparing the different populations of T.
caerulescens have shown that hyperaccumulation of Zn is a
constitutive property, although the traits are probably separate from
those for tolerance (Baker et al., 1994; Meerts and Van Isacker, 1997).
Compared with the nonaccumulating species, T. caerulescens
possesses an enhanced capacity to take up Zn and transport it from
roots to shoots (Baker et al., 1994; Brown et al., 1995a; Shen et al.,
1997). Lasat et al. (1996) found that roots of T.
caerulescens and the nonaccumulator Thlaspi arvense had
similar apparent Km values for
Zn2+, but that the
Vmax in the former was 4.5-fold higher than
that in the latter species, indicating that the hyperaccumulator
T. caerulescens possessed more
Zn2+-transport sites in the plasma membranes of
root cells. Shen et al. (1997) showed that T. caerulescens
was much more effective in exporting the Zn that was accumulated
previously in roots to the shoots than an intermediate accumulator
species, Thlaspi ochrolucum. Organic acids such as malic
acid have been suggested to play a key role in shuttling Zn from
cytoplasm to vacuoles (Mathys, 1977). However, the
low affinity of malate to chelate Zn (stability constant
pK = 3.5 at infinite dilution) does not favor this
hypothesis. Moreover, high concentrations of malate found in the shoot
tissues of T. caerulescens appear to be a constitutive
property (Tolrà et al., 1996; Shen et al., 1997).The extraordinary tolerance of hyperaccumulator plants must also
involve compartmentation of toxic metals at the cellular and
subcellular levels. Vázquez et al. (1992, 1994) studied
localization of Zn in the root and leaf tissues of T.
caerulescens using EDXMA. They compared two methods of sample
preparation and found that Na2S fixation was not
suitable for preventing the loss of metal ions from the samples. Using
cryofixation and freeze substitution, they showed that Zn accumulated
mainly in the vacuoles as electron-dense deposits. Many vacuoles of
leaf-epidermal and subepidermal cells contained globular crystals that
were very rich in Zn. However, it is not known whether the Zn-rich,
globular crystal deposits occur inside of the leaf vacuoles in vivo or
if they are artifacts caused by sample preparation. Also, the technique
used by Vázquez et al. (1992, 1994) allows only semiquantitative
determination of Zn concentrations.In this study we used two techniques to investigate cellular
compartmentation of Zn in the leaves of T. caerulescens. The
first utilized EDXMA of frozen, hydrated tissue to survey the
distribution patterns of Zn and other elements across different leaf
cells. The second method involved sampling sap from single cells using
microcapillaries, followed by fully quantitative determination of Zn
and other elements using EDXMA. 相似文献
11.
Lidia Osuna Jean-N?el Pierre María-Cruz González Rosario Alvarez Francisco J. Cejudo Cristina Echevarría Jean Vidal 《Plant physiology》1999,119(2):511-520
Phosphoenolpyruvate
carboxylase (PEPC) activity was detected in aleurone-endosperm extracts
of barley (Hordeum vulgare) seeds during germination,
and specific anti-sorghum (Sorghum bicolor)
C4 PEPC polyclonal antibodies immunodecorated constitutive
103-kD and inducible 108-kD PEPC polypeptides in western analysis. The
103- and 108-kD polypeptides were radiolabeled in situ after imbibition
for up to 1.5 d in 32P-labeled inorganic phosphate. In
vitro phosphorylation by a Ca2+-independent PEPC protein
kinase (PK) in crude extracts enhanced the enzyme''s velocity and
decreased its sensitivity to l-malate at suboptimal pH and
[PEP]. Isolated aleurone cell protoplasts contained both
phosphorylated PEPC and a Ca2+-independent PEPC-PK that was
partially purified by affinity chromatography on blue dextran-agarose.
This PK activity was present in dry seeds, and PEPC phosphorylation in
situ during imbibition was not affected by the cytosolic
protein-synthesis inhibitor cycloheximide, by weak acids, or by various
pharmacological reagents that had proven to be effective blockers of
the light signal transduction chain and PEPC phosphorylation in
C4 mesophyll protoplasts. These collective data support the
hypothesis that this Ca2+-independent PEPC-PK was formed
during maturation of barley seeds and that its presumed underlying
signaling elements were no longer operative during germination.Higher-plant PEPC (EC 4.1.1.31) is subject to in vivo
phosphorylation of a regulatory Ser located in the N-terminal domain of
the protein. In vitro phosphorylation by a
Ca2+-independent, low-molecular-mass (30–39 kD)
PEPC-PK modulates PEPC regulation interactively by opposing metabolite
effectors (e.g. allosteric activation by Glc-6-P and feedback
inhibition by l-malate; Andreo et al., 1987), decreasing
significantly the extent of malate inhibition of the leaf enzyme
(Carter et al., 1991; Chollet et al., 1996; Vidal et al., 1996; Vidal
and Chollet, 1997). These metabolites control the rate of
phosphorylation of PEPC via an indirect target-protein effect (Wang and
Chollet, 1993; Echevarría et al., 1994; Vidal and Chollet,
1997).Several lines of evidence support the view that this protein-Ser/Thr
kinase is the physiologically relevant PEPC-PK (Li and Chollet, 1993;
Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). The
presence and inducible nature of leaf PEPC-PK have been established
further in various C3, C4,
and CAM plant species (Chollet et al., 1996). In all cases, CHX proved
to be a potent inhibitor of this up-regulation process so that apparent
changes in the turnover rate of PEPC-PK itself or another, as yet
unknown, protein factor were invoked to account for this observation
(Carter et al., 1991; Jiao et al., 1991; Chollet et al., 1996).
Consistent with this proposal are recent findings about PEPC-PK from
leaves of C3, C4, and CAM
plants that determined activity levels of the enzyme to depend on
changes in the level of the corresponding translatable mRNA (Hartwell
et al., 1996).Using a cellular approach we previously showed in
sorghum (Sorghum bicolor) and hairy crabgrass
(Digitaria sanguinalis) that PEPC-PK is
up-regulated in C4 mesophyll cell protoplasts
following illumination in the presence of a weak base
(NH4Cl or methylamine; Pierre et al., 1992;
Giglioli-Guivarc''h et al., 1996), with a time course (1–2 h) similar
to that of the intact, illuminated sorghum (Bakrim et al., 1992) or
maize leaf (Echevarría et al., 1990). This light- and
weak-base-dependent process via a complex transduction chain is likely
to involve sequentially an increase in pHc, inositol
trisphosphate-gated Ca2+ channels of the
tonoplast, an increase in cytosolic Ca2+, a
Ca2+-dependent PK, and PEPC-PK.Considerably less is known about the up-regulation of PEPC-PK and
PEPC phosphorylation in nongreen tissues. A sorghum root PEPC-PK
purified on BDA was shown to phosphorylate in vitro both recombinant
C4 PEPC and the root
C3-like isoform, thereby decreasing the enzyme''s
malate sensitivity (Pacquit et al., 1993). PEPC from soybean root
nodules was phosphorylated in vitro and in vivo by an endogenous PK
(Schuller and Werner, 1993; Zhang et al., 1995; Zhang and Chollet,
1997). A Ca2+-independent nodule PEPC-PK
containing two active polypeptides (32–37 kD) catalyzed the
incorporation of phosphate on a Ser residue of the target enzyme and
was modulated by photosynthate transported from the shoots (Zhang and
Chollet, 1997). Regulatory seryl phosphorylation of a heterotetrameric
(α2β2) banana fruit
PEPC by a copurifying, Ca2+-independent PEPC-PK
was shown to occur in vitro (Law and Plaxton, 1997). Although
phosphorylation was also detected in vivo and found to concern
primarily the α-subunit, PEPC exists mainly in the dephosphorylated
form in preclimacteric, climacteric, and postclimacteric fruit.In a previous study we showed that PEPC undergoes regulatory
phosphorylation in aleurone-endosperm tissue during germination of
wheat seeds (Osuna et al., 1996). Here we report on PEPC and the
requisite PEPC-PK in germinating barley (Hordeum vulgare)
seeds. PEPC was highly phosphorylated by a
Ca2+-independent Ser/Thr PEPC-PK similar to that
found in other plant systems studied previously (Chollet et al., 1996);
however, the PK was already present in the dry seed and its activity
did not require protein synthesis during imbibition. 相似文献
12.
Claudia A. Blindauer Ian Harvey Kerry E. Bunyan Alan J. Stewart Darrell Sleep David J. Harrison Stephen Berezenko Peter J. Sadler 《The Journal of biological chemistry》2009,284(34):23116-23124
Most blood plasma zinc is bound to albumin, but the structure of the binding site has not been determined. Zn K-edge extended x-ray absorption fine structure spectroscopy and modeling studies show that the major Zn2+ site on albumin is a 5-coordinate site with average Zn-O/N distances of 1.98 Å and a weak sixth O/N bond of 2.48 Å, consistent with coordination to His67 and Asn99 from domain I, His247 and Asp249 from domain II (residues conserved in all sequenced mammalian albumins), plus a water ligand. The dynamics of the domain I/II interface, thought to be important to biological function, are affected by Zn2+ binding, which induces cooperative allosteric effects related to those of the pH-dependent neutral-to-base transition. N99D and N99H mutations enhance Zn2+ binding but alter protein stability, whereas mutation of His67 to alanine removes an interdomain H-bond and weakens Zn2+ binding. Both wild-type and mutant albumins promote the safe management of high micromolar zinc concentrations for cells in cultures.Zinc is not only required for hundreds of essential extra- and intracellular proteins and enzymes but is also recruited by toxins such as anthrax lethal factor (1) and staphylococcal enterotoxin (2). There is a need to understand how zinc transport and distribution is controlled (3).Although considerable progress has been made in the identification and study of membrane-bound zinc transporters, the molecular mechanism of extracellular zinc transport is still obscure. The total concentration of zinc in blood is high, ∼15–20 μm (4), and plasma zinc concentrations are maintained at a relatively constant level, except during periods of dietary zinc depletion and acute responses to stress or inflammation, when they are depressed (5). In humans, ∼98% of so-called “exchangeable” zinc in blood plasma (9–14 μm) is bound to serum albumin (6). Studies on perfused rat intestine have implicated albumin in the transport of newly absorbed zinc in portal blood, from the intestine to the liver (5). Albumin has also been shown to promote zinc uptake by endothelial cells, with receptor-mediated endocytosis as the most likely mechanism (7).Albumin, the most abundant protein in blood plasma (∼40 mg ml−1 and 0.6 mm), is synthesized in the liver and secreted into the blood stream as a 585-residue, single-chain protein after loss of a 24-residue propeptide (8). The protein is largely α-helical and folds into three structurally homologous domains (I, II, and III), each of which contains two subdomains (A and B) (see Fig. 1A) (9). There are 35 Cys residues that form six disulfide bridges in each domain, except for domain I, which contains only five bridges and a free thiol at Cys34.Open in a separate windowFIGURE 1.A, domain structure of human serum albumin (9). Domain I (red), domain II (blue), and domain III (green) are held together solely by linker helices and weak interactions. B, the proposed interdomain zinc site on albumin is formed by two residues from domain I (red) and two residues from domain II (blue).Zinc binding to albumin has also been demonstrated in vitro, and the so-called high-affinity site for zinc on albumin displays a binding constant of K ≈ 107 m−1 (10–13). Although over 50 x-ray structures of albumin have been reported to date (9, 14–16), no experimental structural data are available for the zinc site on albumin. On the basis of 111Cd NMR studies, site-directed mutagenesis, and molecular modeling, we have proposed that the major zinc site on albumin, termed site A, is located at the interface of domains I and II and is formed by the side chains of His67 and Asn99 from domain I and by His247 and Asp249 from domain II (Fig. 1B) (17, 18).Here, we report the first direct structural characterization of a zinc site on albumin, using Zn K-edge x-ray absorption fine structure (EXAFS)2 spectroscopy. We also explored the possibility of engineering albumins with increased or decreased zinc-binding affinity by mutating the postulated zinc-binding ligands. NMR methods were used to identify mutated and zinc-binding histidines and to probe the effects of zinc binding on the conformational dynamics of the protein. Finally, we show that albumin at physiological concentrations promotes the culture of hepatocytes at otherwise toxic zinc concentrations. 相似文献
13.
14.
15.
Louis Flamand Fabio Romerio Marvin S. Reitz Robert C. Gallo 《Journal of virology》1998,72(11):8797-8805
16.
17.
18.
19.
Cl��ment Faye Christophe Moreau Emilie Chautard Reidunn Jetne Naomi Fukai Florence Ruggiero Martin J. Humphries Bjorn R. Olsen Sylvie Ricard-Blum 《The Journal of biological chemistry》2009,284(33):22029-22040
Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. α5β1 and αvβ3 integrins form stable complexes with immobilized endostatin (KD = ∼1.8 × 10−8 m, two-state model). Two arginine residues (Arg27 and Arg139) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the αvβ3 integrin at the interface between the β-propeller domain of the αv subunit and the βA domain of the β3 subunit. In addition, we report that α5β1 and αvβ3 integrins bind to heparin/heparan sulfate. The ectodomain of the α5β1 integrin binds to haparin with high affinity (KD = 15.5 nm). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and α5β1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.Endostatin is an endogenous inhibitor of angiogenesis that inhibits proliferation and migration of endothelial cells (1–3). This C-fragment of collagen XVIII has also been shown to inhibit 65 different tumor types and appears to down-regulate pathological angiogenesis without side effects (2). Endostatin regulates angiogenesis by complex mechanisms. It modulates embryonic vascular development by enhancing proliferation, migration, and apoptosis (4). It also has a biphasic effect on the inhibition of endothelial cell migration in vitro, and endostatin therapy reveals a U-shaped curve for antitumor activity (5, 6). Short term exposure of endothelial cells to endostatin may be proangiogenic, unlike long term exposure, which is anti-angiogenic (7). The effect of endostatin depends on its concentration and on the type of endothelial cells (8). It exerts the opposite effects on human umbilical vein endothelial cells and on endothelial cells derived from differentiated embryonic stem cells. Furthermore, two different mechanisms (heparin-dependent and heparin-independent) may exist for the anti-proliferative activity of endostatin depending on the growth factor used to induce cell proliferation (fibroblast growth factor 2 or vascular endothelial growth factor). Its anti-proliferative effect on endothelial cells stimulated by fibroblast growth factor 2 is mediated by the binding of endostatin to heparan sulfate (9), whereas endostatin inhibits vascular endothelial growth factor-induced angiogenesis independently of its ability to bind heparin and heparan sulfate (9, 10). The broad range of molecular targets of endostatin suggests that multiple signaling systems are involved in mediating its anti-angiogenic action (11), and although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanisms of action are not as fully elucidated as they are for other endogenous angiogenesis inhibitors (11).Endostatin binds with relatively low affinity to several membrane proteins including α5β1 and αvβ3 integrins (12), heparan sulfate proteoglycans (glypican-1 and -4) (13), and KDR/Flk1/vascular endothelial growth factor receptor 2 (14), but no high affinity receptor(s) has been identified so far. The identification of molecular interactions established by endostatin at the cell surface is a first step toward the understanding of the mechanisms by which endostatin regulates angiogenesis. We have previously characterized the binding of endostatin to heparan sulfate chains (9). In the present study we have focused on characterizing the interactions between endostatin, α5β1, αvβ3, and αvβ5 integrins and heparan sulfate. Although interactions between several integrins and endostatin have been studied previously in solid phase assays (12) and in cell models (12, 15, 16), no molecular data are available on the binding site of endostatin to the integrins. We found that two arginine residues of endostatin (Arg27 and Arg139) participate in binding to integrins and to heparan sulfate, suggesting that endostatin is not able to bind simultaneously to these molecules displayed at the cell surface. Furthermore, we have demonstrated that α5β1, αvβ3, and αvβ5 integrins bind to heparan sulfate. This may explain why both heparan sulfate and α5β1 integrins are required for the localization of endostatin in lipid rafts, in support of the model proposed by Wickström et al. (15). 相似文献
20.
Laszlo L. P. Hosszu Clare R. Trevitt Samantha Jones Mark Batchelor David J. Scott Graham S. Jackson John Collinge Jonathan P. Waltho Anthony R. Clarke 《The Journal of biological chemistry》2009,284(33):21981-21990
Prion propagation involves a conformational transition of the cellular form of prion protein (PrPC) to a disease-specific isomer (PrPSc), shifting from a predominantly α-helical conformation to one dominated by β-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91–231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrPSc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant β-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrPC conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a β-rich conformation. This precursor state is almost as compact as the folded PrPC structure and, as it assembles, only residues 126–227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.Prion diseases, such as Creutzfeldt-Jacob and Gerstmann-Sträussler-Scheinker in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, are fatal neurological disorders associated with the deposition of an abnormally folded form of a host-encoded glycoprotein, prion (PrP)2 (1). These diseases may be inherited, arise sporadically, or be acquired through the transmission of an infectious agent (2, 3). The disease-associated form of the protein, termed the scrapie form or PrPSc, differs from the normal cellular form (PrPC) through a conformational change, resulting in a significant increase in the β-sheet content and protease resistance of the protein (3, 4). PrPC, in contrast, consists of a predominantly α-helical structured domain and an unstructured N-terminal domain, which is capable of binding a number of divalent metals (5–12). A single disulfide bond links two of the main α-helices and forms an integral part of the core of the structured domain (13, 14).According to the protein-only hypothesis (15), the infectious agent is composed of a conformational isomer of PrP (16) that is able to convert other isoforms to the infectious isomer in an autocatalytic manner. Despite numerous studies, little is known about the mechanism of conversion of PrPC to PrPSc. The most coherent and general model proposed thus far is that PrPC fluctuates between the dominant native state and minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure composed of misfolded PrP monomers (3, 17). This stable, oligomeric species can then bind to, and stabilize, rare non-native monomer conformations that are structurally complementary. In this manner, new monomeric chains are recruited and the system can propagate.In view of the above model, considerable effort has been devoted to generating and characterizing alternative, possibly PrPSc-like, conformations in the hope of identifying common properties or features that facilitate the formation of amyloid oligomers. This has been accomplished either through PrPSc-dependent conversion reactions (18–20) or through conversion of PrPC in the absence of a PrPSc template (21–25). The latter approach, using mainly disulfide-oxidized recombinant PrP, has generated a wide range of novel conformations formed under non-physiological conditions where the native state is relatively destabilized. These conformations have ranged from near-native (14, 26, 27), to those that display significant β-sheet content (21, 23, 28–33). The majority of these latter species have shown a high propensity for aggregation, although not all are on-pathway to the formation of amyloid. Many of these non-native states also display some of the characteristics of PrPSc, such as increased β-sheet content, protease resistance, and a propensity for oligomerization (28, 29, 31) and some have been claimed to be associated with the disease process (34).One such PrP folding intermediate, termed β-PrP, differs from the majority of studied PrP intermediate states in that it is formed by refolding the PrP molecule from the native α-helical conformation (here termed α-PrP), at acidic pH in a reduced state, with the disulfide bond broken (22, 35). Although no covalent differences between the PrPC and PrPSc have been consistently identified to date, the role of the disulfide bond in prion propagation remains disputed (25, 36–39). β-PrP is rich in β-sheet structure (22, 35), and displays many of the characteristics of a PrPSc-like precursor molecule, such as partial resistance to proteinase K digestion, and the ability to form amyloid fibrils in the presence of physiological concentrations of salts (40).The β-PrP species previously characterized, spanning residues 91–231 of PrP, was soluble at low ionic strength buffers and monomeric, according to elution volume on gel filtration (22). NMR analysis showed that it displayed radically different spectra to those of α-PrP, with considerably fewer observable peaks and markedly reduced chemical shift dispersion. Data from circular dichroism experiments showed that fixed side chain (tertiary) interactions were lost, in contrast to the well defined β-sheet secondary structure, and thus in conjunction with the NMR data, indicated that β-PrP possessed a number of characteristics associated with a “molten globule” folding intermediate (22). Such states have been proposed to be important in amyloid and fibril formation (41). Indeed, antibodies raised against β-PrP (e.g. ICSM33) are capable of recognizing native PrPSc (but not PrPC) (42–44). Subsequently, a related study examining the role of the disulfide bond in PrP folding confirmed that a monomeric molten globule-like form of PrP was formed on refolding the disulfide-reduced protein at acidic pH, but reported that, under their conditions, the circular dichroism response interpreted as β-sheet structure was associated with protein oligomerization (45). Indeed, atomic force microscopy on oligomeric full-length β-PrP (residues 23–231) shows small, round particles, showing that it is capable of formation of oligomers without forming fibrils (35). Notably, however, salt-induced oligomeric β-PrP has been shown to be a potent inhibitor of the 26 S proteasome, in a similar manner to PrPSc (46). Impairment of the ubiquitin-proteasome system in vivo has been linked to prion neuropathology in prion-infected mice (46).Although the global properties of several PrP intermediate states have been determined (30–32, 35), no information on their conformational properties on a sequence-specific basis has been obtained. Their conformational properties are considered important, as the elucidation of the chain conformation may provide information on the way in which these chains pack in the assembly process, and also potentially provide clues on the mechanism of amyloid assembly and the phenomenon of prion strains. As the conformational fluctuations and heterogeneity of molten globule states give rise to broad NMR spectra that preclude direct observation of their conformational properties by NMR (47–50), here we use denaturant titration experiments to determine the conformational properties of β-PrP, through the population of the unfolded state that is visible by NMR. In addition, we use circular dichroism and analytical ultracentrifugation to examine the global structural properties, and the distribution of multimeric species that are formed from β-PrP. 相似文献