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1.
Cell death can be divided into the anti-inflammatory process of apoptosis and the
pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell
death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP)
1/3 are major mediators. We previously showed that absence or inhibition of PARP-1
protects mice from nephritis, however only the male mice. We therefore hypothesized that
there is an inherent difference in the cell death program between the sexes. We show here
that in an immune-mediated nephritis model, female mice show increased apoptosis compared
to male mice. Treatment of the male mice with estrogens induced apoptosis to levels
similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in
both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We
also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male
and female mice are prone to different types of cell death. Our data also suggest that
estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore
propose that targeting cell death based on sex will lead to tailored and better treatments
for each gender. 相似文献
2.
3.
4.
5.
Lilly Y. W. Bourguignon Weiliang Xia Gabriel Wong 《The Journal of biological chemistry》2009,284(5):2657-2671
6.
Lisa Placanica Leonid Tarassishin Guangli Yang Erica Peethumnongsin Seong-Hun Kim Hui Zheng Sangram S. Sisodia Yue-Ming Li 《The Journal of biological chemistry》2009,284(5):2967-2977
γ-Secretase is known to play a pivotal role in the pathogenesis of
Alzheimer disease through production of amyloidogenic Aβ42 peptides.
Early onset familial Alzheimer disease mutations in presenilin (PS), the
catalytic core of γ-secretase, invariably increase the
Aβ42:Aβ40 ratio. However, the mechanism by which these mutations
affect γ-secretase complex formation and cleavage specificity is poorly
understood. We show that our in vitro assay system recapitulates the
effect of PS1 mutations on the Aβ42:Aβ40 ratio observed in cell and
animal models. We have developed a series of small molecule affinity probes
that allow us to characterize active γ-secretase complexes. Furthermore
we reveal that the equilibrium of PS1- and PS2-containing active complexes is
dynamic and altered by overexpression of Pen2 or PS1 mutants and that
formation of PS2 complexes is positively correlated with increased
Aβ42:Aβ40 ratios. These data suggest that perturbations to
γ-secretase complex equilibrium can have a profound effect on enzyme
activity and that increased PS2 complexes along with mutated PS1 complexes
contribute to an increased Aβ42:Aβ40 ratio.β-Amyloid
(Aβ)5 peptides
are believed to play a causative role in Alzheimer disease (AD). Aβ
peptides are generated from the processing of the amyloid precursor protein
(APP) by two proteases, β-secretase and γ-secretase. Although
γ-secretase generates heterogenous Aβ peptides ranging from 37 to
46 amino acids in length, significant work has focused mainly on the Aβ40
and Aβ42 peptides that are the major constituents of amyloid plaques.
γ-Secretase is a multisubunit membrane aspartyl protease comprised of at
least four known subunits: presenilin (PS), nicastrin (Nct), anterior
pharynx-defective (Aph), and presenilin enhancer 2 (Pen2). Presenilin is
thought to contain the catalytic core of the complex
(1–4),
whereas Aph and Nct play critical roles in the assembly, trafficking, and
stability of γ-secretase as well as substrate recognition
(5,
6). Lastly Pen2 facilitates the
endoproteolysis of PS into its N-terminal (NTF) and C-terminal (CTF) fragments
thereby yielding a catalytically competent enzyme
(5,
7–10).
All four proteins (PS, Nct, Aph1, and Pen2) are obligatory for
γ-secretase activity in cell and animal models
(11,
12). There are two homologs of
PS, PS1 and PS2, and three isoforms of Aph1, Aph1aS, Aph1aL, and Aph1b. At
least six active γ-secretase complexes have been reported (two
presenilins × three Aph1s)
(13,
14). The sum of apparent
molecular masses of the four proteins (PS1-NTF/CTF ≈ 53 kDa, Nct ≈ 120
kDa, Aph1 ≈ 30 kDa, and Pen2 ≈ 10kDa) is ∼200 kDa. However, active
γ-secretase complexes of varying sizes, ranging from 250 to 2000 kDa,
have been reported
(15–19).
Recently a study suggested that the γ-secretase complex contains only
one of each subunit (20).
Collectively these studies suggest that a four-protein complex around
200–250 kDa may be the minimal functional γ-secretase unit with
additional cofactors and/or varying stoichiometry of subunits existing in the
high molecular weight γ-secretase complexes. CD147 and TMP21 have been
found to be associated with the γ-secretase complex
(21,
22); however, their role in
the regulation of γ-secretase has been controversial
(23,
24).Mutations of PS1 or PS2 are associated with familial early onset AD (FAD),
although it is debatable whether these familial PS mutations act as
“gain or loss of function” alterations in regard to
γ-secretase activity
(25–27).
Regardless the overall outcome of these mutations is an increased ratio of
Aβ42:Aβ40. Clearly these mutations differentially affect
γ-secretase activity for the production of Aβ40 and Aβ42.
Despite intensive studies of Aβ peptides and γ-secretase, the
molecular mechanism controlling the specificity of γ-secretase activity
for Aβ40 and Aβ42 production has not been resolved. It has been
found that PS1 mutations affect the formation of γ-secretase complexes
(28). However, the precise
mechanism by which individual subunits alter the dynamics of γ-secretase
complex formation and activity is largely unresolved. A better mechanistic
understanding of γ-secretase activity associated with FAD mutations has
been hindered by the lack of suitable assays and probes that are necessary to
recapitulate the effect of these mutations seen in cell models and to
characterize the active γ-secretase complex.In our present studies, we have determined the overall effect of Pen2 and
PS1 expression on the dynamics of PS1- and PS2-containing complexes and their
association with γ-secretase activity. Using newly developed
biotinylated small molecular probes and activity assays, we revealed that
expression of Pen2 or PS1 FAD mutants markedly shifts the equilibrium of
PS1-containing active complexes to that of PS2-containing complexes and
results in an overall increase in the Aβ42:Aβ40 ratio in both stable
cell lines and animal models. Our studies indicate that perturbations to the
equilibrium of active γ-secretase complexes by an individual subunit can
greatly affect the activity of the enzyme. Moreover they serve as further
evidence that there are multiple and distinct γ-secretase complexes that
can exist within the same cells and that their equilibrium is dynamic.
Additionally the affinity probes developed here will facilitate further study
of the expression and composition of endogenous active γ-secretase from
a variety of model systems. 相似文献
7.
8.
T. Møller-Nielsen H. Gesser 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1992,162(6):526-534
Summary Isometric force and series membrane potential were recorded in isolated ventricular strips from rainbow trout at 20 and 10 °C. Preparations were electrically stimulated to contract at either 0.5 or 0.2 Hz. Single extrastimulations elicited a twitch force which diminished when the preceding diastole was shortened below the regular value. The stimulation following this extra stimulation evoked no potentiation of force. Apart from a marginal effect on the post extrasystolic force at 20 °C, ryanodine did not affect either of these responses or the steady-state force at 0.5 Hz. At 0.2 Hz the steady-state force was somewhat depressed by ryanodine at 20 but not at 10 °C. In contrast, extrastimulations preceded by diastoles of up to 1 h more than doubled extrasystolic force at 20 °C. This effect was removed by ryanodine. Both the potentiations and the effect of ryanodine were strongly reduced at 10 °C. Apparently, temperature acts on the release of Ca2+ from the sarcoplasmic reticulum, since Ca2+ seems to be taken up at both temperatures. Hence, at both 20 and 10 °C, the contractures evoked in a solution inhibiting sarcolemmal Ca2+ transfer and releasing Ca2+ from the sarcoplasmic reticulum were diminished by pretreatment with 15 mM caffeine. Action potential duration at 20 °C was less than half of that at 10 °C. At both temperatures it tended to be prolonged by periods of prolonged rest. No effect of ryanodine on action potential configuration was detected. The results suggest that trout myocardial sarcoplasmic reticulum, although powerful at unphysiologically low stimulation rates, does not partake in the beat-to-beat regulation of force at heart rates encountered in vivo.Abbreviations ESF
extrasystolic force
- SR
sarcoplasmic reticulum
-
v
F
maximal rate of force development
-
v
R
maximal rate of relaxation
- TPF
time to peak force
-
TR
0.5
time for half relaxation
- TTF
duration of force development 相似文献
9.
Rosanna Pescini Gobert Monique van den Eijnden Cedric Szyndralewiez Catherine Jorand-Lebrun Dominique Swinnen Linfeng Chen Corine Gillieron Fiona Pixley Pierre Juillard Patrick Gerber Caroline Johnson-L��ger Serge Halazy Montserrat Camps Agnes Bombrun Margaret Shipp Pierre-Alain Vitte Vittoria Ardissone Chiara Ferrandi Dominique Perrin Christian Rommel Rob Hooft van Huijsduijnen 《The Journal of biological chemistry》2009,284(17):11385-11395
10.
The V kappa 10 family in BALB/c mice is composed of three members, two of which are utilized in a variety of immune responses. We previously demonstrated that the product of the third gene, V kappa 10C, has never been detected as part of a functional antibody and productive rearrangements are selectively lost during B-cell development. Here we analyzed germline V kappa 10 genes from inbred and wild-derived mice by RFLP and sequencing in order to determine the origin of the V kappa 10C gene, as well as to examine the evolutionary relationships of V kappa 10 genes. Our results demonstrated that the V kappa 10 family is highly conserved across Mus species and subspecies, but that V kappa 10C is rare, being found in only inbred mice of V kappa 10 allelic group b and two of six M. m. domesticus isolates. It was not found in other M. musculus subspecies or M. spretus. V kappa 10A and V kappa 10B were found in all strains, with the exception of one M. m. domesticus isolate, which had only V kappa 10B genes. Overall, V kappa 10A sequences were more highly conserved than V kappa 10B, indicating that different selective pressures may be operating on these genes. The two V kappa 10C sequences from M. m. domesticus were 100% identical to that found in inbred mice. V kappa 10C is more closely related to V kappa 10B than to V kappa 10A and our data suggest that it is a recent duplication of the V kappa 10B gene. 相似文献
11.
David S. Harburger Mohamed Bouaouina David A. Calderwood 《The Journal of biological chemistry》2009,284(17):11485-11497
Integrin activation, the rapid conversion of integrin adhesion receptors
from low to high affinity, occurs in response to intracellular signals that
act on the short cytoplasmic tails of integrin β subunits. Talin binding
to integrin β tails provides one key activation signal, but additional
factors are likely to cooperate with talin to regulate integrin activation.
The integrin β tail-binding proteins kindlin-2 and kindlin-3 were
recently identified as integrin co-activators. Here we report an analysis of
kindlin-1 and kindlin-2 interactions with β1 and β3 integrin tails
and describe the effect of kindlin expression on integrin activation. We
demonstrate a direct interaction of kindlin-1 and -2 with recombinant integrin
β tails in pulldown binding assays. Our mutational analysis shows that
the second conserved NXXY motif (Tyr795), a preceding
threonine-containing region (Thr788 and Thr789) of the
integrin β1A tail, and a conserved tryptophan in the F3 subdomain of the
kindlin FERM domain (kindlin-1 Trp612 and kindlin-2
Trp615) are required for direct kindlin-integrin interactions.
Similar interactions were observed for integrin β3 tails. Using
fluorescence-activated cell sorting we further show that transient expression
of kindlin-1 or -2 in Chinese hamster ovary cells inhibits the activation of
endogenous α5β1 or stably expressed αIIbβ3 integrins.
This inhibition is not dependent on direct kindlin-integrin interactions
because mutant kindlins exhibiting impaired integrin binding activity
effectively inhibit integrin activation. Consistent with previous reports, we
find that when co-expressed with the talin head, kindlin-1 or -2 can activate
αIIbβ3. This effect is dependent on an intact integrin-binding site
in kindlin. Notably however, even when co-expressed with activating levels of
talin head, neither kindlin-1 or -2 can cooperate with talin to activate
β1 integrins; instead they strongly inhibit talin-mediated activation. We
suggest that kindlins are adaptor proteins that regulate integrin activation,
that kindlin expression levels determine their effects, and that kindlins may
exert integrin-specific effects.Integrins are a family of αβ heterodimeric transmembrane
receptors that mediate cell adhesion to extracellular matrix, cell surface, or
soluble protein ligands and modulate a variety of intracellular signaling
cascades. A key feature of integrins is their ability to dynamically regulate
their affinity for extracellular ligands. In a tightly regulated process
generally termed integrin activation, intracellular signals that impinge upon
the β subunit cytoplasmic tail induce conformational rearrangements in
the integrin extracellular domains, increasing the binding affinity for
extracellular ligands
(1-3).
Ligand-bound integrins then recruit additional signaling, adaptor, and
cytoskeletal proteins to the integrin cytoplasmic domains, providing
mechanical connections to the actin cytoskeleton and a link to a variety of
signal transduction pathways
(2-8).Recent years have seen significant advances in our understanding of
integrin activation. Notable among these is the identification of the actin-
and integrin-binding protein talin as a key integrin activator
(1,
9). The 50-kDa talin head
contains the principal integrin-binding site, and expression of the talin head
is sufficient to activate β1 and β3 integrins
(10,
11). The talin head contains a
FERM (four point one ezrin radixin
moesin) domain. FERM domains consist of trefoil arrangement of
three subdomains (F1, F2, and F3). The phosphotyrosine-binding domain-like F3
subdomain of the talin FERM directly binds a conserved NP(I/L)Y motif in
integrin β tails, and this interaction is necessary for integrin
activation in vitro and in vivo
(10,
12-19).
However, although abundant evidence supports the importance of talin binding
to integrin β tails during integrin activation, differences in
sensitivity of integrins to talin activation and submaximal activation by
overexpressed talin suggested that other activating factors may cooperate with
talin (10,
20). In an attempt to identify
and characterize potential co-activators, we investigated the kindlin family
of FERM domain-containing proteins.Kindlin family proteins
(21) were first characterized
in nematodes where the sole Caenorhabditis elegans kindlin, UNC-112,
was identified in an embryonic screen for defective motility and shown to be
essential for the assembly of proper cell-matrix adhesion structures, where it
normally co-localized with β integrin
(22-24).
UNC-112 is conserved across many species, because the nematode, fly, and human
homologs are ∼60% similar (∼41% identical) over their entire length
(24). Humans express three
known homologs of UNC-112: kindlin-1 (Kindlerin, URP1, and FERMT1), kindlin-2
(Mig2 and mig-2), and kindlin-3 (Mig2B and URP2)
(25-27).
Kindlin-1 and -2 are most closely related, sharing 60% identity and 74%
similarity, whereas kindlin-3 shares 53% identity and 69% similarity to
kindlin-1 and 49% identity and 67% similarity to kindlin-2
(28). The kindlin proteins all
contain a predicted Pleckstrin homology domain and a FERM domain that is most
closely related to the talin FERM domain, particularly within the
integrin-binding F3 subdomain
(29). Based on this sequence
similarity we proposed that kindlin FERM domains may directly bind integrin
β tails, and we previously showed that kindlin-1 could be pulled down
from cell lysates using recombinant integrin β1 and β3 tails and
that kindlin-1 co-localized with integrins in focal adhesions
(29). A similar localization
was reported for kindlin-2
(26,
30), and recent reports
provided clear evidence implicating kindlin-2 and kindlin-3 in regulation of
integrin activation
(31-33).
Here, we have used integrin pulldown assays to demonstrate direct binding of
full-length kindlin-1 to the cytoplasmic tails of β1A and β3
integrins and to identify key binding residues within the integrin tails and
the kindlin F3 subdomain. We confirm that these interactions are important for
recruiting kindlin-1 to focal adhesions and show that, contrary to
expectations, overexpressed kindlin-1 or -2 inhibit β1 and β3
integrin activation. Overexpressed kindlin-1 or -2 can, however, cooperate
with expressed talin head to activate β3 but not β1 integrins. We
therefore provide the first data suggesting that kindlin-1 and -2 effects on
integrin activation may show β subunit specificity. 相似文献
12.
Ellen J. Tisdale Fouad Azizi Cristina R. Artalejo 《The Journal of biological chemistry》2009,284(9):5876-5884
Rab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical
protein kinase Cι (aPKCι) for retrograde vesicle formation from
vesicular tubular clusters that sort secretory cargo from recycling proteins
returned to the endoplasmic reticulum. However, the precise role of GAPDH and
aPKCι in the early secretory pathway is unclear. GAPDH was the first
glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly,
aPKC associates directly with MTs. To learn whether Rab2 also binds directly
to MTs, a MT binding assay was performed. Purified Rab2 was found in a
MT-enriched pellet only when both GAPDH and aPKCι were present, and
Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2
amino terminus (residues 2-70), which directly interacts with GAPDH and
aPKCι. Because GAPDH binds to the carboxyl terminus of α-tubulin,
we characterized the distribution of tyrosinated/detyrosinated α-tubulin
that is recruited by Rab2 in a quantitative membrane binding assay.
Rab2-treated membranes contained predominantly tyrosinated α-tubulin;
however, aPKCι was the limiting and essential factor.
Tyrosination/detyrosination influences MT motor protein binding; therefore, we
determined whether Rab2 stimulated kinesin or dynein membrane binding.
Although kinesin was not detected on membranes incubated with Rab2, dynein was
recruited in a dose-dependent manner, and binding was aPKCι-dependent.
These combined results suggest a mechanism by which Rab2 controls MT and motor
recruitment to vesicular tubular clusters.The small GTPase Rab2 is essential for membrane trafficking in the early
secretory pathway and associates with vesicular tubular
clusters
(VTCs)2 located
between the endoplasmic reticulum (ER) and the cis-Golgi compartment
(1,
2). VTCs are pleomorphic
structures that sort anterograde-directed cargo from recycling proteins and
trafficking machinery retrieved to the ER
(3-6).
Rab2 bound to a VTC microdomain stimulates recruitment of soluble factors that
results in the release of vesicles containing the recycling protein p53/p58
(7). In that regard, we have
previously reported that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and
atypical PKC ι (aPKCι) are Rab2 effectors that interact directly
with the Rab2 amino terminus and with each other
(8,
9). Their interaction requires
Src-dependent tyrosine phosphorylation of GAPDH and aPKCι
(10). Moreover, GAPDH is a
substrate for aPKCι (11).
GAPDH catalytic activity is not required for ER to Golgi transport indicating
that GAPDH provides a specific function essential for membrane trafficking
from VTCs independent of glycolytic function
(9). Indeed, phospho-GAPDH
influences MT dynamics in the early secretory pathway
(11).GAPDH was the first glycolytic enzyme reported to co-purify with
microtubules (MTs) (12) and
subsequently was shown to interact with the carboxyl terminus of
α-tubulin (13). The
binding of GAPDH to MTs promotes formation of cross-linked parallel MT arrays
or bundles (14,
15). GAPDH has also been
reported to possess membrane fusogenic activity, which is inhibited by tubulin
(16). Similarly, aPKC
associates directly with tubulin and promotes MT stability and MT remodeling
at specific intracellular sites
(17-21).
It may not be coincidental that these two Rab2 effectors influence MT dynamics
because recent studies indicate that the cytoskeleton plays a central role in
the organization and operation of the secretory pathway
(22).MTs are dynamic structures that grow or shrink by the addition or loss of
α- and β-tubulin heterodimers from the ends of protofilaments
(23). Their assembly and
stability is regulated by a variety of proteins traditionally referred to as
microtubule-associated proteins (MAPs). In addition to the multiple
α/β isoforms that are present in eukaryotes, MTs undergo an
assortment of post-translational modifications, including acetylation,
glycylation, glutamylation, phosphorylation, palmitoylation, and
detyrosination, which further contribute to their biochemical heterogeneity
(24,
25). It has been proposed that
these tubulin modifications regulate intracellular events by facilitating
interaction with MAPs and with other specific effector proteins
(24). For example, the
reversible addition of tyrosine to the carboxyl terminus of α-tubulin
regulates MT interaction with plus-end tracking proteins (+TIPs) containing
the cytoskeleton-associated protein glycine-rich (CAP-Gly) motif and with
dynein-dynactin
(27-29).
Additionally, MT motility and cargo transport rely on the cooperation of the
motor proteins kinesin and dynein
(30). Kinesin is a plus-end
directed MT motor, whereas cytoplasmic dynein is a minus-end MT-based motor,
and therefore the motors transport vesicular cargo toward the opposite end of
a MT track (31).Although MT assembly does not appear to be directly regulated by small
GTPases, Rab proteins provide a molecular link for vesicle movement along MTs
to the appropriate target (22,
32-34).
In this study, the potential interaction of Rab2 with MTs and motor proteins
was characterized. We found that Rab2 does not bind directly to preassembled
MTs but does associate when both GAPDH and aPKCι are present and bound to
MTs. Moreover, the MTs predominantly contained tyrosinated α-tubulin
(Tyr-tubulin) suggesting that a dynamic pool of MTs that differentially binds
MAPs/effector proteins/motors associates with VTCs in response to Rab2. To
that end, we determined that Rab2-promoted dynein/dynactin binding to
membranes and that the recruitment required aPKCι. 相似文献
13.
14.
Sorghum (Sorghum bicolor L. Moench) has two isozymes of the cyanogenic β-glucosidase dhurrinase: dhurrinase-1 (Dhr1) and dhurrinase-2 (Dhr2). A nearly full-length cDNA encoding dhurrinase was isolated from 4-d-old etiolated seedlings and sequenced. The cDNA has a 1695-nucleotide-long open reading frame, which codes for a 565-amino acid-long precursor and a 514-amino acid-long mature protein, respectively. Deduced amino acid sequence of the sorghum Dhr showed 70% identity with two maize (Zea mays) β-glucosidase isozymes. Southern-blot data suggested that β-glu-cosidase is encoded by a small multigene family in sorghum. Northern-blot data indicated that the mRNA corresponding to the cloned Dhr cDNA is present at high levels in the node and upper half of the mesocotyl in etiolated seedlings but at low levels in the root—only in the zone of elongation and the tip region. Light-grown seedling parts had lower levels of Dhr mRNA than those of etiolated seedlings. Immunoblot analysis performed using maize-anti-β-glucosidase sera detected two distinct dhurrinases (57 and 62 kD) in sorghum. The distribution of Dhr activity in different plant parts supports the mRNA and immunoreactive protein data, suggesting that the cloned cDNA corresponds to the Dhr1 (57 kD) isozyme and that the dhr1 gene shows organ-specific expression. 相似文献
15.
16.
Xiaojing Wang Snezana Levic Michael Anne Gratton Karen Jo Doyle Ebenezer N. Yamoah Anthony E. Pegg 《The Journal of biological chemistry》2009,284(2):930-937
Male gyro (Gy) mice, which have an X chromosomal deletion inactivating the
SpmS and Phex genes, were found to be profoundly hearing
impaired. This defect was due to alteration in polyamine content due to the
absence of spermine synthase, the product of the SpmS gene. It was
reversed by breeding the Gy strain with CAG/SpmS mice, a transgenic line that
ubiquitously expresses spermine synthase under the control of a composite
cytomegalovirus-IE enhancer/chicken β-actin promoter. There was an almost
complete loss of the endocochlear potential in the Gy mice, which parallels
the hearing deficiency, and this was also reversed by the production of
spermine from the spermine synthase transgene. Gy mice showed a striking toxic
response to treatment with the ornithine decarboxylase inhibitor
α-difluoromethylornithine (DFMO). Within 2–3 days of exposure to
DFMO in the drinking water, the Gy mice suffered a catastrophic loss of motor
function resulting in death within 5 days. This effect was due to an inability
to maintain normal balance and was also prevented by the transgenic expression
of spermine synthase. DFMO treatment of control mice or Gy-CAG/SpmS had no
effect on balance. The loss of balance in Gy mice treated with DFMO was due to
inhibition of polyamine synthesis because it was prevented by administration
of putrescine. Our results are consistent with a critical role for polyamines
in regulation of Kir channels that maintain the endocochlear potential and
emphasize the importance of normal spermidine:spermine ratio in the hearing
and balance functions of the inner ear.Polyamines are essential for viability in mammals. Knockouts of the genes
for ornithine decarboxylase and S-adenosylmethionine decarboxylase,
which are enzymes needed for the synthesis of putrescine, spermidine, and
spermine, are lethal at early stages of embryonic development
(1,
2). There is convincing
evidence that the formation of hypusine in eIF5A, which requires spermidine as
a precursor, is essential for eukaryotes
(3). However, the function(s)
of spermine is not so well established. Yeast mutants with inactivated
spermine synthase grow at a normal rate
(4). Mammalian cells in culture
also grow normally in the presence of inhibitors of spermine synthase
(5) or after inactivation of
the spermine synthase gene (SpmS)
(6–8).
Inactivation of both of the genes that were originally described as encoding
spermine synthases in plants leads to profound developmental defects
(9–11),
but recently it was discovered that one of these genes actually encodes a
thermospermine synthase, and it appears that the lack of thermospermine may be
responsible for these defects
(12).In contrast, spermine is clearly required for normal development in
mammals. The rare human Snyder-Robinson syndrome is caused by mutations in
SpmS located in the X chromosome that drastically reduces the amount
of spermine synthase (13,
14). This leads to mental
retardation, hypotonia, cerebellar circuitry dysfunction, facial asymmetry,
thin habitus, osteoporosis, and kyphoscoliosis. Male mice, which have an X
chromosomal deletion that includes SpmS and have no detectable
spermine synthase activity, do survive but are only viable on the B6C3H
background
(15–17).
This mouse strain having an X-linked dominant mutation was isolated from a
female offspring of an irradiated mouse and was termed gyro
(Gy)2 based on a
circling behavior pattern in affected males
(18). Subsequent studies have
shown that the Gy mice have a deletion of part of the X chromosome that
inactivates both Phex, a gene that regulates phosphate metabolism,
and SpmS (16,
19). The lack of SpmS
causes a total absence of spermine
(6,
7,
15,
16). Such Gy mice suffer from
hypophosphatemia, have a greatly reduced size, sterility, and neurological
abnormalities, and have a short life span
(6,
16,
18). All of these changes
except the hypophosphatemia are reversed when spermine synthase activity is
restored (20).The original characterization of Gy mice also reported preliminary
indications that these mice had hearing defects lacking the Preyer reflex
(21,
22). This is of particular
interest in the context of polyamine metabolism because a drug,
α-difluoromethylornithine (DFMO, Eflornithine), that targets ornithine
decarboxylase has been shown to cause occasional hearing loss in some patients
(23–26).
Although DFMO was ineffective for cancer treatment, it is an extremely
promising agent for cancer chemoprevention
(27,
28). When combined with
sulindac, DFMO treatment produced a substantial reduction in the recurrence of
colorectal adenomas in a large clinical trial
(27). DFMO is a major drug for
the treatment of African sleeping sickness caused by Trypanosoma
brucei (29,
30). It is also used as a
topically applied cream for treatment of unwanted facial hair in women
(31,
32). DFMO is generally well
tolerated even at high doses, but reversible hearing loss has been reported in
multiple clinical trials (25,
33), and a rarer irreversible
defect has also been reported
(34). These side effects are
not observed at lower doses of DFMO
(26,
27).Ototoxicity has been demonstrated to occur in experimental animals treated
with DFMO including rats (35),
guinea pigs (36), gerbils
(37), and mice
(38). Using
immunohistochemistry, a high level of ornithine decarboxylase was observed in
the inner ear of the rat, with the highest in the organ of Corti and lateral
wall followed by the cochlear nerve
(39). Measurements of
polyamines in the relevant structures are very difficult due to the small
amount of tissue available, but as expected, DFMO treatment reduced polyamine
levels and ornithine decarboxylase activity in the inner ear of the guinea pig
(36). A plausible explanation
for the importance of polyamines in auditory physiology is based on their well
documented role as regulators of potassium channels
(38). The inward rectification
of Kir channels is caused by blockage of the outward current by polyamines
(40–42).
Studies of the cloned mouse cochlear lateral wall-specific Kir4.1 channel
showed that inward rectification was reduced and that there was a marked
reduction in endocochlear potential (EP). It was proposed that DFMO treatment
increases the outward Kir4.1 current, resulting in a drop in EP
(38).In the experiments reported here, we have studied in more detail the role
of polyamines in auditory physiology using Gy mice and crosses of these mice
with transgenic CAG/SpmS mice
(43). These mice express
spermine synthase under the control of a composite cytomegalovirus-IE
enhancer/chicken β-actin promoter, which was designed to provide
ubiquitous expression
(44–46).
Assays of the spermine synthase activity in CAG/SpmS line 8 confirmed that
there was a high level of expression of the transgene in many different organs
and that this level was maintained for at least 1 year
(43). Our studies confirm that
Gy mice are totally deaf and that this condition is reversed by the expression
of the SpmS gene. These changes are due to a virtually complete loss
of the EP in the Gy mice. We have also examined the effect of DFMO on the Gy
mice. Unexpectedly, it was found that these mice show a rapid and profound
toxicity to this drug, leading to death within a few days. Within 5 days of
exposure to DFMO in the drinking water, the DFMO-treated mice suffered a
catastrophic loss of balance due to inner ear effects. This toxicity was also
prevented by the transgenic expression of spermine synthase in the Gy
background. 相似文献
17.
18.
Nurit Beyth Dana Kesler Shvero Nathan Zaltsman Yael Houri-Haddad Itzhak Abramovitz Michael Perez Davidi Ervin I. Weiss 《PloS one》2013,8(11)
With growing concern over bacterial resistance, the identification of new antimicrobial means is paramount. In the oral cavity microorganisms are essential to the development of periradicular diseases and are the major causative factors associated with endodontic treatment failure. As quaternary ammonium compounds have the ability to kill a wide array of bacteria through electrostatic interactions with multiple anionic targets on the bacterial surface, it is likely that they can overcome bacterial resistance. Melding these ideas, we investigated the potency of a novel endodontic sealer in limiting Enterococcus faecalis growth. We used a polyethyleneimine scaffold to synthesize nano-sized particles, optimized for incorporation into an epoxy-based endodontic sealer. The novel endodontic sealer was tested for its antimicrobial efficacy and evaluated for biocompatibility and physical eligibility. Our results show that the novel sealer foundation affixes the nanoparticles, achieving surface bactericidal properties, but at the same time impeding nanoparticle penetration into eukaryotic cells and thereby mitigating a possible toxic effect. Moreover, adequate physical properties are maintained. The nanosized quaternary amine particles interact within minutes with bacteria, triggering cell death across wide pH values. Throughout this study we demonstrate a new antibacterial perspective for endodontic sealers; a novel antibacterial, effective and safe antimicrobial means. 相似文献
19.
20.
Wilke CM Wei S Wang L Kryczek I Kao J Zou W 《Cancer immunology, immunotherapy : CII》2011,60(11):1529-1541
It is generally thought that each cytokine exerts either immune stimulatory (inflammatory) or immune inhibitory (antiinflammatory
or regulatory) biological activities. However, multiple cytokines can enact both inhibitory and stimulatory effects on the
immune system. Two of these cytokines are interleukin (IL)-10 and interferon-gamma (IFNγ). IL-10 has demonstrated antitumor
immunity even though it has been known for years as an immunoregulatory protein. Generally perceived as an immune stimulatory
cytokine, IFNγ can also induce inhibitory molecule expression including B7-H1 (PD-L1), indoleamine 2,3-dioxygenase (IDO),
and arginase on multiple cell populations (dendritic cells, tumor cells, and vascular endothelial cells). In this review,
we will summarize current knowledge of the dual roles of both of these cytokines and stress the previously underappreciated
stimulatory role of IL-10 and inhibitory role of IFNγ in the context of malignancy. Our progressive understanding of the dual
effects of these cytokines is important for dissecting cytokine-associated pathology and provides new avenues for developing
effective immune therapy against human diseases, including cancer. 相似文献