PubMed Health⌕ Search

Biomedical subjects

Tomoko Yamaguchi

Publications and source records attributed to Tomoko Yamaguchi.

At least 19 recordsLinked to original sources
PubMed2006

Roles of CD4+CD25+ T cells in the development of experimental murine allergic conjunctivitis.

BACKGROUND: CD25+ regulatory T (T reg) cells play a suppressive role in experimental autoimmune uveoretinitis as well as experimental airway inflammation but their involvement in the development of allergic conjunctivitis (AC) remains unclear. We therefore investigated whether T reg cells play a role in the development of experimental AC (EC). METHODS: BALB/c and C57BL/6 mice were actively immunized with ragweed (RW). The mice were treated with an anti-CD25 Ab (PC61) or control normal rat IgG (nrIgG) either 2 days prior to active immunization or during the induction phase (days 0, 2, 4, 6 and 8). Ten days after active immunization, the mice were challenged with RW-containing drops. Twenty-four hours after the challenge, the conjunctivas were harvested for histological analysis of eosinophil infiltration, and the spleens were harvested for cell culture for splenocyte transfer. Cultured splenocytes were transferred into syngeneic mice, and 4 days after the transfer, the recipient mice were challenged with RW. Twenty-four hours after the challenge, conjunctivas were collected for histological analysis. RESULTS: Pretreatment with PC61 did not affect EC in either strain of mice; however, treatment with PC61 during the induction phase significantly suppressed EC in C57BL/6 mice. In contrast, transfer of RW-primed splenocytes from mice treated with PC61 induced EC that was significantly more severe regardless of strain and treatment protocol. CONCLUSIONS: The finding that T reg cells play a suppressive role in the development of EC in splenocyte transfer experiments suggests that modulation of T reg cells may be a possible therapy for AC.

Adoptive Transfer↗
PubMed2006

Modulation of murine experimental allergic conjunctivitis by treatment with alpha-galactosylceramide.

When mice are treated with alpha-galactosylceramide (alpha-GalCer), NKT cells are activated and suppress the development of experimental airway inflammation. This suppressive effect is believed to be mediated by the upregulation of IFN-gamma. Here, we investigated whether alpha-GalCer treatment can also modulate the development of experimental allergic conjunctivitis (EC). EC was induced in wild-type and IFN-gamma-deficient Balb/c mice by active immunization with ragweed (RW) followed by challenge with RW in eye drops. The mice were intraperitoneally injected with alpha-GalCer or vehicle at the time of immunization or before RW challenge. Twenty-four hours after RW challenge, conjunctivas, spleens and sera were harvested for histological analysis, flow cytometric, proliferation and cytokine assays, and measurement of immunoglobulin levels, respectively. Treatment with alpha-GalCer at the time of the EC-priming immunization significantly increased Th2 responses and markedly upregulated the severity of the EC. However, treatment with alpha-GalCer just before the Ag challenge that triggers EC in primed animals significantly suppressed the disease. This was associated with an increased frequency of CD4(+)CD25(+) cells, which express Foxp3, in the spleen. alpha-GalCer treatment just prior to Ag challenge also suppressed the development of EC in IFN-gamma-deficient mice, and we found apoptosis and anergy are unlikely to play a major role in the mechanism by which pre-challenge alpha-GalCer treatment suppresses EC. These data suggest that NKT cells can play a downregulatory role in the development of EC and that alpha-GalCer may be useful for treating allergic conjunctivitis.

Allergens↗
PubMed2006

Intracellular Ca2+ contributes to K+-induced increase in renal kallikrein secretion.

We have reported that natriuretic effects of K(+) are involved in enhancement of renal kallikrein-kinin system. The study was aimed to examine 1) comparison of augmentative effects of K(+) on urinary KK excretion with non-specific washout effects by trichlormethiazide (thiazide), polyethyleneglycol 200 (PEG) and rapid physiological saline infusion, 2) contribution of Ca(2+) on the K(+)-induced increase in renal kallikrein secretion. Renal kallikrein activities were measured as fluorescence activities of methylcoumarinylamide-labeled synthetic substrate of tissue kallikrein (TK). Increases in urinary TK excretion were simultaneously observed with diuresis caused by thiazide, PEG, and rapid saline infusion. K(+) infusion increased urinary TK excretion with a diuretic response same as the control. K(+), but not thiazide, showed an early increase in renal TK secretion dose dependently in the kidney slices. Increases in renal TK secretion persisted during treatment with K(+). Neither voltage-dependent Ca(2+)-channel blockers such as verapamil and nifedipine nor simultaneous treatment of EDTA affected on the K(+)-induced increase in renal TK secretion. While, EDTA decreased the K(+)-induced increases in renal TK secretion with time. Caffeine also had an early effect on the increase in renal TK secretion. K(+)-induced increases in renal TK secretion was demonstrated even after treatment with ryanodine or depletion of caffeine-sensitive intracellular Ca(2+) by thapsigargin. It was indicated that the increase in renal TK secretion by K(+) depends on the intracellular Ca(2+) and the caffeine-sensitive release of intracellular Ca(2+) may not be involved in this response. Mechanisms for the K(+)-induced increase in renal TK secretion needs to be further elucidated.

Animals↗
PubMed2006

Role of VLA-4 in the development of allergic conjunctivitis in mice.

PURPOSE: The severity of allergic conjunctivitis (AC) correlates with the degree of eosinophil infiltration into the conjunctiva, which is believed to be mediated by chemokines and adhesion molecules. The adhesion molecule very late antigen (VLA)-4 and its ligand, vascular cell adhesion molecule (VCAM)-1, are known to play important roles in eosinophil infiltration. However, the expression and function of VLA-4 in AC have not been investigated in detail. We sought to characterize VLA-4-expressing cells in the conjunctivas of mice that are developing experimental AC (EC) and to determine whether the interaction between VLA-4 and VCAM-1 is needed for the infiltration of eosinophils into the conjunctiva in AC. METHODS: EC was induced in Balb/c mice by active immunization with ragweed (RW) or adoptive transfer of RW-primed splenocytes, followed by RW challenge. Twenty-four hours after RW challenge, the conjunctivas were harvested. The conjunctivas from naive mice or mice developing EC were evaluated for VLA-4 and VCAM-1 expression by immunohistochemistry and immunofluorescent analyses. To investigate whether the interaction between VLA-4 and VCAM-1 is needed for the genesis of AC, mice developing EC were treated with anti-VLA-4 or anti-VCAM-1 antibodies two h before and after RW challenge. As a control, EC-developing mice were treated with normal rat IgG. Twenty-four hours after RW challenge, the conjunctivas were harvested for histological analysis. RESULTS: Upon induction of EC, VLA-4-expressing cells infiltrated the conjunctiva but the constitutive VCAM-1 expression around conjunctival vessels was not augmented. Immunofluorescent analyses demonstrated that most of the T cells infiltrating the conjunctiva expressed VLA-4 but only half of the infiltrating eosinophils expressed it. Treatment with both anti-VLA-4 and anti-VCAM-1 antibodies significantly suppressed the infiltration of eosinophils into the conjunctiva that was induced by either active immunization or splenocyte transfer. CONCLUSIONS: These results confirm that VLA-4-expressing cells infiltrate the conjunctiva and that the interaction between VLA-4 and VCAM-1 is needed for the development of EC.

Adoptive Transfer↗
PubMed2006

CD8+ T cells play disparate roles in the induction and the effector phases of murine experimental allergic conjunctivitis.

Although CD4+ Th2 cells clearly play an essential role in the development of experimental allergic diseases, the functions CD8+ T cells may have in these diseases have been investigated less extensively and remain controversial. Here, we investigated the roles of CD8+ T cells in the development of experimental allergic conjunctivitis (EC). EC was induced in CD8alpha-deficient (CD8KO) mice and wild-type (WT) mice by active immunization with short ragweed pollen (RW) followed by challenge with RW-containing eye drops. Alternatively, EC was induced by transferring RW-primed splenocytes followed by RW challenge. With regard to actively immunized mice, CD8KO mice showed significantly less severe eosinophil infiltration of the conjunctiva and lower total IgE levels, although the levels of the other Igs were equivalent between the two strains. Cytokine production by cultured splenocytes also did not differ, but the WT conjunctivas showed upregulated IL-5 and IL-6 expression and greater upregulation of IL-4 expression than the conjunctivas of CD8KO mice. Thus, CD8+ T cells may play a significant role during the induction phase by aiding IgE production and the generation of Th2 cytokines in the conjunctiva, thus promoting the development of EC. In contrast, splenocytes from CD8KO mice induced significantly more severe EC in WT mice than cells from WT mice. In addition, transfer of RW-primed splenocytes induced significantly more severe eosinophil infiltration in CD8KO recipient mice. Thus, CD8+ T cells promote the development of EC during the induction phase, but suppress it during the effector phase.

Adoptive Transfer↗
PubMed2006

Cloning and characterization of lipopolysaccharide-induced tumor necrosis factor alpha factor promoter.

We have recently identified lipopolysaccharide tumor-induced tumor necrosis factor alpha factor (LITAF) as a novel transcription factor controlling necrosis factor (TNF)-alpha expression in the human monocytic cell line, THP-1. To characterize the human (h) LITAF promoter, we isolated a 1.2-kb DNA fragment and followed this by a screening of human genomic DNA with a hLITAF cDNA probe. A 34-bp sequence domain located from nucleotides -74 to -43 in the hLITAF promoter exhibited the highest basal reporter gene activity; however, the activity was not elevated by lipopolysaccharide (LPS) stimulation. The sequence domain included a consensus sequence for hepatocyte nuclear factor (HNF)-3alpha, regulating the transcription of many kinds of genes. Interestingly, the DNA sequence position between -542 and -538 in the hLITAF promoter contained the CTCCC motif, which has been reported to act as a specific binding site for hLITAF protein. Electrophoretic mobility shift assays demonstrated that LPS induced the binding of THP-1 nuclear factors to a 22 bp probe containing the CTCCC motif. In addition, hLITAF mRNA and nuclear hLITAF protein increased significantly in the THP-1 cells immediately after LPS stimulation. These results suggest that the consensus sequence for HNF-3alpha, or a nuclear binding protein to the CTCCC motif, may play an important role in regulating LPS-dependent LITAF transcription.

5' Flanking Region↗
PubMed2006

Protein kinase C delta activates topoisomerase IIalpha to induce apoptotic cell death in response to DNA damage.

DNA topoisomerase II is an essential nuclear enzyme that modulates DNA processes by altering the topological state of double-stranded DNA. This enzyme is required for chromosome condensation and segregation; however, the regulatory mechanism of its activation is largely unknown. Here we demonstrate that topoisomerase IIalpha is activated in response to genotoxic stress. Concomitant with the activation, the expression of topoisomerase IIalpha is increased following DNA damage. The results also demonstrate that the proapoptotic kinase protein kinase C delta (PKCdelta) interacts with topoisomerase IIalpha. This association is in an S-phase-specific manner and is required for stabilization and catalytic activation of topoisomerase IIalpha in response to DNA damage. Conversely, inhibition of PKCdelta activity attenuates DNA damage-induced activation of topoisomerase IIalpha. Finally, aberrant activation of topoisomerase IIalpha by PKCdelta is associated with induction of apoptosis upon exposure to genotoxic agents. These findings indicate that PKCdelta regulates topoisomerase IIalpha and thereby cell fate in the genotoxic stress response.

Antigens, Neoplasm↗
PubMed2006

Roles of OX40 in the development of murine experimental allergic conjunctivitis: exacerbation and attenuation by stimulation and blocking of OX40.

PURPOSE: To investigate the roles of interaction between OX40 and OX40 ligand (OX40L) in the development of experimental allergic conjunctivitis (EC) in mice. METHODS: BALB/c mice actively immunized with short ragweed pollen (RW) were intraperitoneally injected on days 0, 2, 4, 6, and 8 with agonistic anti-OX40 Ab, blocking anti-OX40L Ab, or normal rat (nr)IgG. On day 10, the mice were challenged with RW in eye drops, and 24 hours later their conjunctivas, spleens, and blood were harvested for analyses. For examination of the effects of the Abs during the late induction (or effector) phase, actively immunized mice were treated with the Abs just before or at the same time as the challenge. In addition, splenocytes from RW-primed mice were transferred into syngeneic naïve mice, and the recipients were treated with Abs twice (on days 2 and 4). On day 4, the mice were challenged with RW and evaluated. RESULTS: When the treatments were performed during the induction phase, anti-OX40 Ab treatment significantly increased clinical EC and eosinophil infiltration into the conjunctiva, whereas anti-OX40L Ab treatment significantly reduced eosinophil infiltration. Compared with splenocytes from nrIgG-treated mice, splenocytes from anti-OX40 Ab-treated mice proliferated vigorously against RW and produced significantly higher amounts of IL-2, -4, and -5 by RW stimulation but a significantly lesser amount of IFN-gamma after Con A stimulation. In contrast, splenocytes from anti-OX40L Ab-treated mice produced significantly less IL-5 with RW stimulation and IL-2 and IL-5 with Con A stimulation, whereas significantly more IFN-gamma was induced by Con A stimulation. Treatment with anti-OX40 and anti-OX40L Abs during the late induction or effector phase of EC did not affect eosinophil infiltration. CONCLUSIONS: Blocking of the interaction between OX40 and OX40L in vivo inhibits the development of EC. In contrast, forced stimulation of OX40 in vivo significantly exacerbates EC by activating T cells, especially Th2 cells. These effects were noted only in the induction phase of EC, suggesting that the interaction between OX40 and OX40L is important in the generation of Th2 immune responses in the development of EC.

Adoptive Transfer↗
PubMed2005

TLR2 agonist ameliorates murine experimental allergic conjunctivitis by inducing CD4 positive T-cell apoptosis rather than by affecting the Th1/Th2 balance.

Innate immune responses that operate through Toll-like receptors (TLRs) are actively involved in the development of diseases predominantly mediated by adaptive immune responses. This is true also for allergic disease, as TLRs have been found to be involved in the development of allergic airway inflammation. We investigated whether stimulating TLR2 also abrogates murine allergic conjunctivitis by upregulating Th1 responses. We found that treating mice during the efferent phase with the TLR2 agonist Pam3CSK4 significantly suppressed eosinophil infiltration into the conjunctiva. However, Pam3CSK4 treatment inhibited both the Th1 and Th2 responses in the mice, and also suppressed eosinophil infiltration in IFN-gamma knockout mice. Flow cytometric analysis demonstrated that Pam3CSK4 treatment significantly elevated the numbers of annexin V-positive splenocytes, especially CD4 positive T cells. Thus, the stimulation of TLR2 during the efferent phase of murine allergic conjunctivitis suppresses eosinophil infiltration by inducing CD4 positive T-cell apoptosis rather than upregulating Th1 responses.

Animals↗
PubMed2005

The interaction between ICOS and B7RP-1 is not required for the development of experimental murine allergic conjunctivitis.

It is still unclear whether the interaction between inducible costimulator (ICOS) and its ligand, B7 related protein (B7RP)-1, is important for the development of allergic diseases. We investigated whether blocking the ICOS/B7RP-1 interaction affects the development of murine experimental allergic conjunctivitis (EC). EC was induced in Balb/c mice either by active immunization of ragweed (RW) or by transferring RW-primed splenocytes, followed by challenge with RW-containing eye drops. The mice were treated with anti-B7RP-1 antibody (Ab) or normal rat immunoglobulin G (IgG) during either the induction or effector phase. Regardless of the induction method or when the animals were treated, eosinophil infiltration into the conjunctiva was not affected by the anti-B7RP-1 Ab treatment. Splenocyte responses were not largely affected by this treatment. However, serum Ig levels were significantly reduced. These data suggest that blocking the ICOS/B7RP-1 in allergic diseases may not always be therapeutic.

Allergens↗
PubMed2005

Fyn regulates eosinophil infiltration into the conjunctiva by downregulating the Th2 response.

PURPOSE: Under certain circumstances, fyn may serve to negatively regulate the differentiation of naïve helper T (Th) cells into Th2 cells. This study aimed to investigate whether fyn negatively regulates the development of experimental immune-mediated blepharoconjunctivitis (EC), in which Th2 cells play an important role in C57BL/6 mice. METHODS: C57BL/6 background wild-type (WT) or fyn knockout (fyn-/-) mice were subcutaneously immunized with ragweed (RW) adsorbed in aluminum hydroxide. Ten days later the mice were challenged with RW in eye drops, and 24 h after challenge, eyes, blood and spleens were harvested for histology, measurement of serum IgE, and proliferation or cytokine assays, respectively. RW-primed splenocytes from WT and fyn-/- mice were cultured in the presence of RW. Seventy-two hours later, either whole splenocytes or isolated CD4+T cells were transferred into syngeneic WT mice. Four days after the transfer, the recipient mice were challenged with RW and evaluated as described above. RESULTS: Infiltration of eosinophils into the conjunctiva induced by active immunization was significantly increased in fyn-/- mice relative to WT mice. Total serum IgE was also significantly higher in fyn-/- mice than in WT mice. In parallel, a higher level of IL-4 production from splenocytes was induced by concanavalin A stimulation in fyn-/- mice than in WT mice. In contrast to active immunization, transfer of whole splenocytes or separated CD4+T cells derived from WT or fyn-/- mice induced similar levels of eosinophilic infiltration in WT mice. CONCLUSIONS: Fyn regulates infiltration of eosinophils into the conjunctiva through downregulation of Th2 responses. This negative regulation is exerted only during the induction phase of EC.

Animals↗
PubMed2005

Engagement of 4-1BB inhibits the development of experimental allergic conjunctivitis in mice.

The 4-1BB receptor acts as a costimulator in CD8(+) T cell activation. Agonistic stimulation through this molecule by treatment with anti-4-1BB Abs has been demonstrated to inhibit various experimentally induced diseases in animals. However, the effect of anti-4-1BB Abs on experimental allergic diseases has not been reported. We investigated the effect of anti-4-1BB Abs on the development and progression of experimental allergic conjunctivitis in mice. To examine the effects of Abs during the induction or effector phase, actively immunized mice or passively immunized mice by splenocyte transfer were treated with agonistic anti-4-1BB Abs, blocking anti-4-1BB ligand Abs, or normal rat IgG. Eosinophil infiltration into the conjunctiva was significantly reduced in wild-type mice by the anti-4-1BB Ab treatment during either induction or effector phase. Th2 cytokine production by splenocytes and total serum IgE were significantly reduced by the anti-4-1BB Ab treatment, while IFN-gamma production was increased. The anti-4-1BB Ab treatment induced a relative increase of CD8-positive cell numbers in the spleens. Moreover, inhibition of eosinophil infiltration by the treatment with anti-4-1BB Abs was also noted in actively immunized IFN-gamma knockout mice. Taken altogether, in vivo treatment with agonistic anti-4-1BB Abs in either induction or effector phase inhibits the development of experimental allergic conjunctivitis, and this inhibition is likely to be mediated by suppression of Th2 immune responses rather than up-regulation of IFN-gamma.

Animals↗
PubMed2005

The immunization protocol determines whether endogenous interferon-gamma suppresses the infiltration of eosinophils into the conjunctiva.

BACKGROUND: Studies with interferon-gamma knockout (GKO) mice showed that endogenous IFN-gamma suppresses the infiltration of inflammatory cells into the conjunctiva. To examine whether this phenomenon is universally true, we induced conjunctival inflammation by four different immunization protocols. METHODS: Both wild type (WT) and GKO mice (C57BL/6 background) were immunized with ragweed (RW) in aluminum hydroxide (alum). Two different immunization protocols were used: either the emulsion was injected into only the left hind footpad or it was also injected into the tail base (50 microg RW in 2mg alum per injection site). In addition, to compare the effects of the immunization dose of RW and the immunization site, 100 microg RW in 2mg alum was injected into only the left hind footpad and 25 microg RW in 2mg alum per injection site was injected into both the left hind footpad and the tail base. Ten days later, the mice were challenged with 2mg RW in 10 microl PBS. Twenty-four hours later, the conjunctivas were analyzed histologically, and the cellular and humoral immune responses in the spleens and sera were determined, respectively. RESULTS: Similar to a previous report, GKO mice showed significant eosinophilic infiltration into the conjunctiva after the footpad only injection of 50 microg RW. However, injection of 50 microg RW per injection site into the footpad plus the tail base resulted in comparable levels of eosinophilic infiltration in WT and GKO mice. On the contrary, either immunization of 100 microg RW in 2mg alum into only the left hind footpad or that of 25 microg RW in 2mg alum into both the left hind footpad and the tail base induced significant infiltration of eosinophils into the conjunctiva of GKO mice, compared to WT mice. CONCLUSIONS: These results show that the immunization protocol employed has a marked effect on the severity of eosinophilic infiltration. These observations indicate that in interpreting experimental results in the study of EC, the immunization protocol employed must be considered.

Allergens↗
PubMed2005

Genetic background determines susceptibility to experimental immune-mediated blepharoconjunctivitis: comparison of Balb/c and C57BL/6 mice.

Balb/c and C57BL/6 mice have been reported to be biased towards Th2 and Th1 immune responses, respectively. We investigated which strain is more susceptible to the development of experimental immune-mediated blepharoconjunctivitis (EC), which is predominantly mediated by Th2 immune responses. EC was induced by three different methods in Balb/c and C57BL/6 mice using ragweed (RW) as the antigen. The mice were thus either actively immunized with RW, passively immunized by transfer of RW-primed T cells, or passively immunized by transfer of RW-specific IgE, followed by RW challenge in eye drops. Twenty-four hours after the challenge, conjunctivas, sera and spleens were harvested for histological analysis, measurement of serum IgE and assessment of cellular immune responses, respectively. The responses of the Balb/c and C57BL/6 mice were compared. In addition, to assess the involvement of IFN-gamma in the development of EC in the two strains, IFN-gamma knockout (GKO) mice of the two strains were actively immunized and evaluated as above. Regardless of the method of induction, EC, as determined by the degree of eosinophil infiltration into the conjunctiva, was more severe in Balb/c mice than in C57BL/6 mice. Moreover, more IgE was produced by actively immunized Balb/c mice than C57BL/6 mice and RW-primed splenocytes from Balb/c mice produced more IL-4 but less IFN-gamma than those from C57BL/6 mice. EC could be induced in the GKO mice of both strains. However, when their EC was compared to that in WT mice, significantly less infiltration of eosinophils was noted in the Balb/c GKO mice. Taken together, Balb/c mice are more susceptible to EC than C57BL/6 mice and this higher susceptibility might be related to the Th2 immune response bias of Balb/c mice. Furthermore, the involvement of endogenous IFN-gamma in the development of EC in these two strains differs.

Adoptive Transfer↗
PubMed2005

Mice lacking the IFN-gamma receptor or fyn develop severe experimental autoimmune uveoretinitis characterized by different immune responses.

Endogenous interferon (IFN)-gamma negatively regulates experimental autoimmune uveoretinitis (EAU), a Th1-mediated disease. Although it is well known that IFN-gamma exerts its effects by binding to the IFN-gamma receptor (IFN-gammaR), the role that IFN-gammaR plays in the development of EAU has not been investigated. Fyn has been reported to inhibit Th2 differentiation. We aimed to investigate how endogenous IFN-gammaR and fyn, which influence Th1/Th2 differentiation, participate in the development of EAU. Sex-matched 6- to 10-week-old C57BL/6 wild-type (WT), IFN-gammaR knockout (GRKO) and fyn knockout (fyn KO) mice were compared. Mice were immunized subcutaneously with human interphotoreceptor retinoid-binding protein peptide 1-20 emulsified in Freund's complete adjuvant together with an intraperitoneal injection of Bordetella pertussis toxin. Three weeks later, mice were sacrificed, and their eyes and spleens were harvested for histopathologic analyses and examination of cellular immune responses, respectively. Cellular immune responses were evaluated by measuring the proliferative responses and cytokine production [interleukin (IL)-4, IL-5, IL-6, IL-13, IFN-gamma and tumor necrosis factor (TNF)-alpha] of splenocytes. The incidence of EAU was 40.0% in WT mice, 59.3% in GRKO mice and 78.6% in fyn KO mice. The average EAU score was 0.294 in WT mice, 0.917 in GRKO mice and 1.063 in fyn KO mice. Upon EAU induction, significant infiltration of eosinophils into the eyes was observed in GRKO and fyn KO mice compared to WT mice. Splenocytes from GRKO mice proliferated against the antigen and a mitogen more vigorously than those from WT and fyn KO mice. Stimulation of splenocytes with the antigen induced a higher production of IL-4, IL-6, IL-13 and IFN-gamma in GRKO mice compared to WT and fyn KO mice. In contrast, IL-5 and TNF-alpha were most abundantly produced by splenocytes from fyn KO mice compared to WT and GRKO mice. The incidence and mean severity of EAU were significantly higher in GRKO and fyn KO mice than in WT mice, suggesting that endogenous IFN-gammaR and fyn negatively regulate the development of EAU. The different cytokine production patterns by the GRKO and fyn KO mice indicate that the negative regulatory mechanism mediated by IFN-gammaR and fyn may differ.

Animals↗
PubMed2005

JNK phosphorylation of 14-3-3 proteins regulates nuclear targeting of c-Abl in the apoptotic response to DNA damage.

The ubiquitously expressed c-Abl tyrosine kinase localizes to the cytoplasm and nucleus. Nuclear c-Abl is activated by diverse genotoxic agents and induces apoptosis; however, the mechanisms that are responsible for nuclear targeting of c-Abl remain unclear. Here, we show that cytoplasmic c-Abl is targeted to the nucleus in the DNA damage response. The results show that c-Abl is sequestered into the cytoplasm by binding to 14-3-3 proteins. Phosphorylation of c-Abl on Thr 735 functions as a site for direct binding to 14-3-3 proteins. We also show that, in response to DNA damage, activation of the c-Jun N-terminal kinase (Jnk) induces phosphorylation of 14-3-3 proteins and their release from c-Abl. Together with these results, expression of an unphosphorylated 14-3-3 mutant attenuates DNA-damage-induced nuclear import of c-Abl and apoptosis. These findings indicate that 14-3-3 proteins are pivotal regulators of intracellular c-Abl localization and of the apoptotic response to genotoxic stress.

14-3-3 Proteins↗
PubMed2005

Influence of cocoa flavanols and procyanidins on free radical-induced human erythrocyte hemolysis.

Cocoa can be a rich source of antioxidants including the flavan-3-ols, epicatechin and catechin, and their oligomers (procyanidins). While these flavonoids have been reported to reduce the rate of free radical-induced erythrocyte hemolysis in experimental animal models, little is known about their effect on human erythrocyte hemolysis. The major objective of this work was to study the effect of a flavonoid-rich cocoa beverage on the resistance of human erythrocytes to oxidative stress. A second objective was to assess the effects of select purified cocoa flavonoids, epicatechin, catechin, the procyanidin Dimer B2 and one of its major metabolites, 3'-O-methyl epicatechin, on free radical-induced erythrocyte hemolysis in vitro. Peripheral blood was obtained from 8 healthy subjects before and 1, 2, 4 and 8h after consuming a flavonoid-rich cocoa beverage that provided 0.25g/kg body weight (BW), 0.375 or 0.50g/kg BW of cocoa. Plasma flavanol and dimer concentrations were determined for each subject. Erythrocyte hemolysis was evaluated using a controlled peroxidation reaction. Epicatechin, catechin, 3'-O-methyl epicatechin and (-)-epicatechin-(4beta > 8)-epicatechin (Dimer B2) were detected in the plasma within 1 h after the consumption of the beverage. The susceptibility of erythrocytes to hemolysis was reduced significantly following the consumption of the beverages. The duration of the lag time, which reflects the capacity of cells to buffer free radicals, was increased. Consistent with the above, the purified flavonoids, epicatechin, catechin, Dimer B2 and the metabolite 3'-O-methyl epicatechin, exhibited dose-dependent protection against AAPH-induced erythrocyte hemolysis at concentrations ranging from 2.5 to 20 microM. Erythrocytes from subjects consuming flavonoid-rich cocoa show reduced susceptibility to free radical-induced hemolysis (p < 0.05).

Adult↗