Academic literature on the topic 'Â2-cell'

Abstract Background Systemic inflammatory factors such as C-reactive protein (CRP), ¥â2-microglobulin (B2MG), and ferritin were documented an prognostic factor in patients with hematologic malignancies including lymphoma. The purpose of this study was to find the significance of systemic inflammatory factors for predicting survival outcome in patients with diffuse large B cell lymphoma (DLBCL). Methods A total of 188 patients who newly diagnosed DLBCL and received an rituximab combined chemotherapy at the South Korea between September 2004 and April 2012 were enrolled retrospectively in the current study. Pretreatment serum CRP, B2MG, and ferritin were measured within 4 weeks before the beginning of first line chemotherapy. Results The median age of patients was 58 years (range, 14-84 years) and the mean level of serum CRP, B2MG, and ferritin at pre-treatment were 2.37 mg/dL (range: 0.01 – 29.80), 2.64 mg/L (range: 0.71 – 24.01), and 285.01 ng/ml (range: 5.34-4872.40). Systemic inflammatory factors score were given 1 point if serum levels were more than normal range (CRP; 0.8 mg/dL, B2MG; 2.5 mg/L and ferritin; 220 ng/ml). Systemic inflammatory risk were divided into three groups according to systemic inflammatory factors scores ; low risk was 0 score, intermediate risk was 1 or 2 score, and high risk was 3 score respectively. 5-year progression free survival rates (PFS) were 66.9%, 65.0% and 10.6% in risk groups respectively (p < 0.001). 5-year overall survival rates (OS) were 74.4%, 48.8% and 19.8% in risk groups respectively (p < 0.001). Conclusions Systemic inflammatory factor (CRP, B2MG and ferritin) score were associated with survival outcomes in patients with DLBCL treated by R-CHOP. However, further studies are needed to confirm prognostic value of systemic factors such as CRP, B2MG and ferritin. IPI, international prognostic index; LDH, lactate dehydrogenase; ECOG, Eastern Cooperative Oncology Group (ECOG) performance status; CRP, C-reactive protein; BM, bone marrow; ALC, absolute lymphocyte counts; CHOP-like (cyclophosphamide, adriamycin, vincristine, prednisolone); RCHOP (rituximab plus CHOP) Systemic inflammatory factor (normal range); ¥â2-microglobulin (2.5mg/L), CRP (0.8mg/dL), ferritin (220 ng/mL) Systemic inflammatory factor score : given 1 point if more than normal range Low risk ; 0, Intermediate risk ; 1 or 2, High risk ; 3 Disclosures: No relevant conflicts of interest to declare.

The imaging of biological macromolecules at high resolution is predicated on the use of imaging modalities that combine a sufficiently high signal-to-noise ratio (S/N) to observe the desired detail with a sufficiently low dose not to have perturbed the structure at the resolution of that detail. For 3D structure determination the S/N has to be sufficiently high to permit the accurate position and orientation determination for individual molecules or assemblies.For 2D crystal specimens the position is determined a priori by the lattice, orientation by the externally chosen tilt of the specimen, and signal-to-noise in a single unit cell enhanced by the translocational redundancy of the many unit cells of the crystal. This is close to an ideal specimen, which has permitted imaging with resolutions of 3.4 À at doses of 20-35 e/Â2[l]. Specimens with high internal symmetry, icosahedral or helical structures, are examples which confer intermediate structural redundancies to assist in reducing the requires electron dose.

Background: Multiple Myeloma (MM) accounts for 1% of malignant tumors and 10%–15% of hematopoietic neoplasms. Bortezomib, a first in class proteasome inhibitor, induces apoptosis and growth arrest and reverse chemoresistence in Myeloma cell and has demonstrated no irreversible adverse effect on haemopoietic stem cell. Dexamethasone increases the response rate. Thus, Bortezomib plus dexamethasone represent highly effective regimen for previously untreated Multiple Myeloma cases and significantly higher response rates approximately 70%– 90% have been observed.This combination thus may serve the basis of future strands of care in Multiple Myeloma patients. Objective: The aim of the study was to assess the efficacy , safety and tolerability of Bortezomib in newly diagnosed cases of Multiple Myeloma patients in Bangladesh. Materials & Methods: This prospective observational study was carried out in the Haematology department of BSMMU from June 2017 to December 2018. Patients received inj. Bortezomib (1.3mg/m2 ) 4 cycles as an intravenous bolus on days 1,4,8,11 in a three week cycle (twice weekly administration) in indoor and same patients as day care basis in outpatients department. Dexamethasone at 40 mg was given intravenously or orally on the day of and day after inj Bortezomib.A self administered questionnaire containing different set of questions regarding Multiple Myeloma were used for data collection. Results: Among the study population, 93% of patients had anaemia followed by bone pain (86%) and renal impairment (39%). Out of 25 patients,complete response achieved in 13 patients (52%), where 4 patients(16%) showed partial response,6 (24%) showed very good partial response and 2 (8%) patients showed no response. The overall response rate was 92% belonged to partial,very goofd partial and no respone respectively. Death occurred in 3 cases (12%). 5 patients (20%) developed Bortezomib induced peripheral neuropathy.Life threatening intracranial haemorrhage occurred in two patients (8%). Death occurred in 3 cases (12%),2 patients due to intracranial haemorrhage and another from cardiac arrest. In this study,S. creatinine, â2 microglobulin and bony lesion variables showed significant association with treatment response. Conclusion: Bortezomib plus dexamethasone is a highly effective and safe regimen for previously untreated multiple myeloma patients. This novel therapy in myeloma represent a new trearment paradigm targeting both tumor and microenvironment which has markedly improve overall response(OR), long progression free survival (PFS) and overall survival (OS)across in all risk groups. Moreover,it can be administered safely in the outpatient setting provided by clinicians. J Dhaka Medical College, Vol. 28, No.1, April, 2019, Page 34-41

Abstract Background : Prognosis of diffuse large B cell lymphoma (DLBCL) is very varied from cure to death. So the prediction for prognosis of DLBCL is very important to make a decision regarding the treatment. Now, international prognostic index (IPI) risk model is widely known as the powerful prognostic indicator for lymphoma. However, many inflammatory factors have been demonstrated as prognostic factors in patients with DLBCL such as C-reactive protein (CRP), ferritin, ¥â2-microglobulin (B2MG), absolute lymphocyte count (ALC) and albumin etc. But LDH is the only inflammatory factor in those of IPI. So we find a new prognostic risk model (inflammatory factors based modified IPI risk model ; inflammatory-IPI) including other inflammatory prognostic factors for predicting more correct survival outcomes and progression free survival (PFS) in patients with DLBCL treated with rituximab combined cyclophosphamide, adriamycin, vincristine and prednisone (RCHOP). Methods: A total of 278 patients who were newly diagnosed with DLBCL and received RCHOP at hospitals throughout South Korea between January 2007 and December 2014 were enrolled retrospectively in the current study. Baseline serum CRP, ferritin, B2MG, ALC, albumin and factors of IPI were measured within 4 weeks before beginning the first line of chemotherapy. Each inflammatory factor of serum CRP > 1.5mg/dL, ferritin > 500ng/mL, B2MG > 3.5mg/L, ALC level < 1.0x109/L, serum albumin < 3.5g/dL, LDH level > 450 IU/L was defined as an abnormal findings, and if the sum of these abnormal findings are not fewer than 2, it is given 1 point as a factor of inflammatory-IPI instead of LDH. After that, sum of this point and the number of negative prognostic factors present at the time of diagnosis (age > 60 years, ECOG performance status ¡Ã 2, extranodal site ¡Ã 2, stage III/IV disease) is named the inflammatory-IPI. The inflammatory-IPI risk model were divided into four groups seems like IPI according to the scores ; low risk were 0 or 1 score, low-intermediate risk were 2 scores, and high-intermediate risk were 3 scores, high risk were 4 scores respectively. Results: In univariate analysis, the following factors showed significant higher 5 years overall survival rates (OS) : age (p=0.001), stage III/IV disease (p=0.008), LDH level (p<0.001), CRP (p=0.001), ferritin (p<0.001), B2MG (p<0.001), revised IPI (p<0.001). These factors also showed significant higher 5 years PFS. But some other factors did not showed significant OS and PFS in our data : ECOG, extranodal involvement, ALC, albumin. The studied groups were comprised of 117 patients in low risk group, 63 patients in low-intermediate group, 56 patients in high-intermediate group, 40 patients in high risk group according to inflammatory-IPI scores including sum of abnormal inflammatory findings. The OS and PFS according to the inflammatory-IPI showed significant differences (p<0.001 and p<0.001, respectively) (Figure 1). Conclusions: The survival outcomes showed significant differences between risk groups which were categorized by inflammatory-IPI. This new inflammatory-IPI risk model could be supplemented the deficiency of standard IPI by including other prognostic inflammatory factors as well as LDH. It will be helpful to predict prognosis and make treatment planning in patients with DLBCL treated with RCHOP. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Abstract Purpose/Objective Clinical and experimental studies have established that allogeneic blood transfusion (ABT) can cause immunosuppression. To identify immune parameters that contribute to this effect, we determined the effect of ABT on peripheral blood (PB) cytokine profiles and Treg numbers and function in a cohort of patients with no underlying pathologies. Materials and Methods Heparinized PB samples were collected from 46 patients (7M/39F, 28-88 yo) that underwent joint replacement surgery. The samples were collected immediately before surgery (BS), and after surgery (AS) on days 0, 7, 1 month, and 3 months to 1 year. Thirty six patients received ABT and 10 did not. PBMC were isolated, and the numbers and % of CD4+CD25+Foxp3+ Tregs and CD4+CD25high/+CD127low/- Tregs were determined by FACS. Tregs and T effectors (Teff) were isolated from patients on days 0-7 and Treg functional assays were performed by culturing Tregs with PHA-stimulated Teff at different ratios for 72h with CFSE, and analyzed by FACS for proliferation. Cytokine levels were determined in plasma by a cytometric bead array assay for IL-2, IL-4, IL-5, IL-6, IL-10, TNF-á, IFN-ã, and ELISA for TNF-á, TNF-RI(p55/p60) and II(p75/p80), TGF-â1 and â2. Results Both, CD4+CD25+Foxp3+ and CD4+CD25high/+CD127low/- Treg populations increased significantly on d0 AS and decreased on d7 AS to below BS levels, to achieve homeostasis >3 months in transfused patients. In contrast, Treg levels remained the same between BS and AS in non-transfused patients. Functional assays showed that Tregs were functional post-ABT and could suppress Teff proliferation efficiently. In culture, isolated Tregs secreted TGF-â1. All cytokines and TNF-RI and II plasma levels increased on d0 AS (IL-6, TNF-RI and II significantly), in transfused patients immediately AS, and decreased by d7 AS. In contrast, TGF-â1 levels decreased on d0 AS and increased by d7. No differences in cytokine or receptor levels were observed in non-transfused patients AS. Conclusion ABT induces the immediate production of plasma cytokines, especially pro-inflammatory IL-6 and TNF-RI and II that decreases by d7. In parallel, ABT induces the proliferation of Tregs. These Tregs are functional, and secrete TGF-â1 that reaches BS plasma levels by d7. The Tregs seem to be Th3 inducible Tregs. Homeostasis is reached by 3 months post-ABT. Disclosures: No relevant conflicts of interest to declare.

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75
Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75.
Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins.