Intracellular Phosphorylation Research Articles

Creatine loading elevates the intracellular phosphorylation potential and alters adaptive responses of rat fast-twitch muscle to chronic low-frequency stimulation.

This study tested the hypothesis that elevating the intracellular phosphorylation potential (IPP = [ATP]/[ADP]free) within rat fast-twitch tibialis anterior muscles by creatine (Cr) loading would prevent fast-to-slow fibre transitions induced by chronic low-frequency electrical stimulation (CLFS, 10 Hz, 12 h/day). Creatine-control and creatine-CLFS groups drank a solution of 1% Cr + 5% dextrose, ad libitum, for 10 days before and during 10 days of CLFS; dextrose-control and dextrose-CLFS groups drank 5% dextrose. Cr loading increased total Cr (P < 0.025), phosphocreatine (PCr) (P < 0.003), and the IPP (P < 0.0008) by 34%, 45%, and 64%, respectively. PCr and IPP were 46% (P < 0.002) and 76% (P < 0.02) greater in creatine-CLFS than in dextrose-CLFS. Higher IPP was confirmed by a 58% reduction in phospho-AMP-activated protein kinase α (Thr172) (P < 0.006). In dextrose-CLFS, myosin heavy chain (MyHC) I and IIa transcripts increased 32- and 38-fold (P < 0.006), respectively, whereas MyHC-IIb mRNA decreased by 75% (P < 0.03); the corresponding MyHC-I and MyHC-IIa protein contents increased by 2.0- (P < 0.03) and 2.7-fold (P < 0.05), respectively, and MyHC-IIb decreased by 30% (P < 0.03). In contrast, within creatine-CLFS, MyHC-I and MyHC-IIa mRNA were unchanged and MyHC-IIb mRNA decreased by 75% (P < 0.003); the corresponding MyHC isoform contents were not altered. Oxidative reference enzymes were similarly elevated (P < 0.01) in dextrose-CLFS and creatine-CLFS, but reciprocal reductions in glycolytic reference enzymes occurred only in dextrose-CLFS (P < 0.02). Preservation of the glycolytic potential and greater SERCA2 and parvalbumin contents in creatine-CLFS coincided with prolonged time to peak tension and half-rise time (P < 0.01). These results highlight the IPP as an important physiological regulator of muscle fibre plasticity and demonstrate that training-induced changes typically associated with improvements in muscular endurance or increased power output are not mutually exclusive in Cr-loaded muscles.

Population pharmacokinetic modeling of plasma and intracellular ribavirin concentrations in patients with chronic hepatitis C virus infection.

Ribavirin, a guanosine analog, is a broad-spectrum antiviral agent. Ribavirin has been a fundamental component of the treatment of hepatitis C virus (HCV) infection for decades, but there is a very limited understanding of the clinical pharmacology of this drug. Furthermore, it is associated with a major dose-limiting toxicity, hemolytic anemia. Ribavirin undergoes intracellular phosphorylation by host enzymes to ribavirin monophosphate (RMP), ribavirin diphosphate (RDP), and ribavirin triphosphate (RTP). The intracellular forms have been associated with antiviral and toxic effects in vitro, but the kinetics of these phosphorylated moieties have not been fully elucidated in vivo. We developed a model to characterize the plasma pharmacokinetics of ribavirin and the difference between intracellular phosphorylation kinetics in red cells (nonnucleated) and in peripheral blood mononuclear cells (nucleated). A time-independent two-compartment model with first-order absorption described the plasma data well. The cellular phosphorylation kinetics was described by a one-compartment model for RMP, with the formation rate driven by plasma concentrations and the first-order degradation rate. RDP and RTP rapidly reached equilibrium with RMP. Concomitant telaprevir use, inosine triphosphatase genetics, creatinine clearance, weight, and sex were significant covariates. The terminal ribavirin half-life in plasma and phosphorylated anabolites in cells was approximately 224 h. We found no evidence of time-dependent kinetics. These data provide a foundation for uncovering concentration-effect associations for ribavirin and determining the optimal dose and duration of this drug for use in combination with newer direct-acting HCV agents. (This study has been registered at ClinicalTrials.gov under registration no. NCT01097395.).

CD4+CD25+ Invariant Natural Killer T Cells Are Enriched in Cord Blood: Implication on Immune-Regulation

unstimulated control. The surface expression of CCR5 was measured separately by flow cytometry. Results: Twenty-four reduced-intensity alloSCT recipients received MVC 300 mg b.i.d. orally from day -3 to day +90 on an ongoing phase II study. Recipient bloodwas tested on days -6 (beforeMVC), 0 and 14, and donor bloodwas tested on day 0. We observed significant variability in surface CCR5 expression on T-cells in donors (range 0e9%) and recipients (range 0e49%). Recipient surface CCR5 expression increased from d-6 to d0 on both CD4 (mean 3.6 to 7.3%, p1⁄40.005) and CD8 (mean 13.5 to 30.6%, p1⁄40.005) T-cells, consistent with blockade of receptor internalization. CD8 T-cell pCCR5 levels increased in response to CCL4 stimulation on d -6 (p1⁄40.01) and decreased in response to MVC (p1⁄40.002), compared to unstimulated blood (Fig. 2). A decrease in pCCR5 levels in response to MVC was observed evenwithout CCL4 stimulation, reflecting loss of background CCR5 activity (p1⁄40.0002). In contrast, d0 pCCR5 levels failed to increase in response to CCL4 (p1⁄40.32), nor did they decrease in response to additional MVC (p1⁄40.13), mirroring effective in vivo blockade of CCR5. The magnitude of increase in CD8 T-cell pCCR5 levels in response to CCL4 stimulation on d-6 correlated with CCR5 surface expression (r1⁄40.55, p1⁄40.02). The same was true of CD4 T-cells (r1⁄40.76, p1⁄40.0004). On d0, however, the ability to stimulate CCR5 inversely correlated with CCR5 surface expression for both CD8 (r1⁄4-0.46, p1⁄40.04) and CD4 (r1⁄4-0.62, p1⁄40.004), suggesting that greater CCR5 surface upregulation may serve as an indicator for complete receptor blockade. Conclusions: Intracellular phosphorylation of CCR5 in response to stimulation is efficiently blocked by MVC in patients undergoing alloSCT, as measured by a novel phosphoflow assay. Upregulation of surface CCR5 may indicate effective CCR5 blockade and should be explored as a biomarker for drug response.

Fractalkine promotes chemotaxis of bone marrow-derived mesenchymal stem cells towards ischemic brain lesions through Jak2 signaling and cytoskeletal reorganization.

The fractalkine (FKN)-CX3CR1 (FKN receptor) axis reportedly plays an important role in the progression of many neural pathologies. However, its role in the recruitment of bone marrow-derived progenitor cells for neurogenesis remains elusive. The chemokine-based mechanism underlying the migration of bone marrow-derived mesenchymal stem cells (BMSCs) was investigated in a double-chamber transmigration model with recombinant FKN and endogenous FKN extract, and the results confirmed the involvement of FKN in migration. This chemotactic response was CX3CR1-dependent and FKN-sensitive. Western blotting, immunoprecipitation and transmigration assays revealed that the Janus kinase (Jak)2-signal transducer and activator of transcription (Stat)5α-extracellular signal-related kinase (ERK)1/2 pathway was activated by FKN. Confocal laser scanning microscopy was used to demonstrate cytoskeletal reorganization caused by remodeling of the surface receptor integrin α5β1, intracellular phosphorylation of Fak and Pax, and upregulation of intercellular adhesion molecule-1 during BMSC migration. Moreover, significant inhibition of signaling and migration was detected after treatment of cells with Jak2-interfering RNA or the antagonist AG490. In addition, the results of a fluorescence immunohistochemical analysis of an in vivo chemotactic model, developed via transplantation of BMSCs into transient middle cerebral artery-occluded rats, were consistent with the in vitro results. These findings suggest that FKN activates Jak2-Stat5α-ERK1/2 signaling through CX3CR1, thereby triggering integrin-dependent machinery reorganization to allow chemotactic migration of BMSCs towards an ischemic cerebral lesion.

Subcellular distribution of FTY720 and FTY720-phosphate in immune cells - another aspect of Fingolimod action relevant for therapeutic application.

FTY720 (Fingolimod; Gilenya®) is an immune-modulatory prodrug which, after intracellular phosphorylation by sphingosine kinase 2 (SphK2) and export, mimics effects of the endogenous lipid mediator sphingosine-1-phosphate. Fingolimod has been introduced to treat relapsing-remitting multiple sclerosis. However, little has been published about the immune cell membrane penetration and subcellular distribution of FTY720 and FTY720-P. Thus, we applied a newly established LC-MS/MS method to analyze the subcellular distribution of FTY720 and FTY720-P in subcellular compartments of spleen cells of wild type, SphK1- and SphK2-deficient mice. These studies demonstrated that, when normalized to the original cell volume and calculated on molar basis, FTY720 and FTY720-P dramatically accumulated several hundredfold within immune cells reaching micromolar concentrations. The amount and distribution of FTY720 was differentially affected by SphK1- and SphK2-deficiency. On the background of recently described relevant intracellular FTY720 effects in the nanomolar range and the prolonged application in multiple sclerosis, this data showing a substantial intracellular accumulation of FTY720, has to be considered for benefit/risk ratio estimates.

Cytokine-stimulated Phosphoflow of PBMC Using CyTOF Mass Cytometry

Phosphorylation of tyrosine, serine, and threonine residues is critical for the control of protein activity involved in various cellular events. An assortment of kinases and phosphatases regulate intracellular protein phosphorylation in many different cell signaling pathways. These pathways include T and B cell signaling, regulating growth and cell cycle control, plus cytokine, chemokine, and stress responses. Phosphoflow assays combine phosphoprotein-specific antibodies with the power of flow cytometry to enhance phosphoprotein study. In our assay, peripheral blood mononuclear cells are stimulated by cytokines, fixed, surface-stained with a cocktail of antibodies labeled with MAXPAR (brand name) metal-chelating polymers and permeabilized with methanol. They are then stained with intracellular phospho-specific antibodies. We use a CyTOF™ mass cytometer to acquire the ICP-MS (inductively coupled plasma mass spectrometry) data. The current mass window selected is approximately AW 103-203, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type. Median values are reported to quantitate the level of phosphorylation of each protein in response to stimulation. Comparing the level of phosphorylation between samples can offer insight to the status of the immune system.

Cytokine-Stimulated Phosphoflow of Whole Blood Using CyTOF Mass Cytometry

The ability to assess the function of a range of cytokine, antigen receptor, and Toll-like receptor (TLR) signaling pathways in a range of immune cells could provide a kind of fingerprint of the state of the human immune system. The mass cytometry or CyTOF, platform allows for the parallel application of about 40 labeled antibodies to a single sample, creating the possibility to read out many cell types and signaling pathways in a single small blood sample. We developed such a mass cytometry panel, consisting of 22 antibodies to cell surface lineage markers and 8 antibodies to phospho-specific epitopes of signaling proteins. These antibodies were chosen to discriminate all major white blood cell lineages, to a level of detail that includes subsets such as naïve, central memory, effector memory, and late effector CD4+ and CD8+T cells, naïve, transitional, and switched memory B cells, plasmablasts, myeloid and plasmacytoid dendritic cells, CD16+ and CD16+CD56+ NK cells, CD16+ and classical monocytes etc. 32 such cell subsets are defined in our standard gating scheme. The eight phospho-specific antibodies were chosen to represent major signaling nodes responsive to cytokine, TLR, and antigen receptor signaling. This antibody panel is used with 8 standard stimulation conditions (unstimulated, IFNa, IL-6, IL-7, IL-10, IL-21, LPS, PMA+ ionomycin), although other stimuli can be added. Comparison of healthy controls to subjects with immune deficiencies of unknown etiology may help elucidate the mechanisms of such deficiencies. Phosphorylation of tyrosine, serine, and threonine residues is critical for the control of protein activity involved in various cellular events. An assortment of kinases and phosphatases regulate intracellular protein phosphorylation in many different cell signaling pathways, such as T and B cell signaling, those regulating apoptosis, growth and cell cycle control, plus those involved with cytokine, chemokine, and stress responses. Phosphoflow assays combine phospho-specific antibodies with the power of flow cytometry to enhance phospho protein study. In our assay, peripheral blood mononuclear cells are stimulated by cytokines, fixed, surface-stained with a cocktail of antibodies labeled with MAXPAR (Brand Name) metal-chelating polymers and permeabilized with methanol. They are then stained with intracellular phospho-specific antibodies. We use a CyTOF™ mass cytometer to acquire the ICP-MS data. The current mass window selected is approximately AW 103-203, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type. Median values are reported to quantitate the level of phosphorylation of each protein in response to stimulation. Comparing the level of phosphorylation between samples can offer insight to the status of the immune system. Whole blood stimulation is the closest to the in vivo condition and it allows for assessment of granulocyte population as well as lymphocytes and monocytes.

マルチPK抗体とPhos-tagを利用した細胞内リン酸化動態の新規解析法

マルチPK抗体とPhos-tagを利用した細胞内リン酸化動態の新規解析法

Measurement of intracellular ribavirin mono-, di- and triphosphate using solid phase extraction and LC-MS/MS quantification.

Ribavirin (RBV) is a nucleoside analog used to treat a variety of DNA and RNA viruses. RBV undergoes intracellular phosphorylation to a mono- (MP), di- (DP), and triphosphate (TP). The phosphorylated forms have been associated with the mechanisms of antiviral effect observed in vitro, but the intracellular pharmacology of the drug has not been well characterized in vivo. A highly sensitive LC-MS/MS method was developed and validated for the determination of intracellular RBV MP, DP, and TP in multiple cell matrix types. For this method, the individual MP, DP, and TP fractions were isolated from lysed intracellular matrix using strong anion exchange solid phase extraction, dephosphorylated to parent RBV, desalted and concentrated and quantified using LC-MS/MS. The method utilized a stable labeled internal standard (RBV-(13)C5) which facilitated accuracy (% deviation within ±15%) and precision (coefficient of variation of ≤15%). The quantifiable linear range for the assay was 0.50 to 200 pmol/sample. The method was applied to the measurement of RBV MP, DP, and TP in human peripheral blood mononuclear cells (PBMC), red blood cells (RBC), and dried blood spot (DBS) samples obtained from patients taking RBV for the treatment of chronic Hepatitis C virus infection.

A Novel Chemical Approach to Expand Platelets Using Immortalized Megakaryocyte Progenitor Cells Derived from Human Induced Pluripotent Stem Cells

Blood platelets can be obtained only by blood donation and reveal short-shelf life by the reason that they must be maintained with plasma at 20-24 degrees with shaking. These factors lead to shortage of donor platelets for clinical use. To overcome this issue, we developed a clinically applicable strategy for the derivation of functional platelets from human pluripotent stem cells (PSCs).We previously reported in vitro culture methods for producing functional platelets from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) (Takayama et al. Blood 2008, J Exp Med 2010). We also have established immortalized megakaryocyte progenitor cell lines (imMKCLs) with long-term expansion capability from hiPSC-derived hematopoietic progenitors with three defined factors, c-MYC, BMI1 and, BCL-XL (Nakamura et al. Cell Stem Cell, 2014). Although imMKCLs can be promising source of functional platelets for transfusion, further inventive efforts are needed to expand imMKCLs more efficiently towards clinical application.Thrombopoietin (TPO) is a cytokine initially identified as the primary regulator of megakaryocyte differentiation and platelet production. TPO is also an essential supplement for expanding platelets from imMKCLs. Recently, several nonpeptidyl small-molecule compounds have been developed to activate the TPO receptor, c-MPL and promote platelet production such as SB-497115 (Eltrombopag), an orally available drug for thrombocytopenia. Chemically synthesized c-MPL agonists reveal the advantage in terms of biological safety, low-immunogenicity or, low-cost manufacturing as compared to peptide-based ligands for platelet production. To obtain a c-MPL agonist that expands imMKCLs more efficiently and cost effectively than recombinant human TPO (rhTPO), we firstly screened small-molecule c-MPL agonists by evaluating its effects on platelet production from hiPSCs. Consequently, we identified “MK-001”, as the most potent compound that increases platelet productivity, as evidenced by the effects of MK-001 on the proliferation, differentiation, cell signaling, and platelet production from imMKCLs. We also studied the functionality of imMKCL-derived platelets.imMKCLs were cultured for 15 days with passage every 3 or 4 days with rhTPO in the presence of either rhTPO, Eltrombopag or MK-001 employing the same method as previously described (Nakamura et al. Cell Stem Cell, 2014). Total cell number was measured by Trypan Blue staining and automated cell counter. On day11, the number of total cells cultured with 200ng/mL of MK-001 was increased >1.5-fold compared with that of 50ng/mL of rhTPO (p<0.01). Subsequently, c-MYC, BMI1, and BCL-XL genes were turned off for inducing platelet yield. After another 4-days of culture, matured megakaryocytes and platelets were collected and analyzed by flow cytometer. Total number of CD41+CD42b+ platelets with MK-001 was increased >2-fold compared with that of rhTPO (p<0.001), whereas 1000ng/mL of Eltrombopag had little effect on platelet production. imMKCLs cultured with MK-001 contained a lot of large multinucleated cells and showed high levels of DNA content as well as those cultured with rhTPO. Intracellular phosphorylation analysis (BD Phosflow assay) revealed that MK-001 activated phosphorylation of components of three major TPO signaling pathways, JAK/STAT, MAPK, and PI3K/AKT within imMKCLs. PAC-1 bindings to platelets produced with MK-001, specific to an activated aIIbb3 integrin by adenosine 5-diphosphate (ADP), thrombin, or phorbol 12-myristate 13-acetate (PMA), were the same levels as those produced with rhTPO. These results indicated MK-001 promotes the production of functional platelets from imMKCLs more efficiently than rhTPO or Eltrombopag.In conclusion, the c-MPL agonist MK-001 could be applicable as an indispensable tool for expansion of platelets from hiPSCs with a combination of imMKCL system. DisclosuresNakauchi:Nissan Chemical Industries: Research Funding. Eto:Nissan Chemical Industries: Research Funding.

Mdivi-1 Protects Against Ischemic Brain Injury via Elevating Extracellular Adenosine in a cAMP/CREB-CD39-Dependent Manner.

This study aimed to examine whether the neuroprotective effects of Mdivi-1 are attributable to extracellular ATP and adenosine. Mdivi-1 was administered prior to or post middle cerebral artery occlusion (MCAO). The extracellular adenosine was measured by in vivo microdialysis and high-pressure liquid chromatography (HPLC) in MCAO mouse model. Western blot was done to determine the influence of Mdivi-1 on the expression of CD39 and CREB phosphorylation both in vivo and in the cultured astrocytes. Intracellular cAMP and protein kinase A (PKA) activity were detected in primary astrocytes. Results showed that Mdivi-1 significantly reduced infarct volume and neurological scores when administered either prior to or post MCAO. Interestingly, pretreatment with Mdivi-1 resulted in marked increase of extracellular adenosine and concomitant decrease in ATP. The expression of CD39, but not CD73, was upregulated by Mdivi-1, which was associated with the elevated phosphorylated cAMP response element-binding protein (CREB), a transcription factor potentially regulating CD39 expression. In primary astrocytes, Mdivi-1 treatment induced increases in intracellular cAMP, PKA activity and CREB phosphorylation, and PKA-specific inhibitor completely reversed Mdivi-1-induced CD39 expression. Our results demonstrate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by cAMP/PKA/CREB cascade. Figure Potential mechanisms by which Mdivi-1 mediates the neuroprotection on cerebral ischemic stroke. Results from the present study indicate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by the cAMP/PKA/CREB cascades.

Interaction of interferon beta-1b and CXCR4 on T cells in multiple sclerosis

presence of UO126. EPE peptide led to inhibition of proliferation in Th17 cells only at higher concentrations. The production of IL-17 was reduced with UO126, yet not affected by the EPE peptide. Interestingly, granulocyte–macrophage colony-stimulating factor (GM-CSF), a cytokine influencing the encephalitogenicity of Th17 cells, was diminished after treatment with EPE or UO126. We observed an increase in intracellular ERK phosphorylation in CD4 T cells in the course of Th17 differentiation. We are currently testing whether the inhibitory peptide EPE affects EAE disease outcome in a T cell adoptive transfer model. Taken together, our findings indicate that ERK translocation promotes encephalitogenicity in T cells by facilitating GM-CSF production.

CCN1 secreted by tonsil-derived mesenchymal stem cells promotes endothelial cell angiogenesis via integrin αv β3 and AMPK.

CCN1 is highly expressed in cancer cells and has been identified in the secretome of bone marrow-derived mesenchymal stem cells (BM-MSC). Although secreted CCN1 is known to promote angiogenesis, its underlying mechanism remains unclear. Here, we examined whether our recently-established tonsil-derived MSC (T-MSC) secrete CCN1 and, if any, how CCN1 promotes the angiogenesis of human umbilical vein endothelial cells (HUVEC). Compared with untreated control T-MSC, a higher level of CCN1 was secreted by T-MSC treated with activin A and sonic hedgehog, drugs known to induce endodermal differentiation. Expectedly, conditioned medium collected from differentiated T-MSC (DCM) significantly increased HUVEC migration and tube formation compared with that from control T-MSC (CCM), and these stimulatory effects were reversed by neutralization with anti-CCN1 antibody. Treatment with recombinant human CCN1 (rh-CCN1) alone also mimicked the stimulatory effects of DCM. Furthermore, treatment with either DCM or rh-CCN1 increased the phosphorylation of AMP kinase (AMPK), and ectopic expression of siRNA of the AMPK gene inhibited all observed effects of both DCM and rh-CCN1. However, no alteration of intracellular ATP levels or phosphorylation of LKB1, a well-known upstream factor of AMPK activation, was observed under our conditions. Finally, the neutralization of integrin α(v) β(3) with anti-integrin α(v) β(3) antibody almost completely reversed the effects of CCN1 on AMPK phosphorylation, and EC migration and tube formation. Taken together, we demonstrated that T-MSC increase the secretion of CCN1 in response to endodermal differentiation and that integrin α(v) β(3) and AMPK mediate CCN1-induced EC migration and tube formation independent of intracellular ATP levels alteration.

Nucleoside mono- and diphosphate prodrugs of 2',3'-dideoxyuridine and 2',3'-dideoxy-2',3'-didehydrouridine.

Despite their close structural similarity to nucleoside analogues such as the anti-HIV drugs AZT and d4T, 2',3'-dideoxyuridine (ddU) and 2',3'-dideoxy-2',3'-didehydrouridine (d4U) are entirely inactive against HIV in their nucleoside form. However, it has been shown that the corresponding triphosphates of these two nucleosides can effectively block HIV reverse transcriptase. Herein we report on two types of nucleotide prodrugs (cycloSal and DiPPro nucleotides) of ddU and d4U to investigate their ability to overcome insufficient intracellular phosphorylation, which may be the reason behind their low anti-HIV activity. The release of the corresponding mono- and diphosphates from these compounds was demonstrated by hydrolysis studies in phosphate buffer (pH 7.3) and human CD4 (+) T-lymphocyte CEM cell extracts. Surprisingly, however, these compounds showed low or no anti-HIV activity in tests with human CD4 (+) T-lymphocyte CEM cells. Studies of the conversion of ddUDP and d4UDP into their triphosphate metabolites by nucleoside diphosphate kinase (NDPK) showed nearly no conversion of either diphosphate, which may be the reason for low intracellular triphosphate levels that result in low antiviral activity.

Interstitial diffusion and the relationship between compartment modelling and multi-scale spatial-temporal modelling of 18F-FLT tumour uptake dynamics

Tumour cell proliferation can be imaged via positron emission tomography of the radiotracer 3ʹ-deoxy-3ʹ-18F-fluorothymidine (18F-FLT). Conceptually, the number of proliferating cells might be expected to correlate more closely with the kinetics of 18F-FLT uptake than with uptake at a fixed time. Radiotracer uptake kinetics are standardly visualized using parametric maps of compartment model fits to time-activity-curves (TACs) of individual voxels. However the relationship between the underlying spatiotemporal accumulation of FLT and the kinetics described by compartment models has not yet been explored.In this work tumour tracer uptake is simulated using a mechanistic spatial-temporal model based on a convection-diffusion-reaction equation solved via the finite difference method. The model describes a chain of processes: the flow of FLT between the spatially heterogeneous tumour vasculature and interstitium; diffusion and convection of FLT within the interstitium; transport of FLT into cells; and intracellular phosphorylation. Using values of model parameters estimated from the biological literature, simulated FLT TACs are generated with shapes and magnitudes similar to those seen clinically. Results show that the kinetics of the spatial-temporal model can be recovered accurately by fitting a 3-tissue compartment model to FLT TACs simulated for those tumours or tumour sub-volumes that can be viewed as approximately closed, for which tracer diffusion throughout the interstitium makes only a small fractional change to the quantity of FLT they contain. For a single PET voxel of width 2.5–5 mm we show that this condition is roughly equivalent to requiring that the relative difference in tracer uptake between the voxel and its neighbours is much less than one.

Dynamic quantification of intracellular calcium and protein tyrosine phosphorylation in cryopreserved boar spermatozoa during short-time incubation with oviductal fluid

Freshly ejaculated boar spermatozoa require several hours of exposure to capacitating conditions to undergo capacitation. We hypothesized that cryopreserved boar spermatozoa might elicit a capacitation response after a relatively shorter time of exposure to capacitating conditions. Washed, frozen-thawed boar spermatozoa were incubated separately with pre-ovulatory isthmic oviductal fluid (EODF), post-ovulatory ODF (MODF), capacitation medium (CM), and noncapacitating medium (NCM) for 60 minutes. Aliquots of spermatozoa were taken at 0, 5, 15, 30, and 60 minutes during incubation and sperm kinematics, intracellular calcium [Ca2+]i content, and protein tyrosine phosphorylation (PTP) were studied. The proportion of motile spermatozoa increased significantly after 5 minutes of incubation with EODF. A similar increase was not observed in the other groups. During the initial 5 minutes of incubation, the proportion of spermatozoa with high [Ca2+]i decreased significantly in all four groups. The proportion of tyrosine phosphorylated spermatozoa increased from 6.49 ± 1.93% to 15.42 ± 3.58% and 18.41 ± 1.57% in EODF and MODF groups, respectively, at 5 minutes of incubation. Neither CM nor NCM elicited any immediate effect on PTP in spermatozoa. There was a positive and significant correlation between [Ca2+]i and sperm motility (P = 0.009). It may be concluded that frozen-thawed boar spermatozoa undergo capacitation-associated changes after a relatively short exposure to EODF, and there are some subpopulations of spermatozoa that undergo PTP despite possessing low [Ca2+]i.

Effect of the PTEN gene on adhesion, invasion and metastasis of osteosarcoma cells.

The phosphatase and tensin homolog (PTEN) gene, an important tumor-suppressor gene, has been demonstrated to have the potential for inhibiting proliferation, migration and invasion in various types of cancer cells. The aim of the present study was to investigate the effect of PTEN expression on osteosarcoma (OS) cells. The wild-type PTEN plasmid was transfected into OS U2-OS cells. The effects of PTEN on the adhesion, migration and invasion of U2-OS cells were evaluated by cell adhesion analysis, invitro scratch and Transwell assays, respectively. The levels of MMP-2 and MMP-9, and focal adhesion kinase (FAK) protein regulated by PTEN were detected via western blot analysis. Meanwhile, the level of intracellular FAK phosphorylation was observed. The results from the present study showed that overexpression of PTEN transcription and protein were observed in U2-OS cells following PTEN transfection. Furthermore, the migration, invasion and adhesion capabilities of the cells with PTEN transfection were significantly decreased compared to these capacities in the cells without PTEN. Meanwhile, it was shown that there was downregulation of MMP-9, FAK and p-FAK concomitant with the elevation of the intracellular PTEN level. It is therefore evident that the upregulation of PTEN may attenuate the adhesion, migration and invasion capabilities of OS cells. The mechanisms of the effects of PTEN on OS cells may be correlated with a reduction in the related genes by PTEN regulation.

Differential roles of ATF-2 in survival and DNA repair contributing to radioresistance induced by autocrine soluble factors in A549 lung cancer cells

Radioresistance is one of the obstacles to the effective radiotherapy for non-small cell lung cancer. Soluble factors in the tumour microenvironment are often implicated in radioresistance but the underpinning mechanism(s) remain largely elusive. We herein studied the wholesome effect of autocrine cytokines and growth factors in the form of self-conditioned medium (CM) on the radiosensitivity of A549 cells. A549 cells grown in CM exhibited radioresistance which was associated with increased survival and DNA repair. CM induced pro-survival pathways through increased intracellular cAMP and phosphorylation of JNK and p38. Downstream to JNK/p38 signalling, ATF-2 phosphorylated at Thr69/71 was accompanied with its increased transcriptional activity in CM treated cells. Pre-treatment with cAMP inhibitor and silencing of ATF-2 abrogated the CM-induced survival. Interestingly, in cells treated with CM followed by radiation, ATF-2 was found to be switched over from transcription factor to DNA damage response protein. In CM treated cells, after γ-radiation p-ATF-2Thr69/71 and subsequently the transcriptional activity of ATF-2 were declined with simultaneous rise in p-ATF-2Ser490/498. Immunoprecipitation/immunoblotting and inhibitor studies showed that phosphorylation of ATF-2 at Ser490/498 was mediated by ATM. Moreover, p-ATF-2Ser490/498 was found to be co-localised with γ-H2AX in DNA repair foci in CM-treated cells. The DNA repair activity of ATF-2 was assisted with higher activity MRN complex in cells grown in CM. Our study revealed that, autocrine soluble factors regulate dual but differential role of ATF-2 as a transcription factor or DNA repair protein, which collectively culminate in radioresistance of A549 cells.

Semi-interpenetrating network (sIPN) co-electrospun gelatin/insulin fiber formulation for transbuccal insulin delivery.

This work was aimed at developing a semi-interpenetrating network (sIPN) co-electrospun gelatin/insulin fiber scaffold (GIF) formulation for transbuccal insulin delivery. Gelatin was electrospun into fibers and converted into an sIPN following eosin Y-initiated polymerization of polyethylene glycol diacrylate (PEG-DA). The cytocompatibility, degradation rate and mechanical properties were examined in the resulting sIPNs with various ratios of PEG-DA to eosin Y to find a suitable formulation for transbuccal drug delivery. Insulin was co-electrospun with gelatin into fibers and converted into an sIPN-GIF using this suitable formulation. The in vitro release kinetics of insulin was evaluated using ELISA. The bioactivity of released insulin was analyzed in 3T3-L1 preadipocytes using Western blotting and Oil Red O staining. The transbuccal permeability of released insulin was determined using an in vitro porcine oral mucosa model. The sIPN-GF formulation of GF cross-linked by PEG-DA (1% w/v) with eosin Y (5% v/v) possessed no cytotoxic effect, a moderate degradation rate with degradation half-life of 49min, and a significant enhancement in mechanical properties. This formulation was used to fabricate sIPN-GIF. Insulin release was extended up to 4h by sIPN-GIF. The released insulin successfully triggered intracellular AKT phosphorylation and induced adipocyte differentiation in 3T3-L1 preadipocytes. The transbuccal permeability of released insulin was determined on the order of 10(-7)cm/s. Insulin can be fabricated into an sIPN-GIF formulation following co-electrospinning and cross-linking without losing bioactivity. It proved the potential of this new formulation for transbuccal insulin delivery.

Chronopharmacology and mechanism of antitumor effect of erlotinib in Lewis tumor-bearing mice.

The epidermal growth factor receptor (EGFR), a ubiquitously expressed receptor tyrosine kinase, is recognized as a key mediator of tumorigenesis in many human epithelial tumors. Erlotinib is tyrosine kinase inhibitor approved by FDA for use in oncology. It inhibits the intracellular phosphorylation of tyrosine kinase associated with the EGFR to restrain the development of the tumor. To investigate the antitumor effect of erlotinib at different dosing times and the underlying molecular mechanism via the PI3K/AKT pathway, we established a mouse model of Lewis lung cancer xenografts. The tumor-bearing mice were housed four or five per cage under standardized light-dark cycle conditions (light on at 7:00 AM, 500 Lux, off at 7:00 PM, 0 Lux) with food and water provided ad libitum. The mice were observed for quality of life, their body weight and tumor volume measured, and the tumor growth curves drawn. After being bled, the mice were sacrificed by cervical dislocation. The tumor masses were removed at different time points and weighed. The mRNA expression of EGFR, AKT, Cyclin D1 and CDK-4 were assayed by quantitative real-time PCR (qRT-PCR). Protein expression levels of AKT, P-AKT and Cyclin D1 were determined by Western blot analysis. The results suggest that erlotinib has a significant antitumor effect on xenografts of non-small cell lung cancer in mice, and its efficacy and toxicity is dependent on the time of day of administration. Its molecular mechanism of action might be related to the EGFR-AKT-Cyclin D1-CDK-4 pathway which plays a crucial role in the development of pathology. Therefore, our findings suggest that the time of day of administration of Erlotinib may be a clinically important variable.