Synthesis of Saxitoxin Biosynthetic Intermediates: Reveal the Mechanism for Formation of its Tricyclic Skeleton in Biosynthesis.

The synthesis and biosynthesis of the complex saxitoxin (STX) structure have garnered significant interest. Previously, we hypothesized that the tricyclic skeleton of STX originates from the monocyclic precursor 11-hydroxy-IntC'2 during biosynthesis, although direct evidence has been lacking. In this study, we identified conditions to synthesize a proposed tricyclic biosynthetic intermediate, 12,12-dideoxy-decarbamoyloxySTX (dd-doSTX), along with its 6-epimer (6-epi-dd-doSTX) and a bicyclic compound, in a single step from di-Boc protected 11-hydroxy-IntC'2. The reaction mechanism involves successive aza-Michael addition of a guanidino amine to the conjugated olefin. Notably, both dd-doSTX and 6-epi-dd-doSTX were detected in a toxin-producing cyanobacterium, suggesting that the biosynthetic enzymes may generate these compounds via similar mechanisms.

Novel and highly reliable leak check tests for drop- and external pressure-type cell-free and concentrated ascites reinfusion therapy.

INTRODUCTION: For safe management of cell-free and concentrated ascites reinfusion therapy (CART), a highly reliable leak test for detecting ascites filter damage is essential. However, such a test has not been established for drop-type CART. METHODS: We devised two novel leak tests for drop- and external pressure-type CART, manual or pump pressurization methods, using high-pressure loading and pressure monitoring, and investigated their reliability. RESULTS: Both methods could easily load and maintain sufficiently high pressure (>400 Torr) on the hollow fibers for 2 min. No result deviation was noted between different operators. The pressure drops in both methods were identical and significantly lower than those in the leak test using a special CART machine, the e-CART. CONCLUSION: The reliability of our revised leak test is equivalent to that of the automatic leak test of e-CART. These highly reliable leak tests may contribute to safety in patients undergoing drop- and external pressure-type CART.

Disease-related protein co-expression networks are associated with the prognosis of resectable node-positive pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a multifactorial disease, the molecular profile of which remains unclear. This study aimed at unveiling the disease-related protein networks associated with different outcomes of resectable, node-positive PDAC cases. We assessed laser-microdissected cancerous cells from PDAC tissues of a poor outcome group (POG; n = 4) and a better outcome group (BOG; n = 4). Noncancerous pancreatic duct tissues (n = 5) were used as the reference. We identified four representative network modules by applying a weighted network correlation analysis to the obtained quantitative PDAC proteome datasets. Two network modules that were significant for POG were associated with the heat shock response to hypoxia-related stress; in the latter, a large involvement of the non-canonical Hedgehog pathway (regulated by GLI1), the internal ribosome entry site-mediated cap-independent translation, the inositol requiring enzyme 1-alpha (IRE1α)/X-box binding protein 1 pathway of the unfolding protein response (UPR), and the aerobic glycolysis was observed. By contrast, the BOG characteristic module was involved in the inactivation of the UPR pathway via the synoviolin 1-dependent proteasomal degradation of IRE1α, the activation of SOX2, and the loss of PALB2 (partner and localizer of BRCA2) function, all potentially suppressing malignant tumor development. Our findings might facilitate future therapeutic strategies for PDAC.

Visualized procollagen Iα1 demonstrates the intracellular processing of propeptides.

The processing of type I procollagen is essential for fibril formation; however, the steps involved remain controversial. We constructed a live cell imaging system by inserting fluorescent proteins into type I pre-procollagen α1. Based on live imaging and immunostaining, the C-propeptide is intracellularly cleaved at the perinuclear region, including the endoplasmic reticulum, and subsequently accumulates at the upside of the cell. The N-propeptide is also intracellularly cleaved, but is transported with the repeating structure domain of collagen into the extracellular region. This system makes it possible to detect relative increases and decreases in collagen secretion in a high-throughput manner by assaying fluorescence in the culture medium, and revealed that the rate-limiting step for collagen secretion occurs after the synthesis of procollagen. In the present study, we identified a defect in procollagen processing in activated hepatic stellate cells, which secrete aberrant collagen fibrils. The results obtained demonstrated the intracellular processing of type I procollagen, and revealed a link between dysfunctional processing and diseases such as hepatic fibrosis.

Comparative proteome analysis of the ligamentum flavum of patients with lumbar spinal canal stenosis.

Background: Thickening of the ligamentum flavum is considered to be the main factor for lumbar spinal canal stenosis (LSCS). Although some mechanisms have been speculated in the thickening of the ligamentum flavum, there are only a few comprehensive approaches to investigate its pathology. The objective of this study was to investigate the pathology of thickened ligamentum flavum in patients with LSCS based on protein expression levels using shotgun proteome analysis. Methods: Ligamentum flavum samples were collected from four patients with LSCS (LSCS group) and four patients with lumbar disc herniation (LDH) as controls (LDH group). Protein mixtures were digested and analyzed by liquid chromatography/mass spectrometry analysis. To compare protein expression levels between the LSCS and LDH groups, the mean Mascot score was compared. Biological processes were assessed using Gene Ontology analysis. Results: A total of 1151 proteins were identified in some samples of ligamentum flavum. Among these, 145 proteins were detected only in the LSCS group, 315 in the LDH group, and 691 in both groups. The demonstrated biological processes occurring in the LSCS group included: extracellular matrix organization, regulation of peptidase activity, extracellular matrix disassembly, and negative regulation of cell growth. Proteins related to fibrosis, chondrometaplasia, and amyloid deposition were found highly expressed in the LSCS group compared with those in the LDH group. Conclusions: Tissue repair via fibrosis, chondrometaplasia, and amyloid deposits may be important pathologies that occur in the thickened ligamentum flavum of patients with LSCS.

Non-metal sensory electrode design and protocol of DNA-nucleobases in living cells exposed to oxidative stresses.

Sensory protocols for evaluation of DNA distortion due to exposure to various harmful chemicals and environments in living cells are needed for research and clinical investigations. Here, a design of non-metal sensory (NMS) electrode was built by using boron-doped carbon spherules for detection of DNA nucleobases, namely, guanine (Gu), adenine (Ad), and thymine (Th) in living cells. The key-electrode based nanoscale NMS structures lead to voids with a facile diffusion, and strong binding events of the DNA nucleobases. Furthermore, the NMS geometric structures would significantly create electrode surfaces with numerous centrally active sites, curvature topographies, and anisotropic spherules. The NMS shows potential as sensitive protocol for DNA-nucleobases in living cells exposed to oxidative stresses. In one-step signaling assay, NMS shows high signaling transduction of Gu-, Ad-, and Th-DNA nucleobases targets with ultra-sensitive and low detection limits of 3.0, 0.36, and 0.34 nM, respectively, and a wide linear range of up to 1 µM. The NMS design and protocol show evidence of the role of surface construction features and B-atoms incorporated into the graphitic carbon network for creating abundant active sites with facile electron diffusion and heavily target loads along with within-/out-plane circular spheres. Indeed NMS, with spherule-rich interstitial surfaces can be used for sensitive and selective evaluation of damaged-DNA to various dysfunctional metabolism in the human body.

Three-Dimensional Circular Surface Curvature of a Spherule-Based Electrode for Selective Signaling and Dynamic Mobility of Norepinephrine in Living Cells.

A highly sensitive protocol for signaling norepinephrine (NEP) in human fluids and neuronal cell line models should be established for clinical investigation of some neuronal diseases. A metal-free electrode catalyst was designed based on a sulfur-doped carbon spheroidal surface (S-CSN) and employed as a transducing element for selective signaling of NEP in biological samples. The designed electrode of S-CSN features a spherical construct and curvature surface to form a spheroidal nanolayer with an average layer size of <2 nm. S-CSN shows surface topography of a circular surface curvature with a rugged surface texture, ridge ends, and free open spaces between interlayers. The rich-space diversity surfaces offer highly active surface with facile molecular/electron diffusion, multi-diffusive centers, and high target loading along with in-/out-of-plane circular spheres of the S-CSN surface. The active doping of S atoms onto the carbon-based electrode creates an active transducing element with many active sites, strong binding to targeted molecules, facile diffusion of charges/molecules, long-term durability, and dense reactive exposure sites for signaling NEP at ultratrace levels. S-CSN could be a sensitive and selective nanosensor for signaling NEP and establishing a sensing protocol with high stability and reproducibility. The sensory protocol based on S-CSN exhibits high sensitivity and selectivity with a low detection limit of 0.001 µM and a wide linear range of 0.01-0.8 µM. The in vitro sensory protocol for NEP secreted from living cells (neuronal cell line model) under stimulated agents possesses high sensitivity, low cytotoxicity, and high biocompatibility. These results confirm the successful establishment of NEP sensor in human blood samples and neuronal cells for clinical investigation.

Identification of a Novel Saxitoxin Analogue, 12ß-Deoxygonyautoxin 3, in the Cyanobacterium, Anabaena circinalis (TA04).

Saxitoxin (STX) and its analogues, the potent voltage-gated sodium channel blockers, are biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates in the extract of the cyanobacterium, Anabaena circinalis (TA04), that are primarily produced during the early and middle stages in the biosynthetic pathway to produce STX. These findings allowed us to propose a putative biosynthetic pathway responsible for STX production based on the structures of these intermediates. In the present study, we identified 12ß-deoxygonyautoxin 3 (12ß-deoxyGTX3), a novel STX analogue produced by A. circinalis (TA04), by comparing the retention time and MS/MS fragmentation pattern with those of synthetic standards using LC-MS. The presence of this compound in A. circinalis (TA04) is consistent with stereoselective enzymatic oxidations at C11 and C12, and 11-O-sulfation, during the late stage of STX biosynthesis, as proposed in previous studies.

PDZK1-interacting protein 1 (PDZK1IP1) traps Smad4 protein and suppresses transforming growth factor-ß (TGF-ß) signaling.

Transforming growth factor (TGF)-ß signaling in humans is stringently regulated to prevent excessive TGF-ß signaling. In tumors, TGF-ß signaling can both negatively and positively regulate tumorigenesis dependent on tumor type, but the reason for these opposite effects is unclear. TGF-ß signaling is mainly mediated via the Smad-dependent pathway, and herein we found that PDZK1-interacting protein 1 (PDZK1IP1) interacts with Smad4. PDZK1IP1 inhibited both the TGF-ß and the bone morphogenetic protein (BMP) pathways without affecting receptor-regulated Smad (R-Smad) phosphorylation. Rather than targeting R-Smad phosphorylation, PDZK1IP1 could interfere with TGF-ß- and BMP-induced R-Smad/Smad4 complex formation. Of note, PDZK1IP1 retained Smad4 in the cytoplasm of TGF-ß-stimulated cells. To pinpoint PDZK1IP1's functional domain, we created several PDZK1IP1 variants and found that its middle region, from Phe40 to Ala49, plays a key role in its Smad4-regulating activity. PDZK1IP1 knockdown enhanced the expression of the TGF-ß target genes Smad7 and prostate transmembrane protein androgen-induced (TMEPAI) upon TGF-ß stimulation. In contrast, PDZK1IP1 overexpression suppressed TGF-ß-induced reporter activities, cell migration, and cell growth inhibition. In a xenograft tumor model in which TGF-ß was previously shown to elicit tumor-promoting effects, PDZK1IP1 gain of function decreased tumor size and increased survival rates. Taken together, these findings indicate that PDZK1IP1 interacts with Smad4 and thereby suppresses the TGF-ß signaling pathway.

Slender transradial iliac artery stenting using a 4.5 French guiding sheath.

We previously reported safety and usefulness of transradial iliac artery stenting using 6 Fr guiding sheath. However, radial artery occlusion was a major limitation of this procedure. We analyzed the safety and utility of slender transradial iliac artery stenting using a 4.5 Fr guiding sheath to prevent radial artery occlusion. We performed transradial iliac artery stenting in left radial artery, using a 4.5 Fr sheath incorporating a shaft length of 110 cm, for 34 lesions in 29 patients. Transradial intervention was attempted at the discretion of the operator. Clinical data were analyzed retrospectively. Cases with scheduled multiple sheath insertions for a bidirectional approach were excluded. Twenty-three (79.3%) patients were male. Diabetes mellitus, hypertension, dyslipidemia, and smoking habit were present in 11 (37.9%), 27 (93.1%), 19 (65.5%), and 24 (82.8%) patients, respectively. Nine lesions (26.5%) were diagnosed as chronic total occlusion. All lesions were successfully treated using a total of 40 stents incorporating a 4.5 Fr radial access system. Ankle-brachial index (ABI) significantly improved from 0.68 ± 0.15 to 0.99 ± 0.17 (p < 0.0001) after the procedure. No patients had procedural or access site-related complications such as hematoma, major bleeding, blood transfusion, stroke, cholesterol embolism, aortic dissection, or arterial perforation. Radial artery occlusion was absent in all cases. ABI value was well maintained at 0.98 ± 0.13 at 1 year, and no target lesion revascularizations were reported. Slender transradial iliac artery stenting using a 4.5 Fr guiding sheath is safe, feasible, and less invasive, and shows no incidence of radial artery occlusion, in carefully selected patient populations.

A Glutathione-Responsive Short Sequence of Metal-Organic Complex Array.

A short metal-organic complex array (MOCA) containing a sequence of RPtRRu (1Cl ) was found to exhibit unique responses to a major biothiol, glutathione (GSH). Upon binding of GSH to 1Cl , the resultant 1:1 complex (1GS ) formed nanofibrous assemblies that suggested supramolecular polymerization through the double-salt-bridge structure formation. The binding behavior of this MOCA sequence to calf thymus DNA was also dependent on GSH; a larger conformational change of DNA was observed upon binding with 1GS , relative to that with 1Cl .

Retained Myogenic Potency of Human Satellite Cells from Torn Rotator Cuff Muscles Despite Fatty Infiltration.

Rotator cuff tears (RCTs) are a common shoulder problem in the elderly that can lead to both muscle atrophy and fatty infiltration due to less physical load. Satellite cells, quiescent cells under the basal lamina of skeletal muscle fibers, play a major role in muscle regeneration. However, the myogenic potency of human satellite cells in muscles with fatty infiltration is unclear due to the difficulty in isolating from small samples, and the mechanism of the progression of fatty infiltration has not been elucidated. The purpose of this study was to analyze the population of myogenic and adipogenic cells in disused supraspinatus (SSP) and intact subscapularis (SSC) muscles of the RCTs from the same patients using fluorescence-activated cell sorting. The microstructure of the muscle with fatty infiltration was observed as a whole mount condition under multi-photon microscopy. Myogenic differentiation potential and gene expression were evaluated in satellite cells. The results showed that the SSP muscle with greater fatty infiltration surrounded by collagen fibers compared with the SSC muscle under multi-photon microscopy. A positive correlation was observed between the ratio of muscle volume to fat volume and the ratio of myogenic precursor to adipogenic precursor. Although no difference was observed in the myogenic potential between the two groups in cell culture, satellite cells in the disused SSP muscle showed higher intrinsic myogenic gene expression than those in the intact SSC muscle. Our results indicate that satellite cells from the disused SSP retain sufficient potential of muscle growth despite the fatty infiltration.

Technical Performance and Clinical Effectiveness of Drop Type With Adjustable Concentrator-Cell Free and Concentrated Ascites Reinfusion Therapy.

Cell-free and concentrated ascites reinfusion therapy (CART) is a very useful treatment method for refractory ascites but is difficult for many hospitals to employ due to its need for specialized equipment. We have therefore developed drop-type with adjustable concentrator CART (DC-CART) that uses a drop-type filtration mechanism and requires only a simple pump and pressure monitor for its concentration process. Easy adjustment of ascites concentration is possible through a recirculation loop, and filter membrane washing is aided by DC-CART's external pressure-type filtration to enable the processing of any quality or quantity of ascites. Moreover, the absence of a roller pump before filtration avoids inflammatory substance release from compressed cells. A total of 268 sessions of DC-CART using ascites from 98 patients were performed with good clinical results at our hospitals between January 2012 and June 2016. This report presents the detailed methods of DC-CART and summarizes its clinical effectiveness using patient ascites and blood data obtained from 59 sessions between March 2015 and February 2016. This novel technique successfully processed refractory ascites in numerous diseases with no serious adverse events. DC-CART could concentrate large amounts of ascites (from median weight: 4900 g [max: 20 200 g] to median weight: 695 g; median concentration ratio: 7.4), and a high amount of protein (median weight: 73 g [max: 294 g]) could be reinfused. Serum albumin levels were significantly increased (P = 0.010) and kidney function and systemic hemodynamics were well maintained in treated subjects. Additional concentration of ascites and adjustment of ascites volume were easily performed by recirculation (from median weight: 615 g to median weight: 360 g; median concentration ratio: 1.5). Time was needed during DC-CART for filter membrane cleaning, especially for viscous ascites. Overall, DC-CART represents a safe and useful treatment method for various forms of refractory ascites that can be performed at a wide range of health care institutions.

Transradial intervention for chronic total occlusion at the external iliac artery using a bidirectional approach through a single guiding catheter.

A 64-year-old male with intermittent claudication due to long chronic total occlusion of external iliac artery was successfully treated with a bi-directional approach. The retrograde guidewire was inserted into the ipsilateral internal iliac artery to the distal femoral artery through a collateral channel. The procedure was performed with a single guiding catheter through a single puncture of the left radial artery. Avoid puncture of the femoral artery may be less invasive with fewer bleeding complications.

Selective DNA Recognition and Cytotoxicity of Water-Soluble Helical Metallosupramolecular Polymers.

Water-soluble helical Fe(II)-based metallosupramolecular polymers ((P)- and (M)-polyFe) were synthesized by 1:1 complexation of Fe(II) ions and bis(terpyridine)s bearing a (R)- and (S)-BINOL spacer, respectively. The binding affinity to calf thymus DNA (ct-DNA) was investigated by titration measurements. (P)-PolyFe with the same helicity as B-DNA showed 40-fold higher binding activity (Kb = 13.08 × 107 M-1) to ct-DNA than (M)-polyFe. The differences in binding affinity were supported by electrochemical impedance spectroscopy analysis. The charge-transfer resistance (Rct) of (P)-polyFe increased from 2.5 to 3.9 kΩ upon DNA binding, while that of (M)-polyFe was nearly unchanged. These results indicate that ionically strong binding of (P)-polyFe to DNA chains decreased the mobility of ions in the conjugate. Unique rod-like images were obtained by atomic force microscopy measurement of the DNA conjugate with (P)-polyFe, likely because of the rigid binding between DNA chains and the polymer. Differences in polymer chirality lead to significantly different cytotoxicity levels in A549 cells. (P)-PolyFe showed higher binding affinity to B-DNA and much higher cytotoxicity than (M)-polyFe. The helicity in metallosupramolecular polymer chains was important not only for chiral recognition of DNA but also for coordination to a biological target in the cellular environment.

Mass spectrometry-based proteomic analysis of formalin-fixed paraffin-embedded extrahepatic cholangiocarcinoma.

BACKGROUND: Extrahepatic cholangiocarcinoma is very difficult to diagnose at an early stage, and has a poor prognosis. Novel markers for diagnosis and optimal treatment selection are needed. However, there has been very limited data on the proteome profile of extrahepatic cholangiocarcinoma. This study was designed to unravel the proteome profile of this disease and to identify overexpressed proteins using mass spectrometry-based proteomic approaches. METHODS: We analyzed a discovery set of formalin-fixed paraffin-embedded tissues of 14 extrahepatic cholangiocarcinomas using shotgun mass spectrometry, and compared proteome profiles with those of seven controls. Then, selected candidates were verified by quantitative analysis using scheduled selected reaction monitoring-based mass spectrometry. Furthermore, immunohistochemical staining used a validation set of 165 cases. RESULTS: In total, 1,992 proteins were identified and 136 proteins were overexpressed. Verification of 58 selected proteins by quantitative analysis revealed 11 overexpressed proteins. Immunohistochemical validation for 10 proteins showed positive rates of S100P (84%), CEAM5 (75%), MUC5A (62%), OLFM4 (60%), OAT (42%), CAD17 (41%), FABPL (38%), AOFA (30%), K1C20 (25%) and CPSM (22%) in extrahepatic cholangiocarcinomas, which were rarely positive in controls. CONCLUSIONS: We identified 10 proteins associated with extrahepatic cholangiocarcinoma using proteomic approaches. These proteins are potential targets for future diagnostic biomarkers and therapy.

The co-transplantation of bone marrow derived mesenchymal stem cells reduced inflammation in intramuscular islet transplantation.

AIMS/HYPOTHESIS: Although the muscle is one of the preferable transplant sites in islet transplantation, its transplant efficacy is poor. Here we attempted to determine whether an intramuscular co-transplantation of mesenchymal stem cells (MSCs) could improve the outcome. METHODS: We co-cultured murine islets with MSCs and then analyzed the morphological changes, viability, insulin-releasing function (represented by the stimulation index), and gene expression of the islets. We also transplanted 500 islets intramuscularly with or without 5 × 105 MSCs to diabetic mice and measured their blood glucose level, the glucose changes in an intraperitoneal glucose tolerance test, and the plasma IL-6 level. Inflammation, apoptosis, and neovascularization in the transplantation site were evaluated histologically. RESULTS: The destruction of islets tended to be prevented by co-culture with MSCs. The stimulation index was significantly higher in islets co-cultured with MSCs (1.78 ± 0.59 vs. 7.08 ± 2.53; p = 0.0025). In terms of gene expression, Sult1c2, Gstm1, and Rab37 were significantly upregulated in islets co-cultured with MSCs. Although MSCs were effective in the in vitro assays, they were only partially effective in facilitating intramuscular islet transplantation. Co-transplanted MSCs prevented an early inflammatory reaction from the islets (plasma IL-6; p = 0.0002, neutrophil infiltration; p = 0.016 inflammatory area; p = 0.021), but could not promote neovascularization in the muscle, resulting in the failure of many intramuscular transplanted islets to engraft. CONCLUSIONS: In conclusion, co-culturing and co-transplanting MSCs is potentially useful in islet transplantation, especially in terms of anti-inflammation, but further augmentation for an anti-apoptosis effect and neovascularization is necessary.

Chondrogenic and fibrotic process in the ligamentum flavum of patients with lumbar spinal canal stenosis.

STUDY DESIGN: A histological, biological, and immunohisto-chemical study of human lumbar ligamentum flavum. OBJECTIVE: To analyze changes in the hypertrophied ligamentum flavum and clarify their etiology. SUMMARY OF BACKGROUND DATA: Hypertrophy of the ligamentum flavum has been considered a major contributor to the development of lumbar spinal canal stenosis (LSCS). Although previous studies have reported some factors related to ligamentum flavum hypertrophy, its etiology is still unclear. METHODS: Ligamentum flavum samples were collected from 20 patients with LSCS (LSCS group) and 10 patients with lumbar disc herniation (LDH group) as a control. The thickness of the ligamentum flavum was measured histologically. The amounts of elastic fibers and proteoglycans were assessed by Elastica-Masson staining and alcian blue staining, respectively. Gene and protein expressions related to fibrosis, inflammation, and chondrogenesis were analyzed by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. The total genes of the 2 groups were compared by DNA microarray analysis. RESULTS: The ligamentum flavum was significantly thicker in the LSCS group, which had a smaller amount of elastic fibers and a larger amount of proteoglycans. The gene expression related to fibrosis was significantly higher in the LSCS group; however, the immunoreactivities of collagen types I and III were weaker on the dorsal side of the ligamentum flavum in the LSCS group. The gene expression related to chondrogenesis and proteoglycan synthesis was significantly higher in the LSCS group. There was no significant difference in the gene expression related to inflammation between the 2 groups. CONCLUSION: Synthesis of the collagenous fibers and degradation of the elastic and collagenous fibers are both accelerated in the ligamentum flavum of patient with LSCS, which may be the reason for hypertrophy of the tissue. In addition, chondrogenesis and proteoglycan synthesis may have critical roles in the pathogenesis of the ligamentum flavum hypertrophy. LEVEL OF EVIDENCE: 5.

Phosphatase CD45 both positively and negatively regulates T cell receptor phosphorylation in reconstituted membrane protein clusters.

T cell receptor (TCR) phosphorylation requires the kinase Lck and phosphatase CD45. CD45 activates Lck by dephosphorylating an inhibitory tyrosine of Lck to relieve autoinhibition. However, CD45 also dephosphorylates the TCR, and the spatial exclusion of CD45 from TCR clustering in the plasma membrane appears to attenuate this negative effect of CD45. To further investigate the role of CD45 in signal initiation, we reconstituted membrane TCR clusters in vitro on supported lipid bilayers. Fluorescence microscopy of single clusters showed that incorporation of CD45 enhanced phosphorylation of TCR clusters, but only when Lck co-clustered with TCR. We found that clustered Lck autophosphorylated the inhibitory tyrosine and thus could be activated by CD45, whereas diffusive Lck molecules did not. In the TCR-Lck clusters and at low CD45 density, we speculate that the effect of Lck activation may overcome dephosphorylation of TCR, resulting in a net positive regulation. The CD45 density in physiological TCR clusters is also low because of the exclusion of CD45. Thus, we propose that the spatial organization of TCR/Lck/CD45 in T cell membranes is important not only for modulating the negative role of CD45 but also for creating conditions in which CD45 has a positive role in signal initiation.

Comprehensive genetic analysis of early host body reactions to the bioactive and bio-inert porous scaffolds.

To design scaffolds for tissue regeneration, details of the host body reaction to the scaffolds must be studied. Host body reactions have been investigated mainly by immunohistological observations for a long time. Despite of recent dramatic development in genetic analysis technologies, genetically comprehensive changes in host body reactions are hardly studied. There is no information about host body reactions that can predict successful tissue regeneration in the future. In the present study, porous polyethylene scaffolds were coated with bioactive collagen or bio-inert poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) and were implanted subcutaneously and compared the host body reaction to those substrates by normalizing the result using control non-coat polyethylene scaffold. The comprehensive analyses of early host body reactions to the scaffolds were carried out using a DNA microarray assay. Within numerous genes which were expressed differently among these scaffolds, particular genes related to inflammation, wound healing, and angiogenesis were focused upon. Interleukin (IL)-1ß and IL-10 are important cytokines in tissue responses to biomaterials because IL-1ß promotes both inflammation and wound healing and IL-10 suppresses both of them. IL-1ß was up-regulated in the collagen-coated scaffold. Collagen-specifically up-regulated genes contained both M1- and M2-macrophage-related genes. Marked vessel formation in the collagen-coated scaffold was occurred in accordance with the up-regulation of many angiogenesis-inducible factors. The DNA microarray assay provided global information regarding the host body reaction. Interestingly, several up-regulated genes were detected even on the very bio-inert PMB-coated surfaces and those genes include inflammation-suppressive and wound healing-suppressive IL-10, suggesting that not only active tissue response but also the inert response may relates to these genetic regulations.