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Context: Dipeptidyl peptidase 4 (CD26/DPP4) is expressed on blood T cells and also circulates in a soluble form (sCD26/DPP4)., Objective: We aimed to evaluate blood T cell and circulating CD26/DPP4 and its association with metabolic parameters in patients with type 2 diabetes mellitus (T2DM)., Designs: We measured CD26/DPP4 expression (percentage of CD26(+) cells using flow cytometry) on CD4(+) and CD8(+) T cells, serum CD26/DPP4 level and activity, and various metabolic parameters in T2DM patients not on DPP4 inhibitor therapy (n = 148). Nondiabetic subjects (n = 50) were included as a control group., Results: Compared with the healthy controls, CD26/DPP4 expression on CD4(+) T cells and CD8(+) T cells was higher in T2DM patients. Serum CD26/DPP4 levels and enzymatic activities were also higher in patients with T2DM than in the control group only when metformin and/or thiazolidinedione-treated T2DM patients were excluded; metformin and/or thiazolidinedione-treated T2DM patients had lower values compared with other T2DM patients. Various parameters in T2DM patients were related to CD26/DPP4 expression on the T cells (hemoglobin A1c), serum sCD26/DPP4 (hemoglobin A1c and insulin resistance assessed by updated homeostasis model assessment), and serum CD26/DPP4 activity (insulin resistance assessed by updated homeostasis model assessment, γ-glutamyl transferase, and alanine aminotransferase) by multivariate analyses. After active glucose control for 12 weeks in drug-naive T2DM patients (n = 50), CD26/DPP4 expression on blood T cells was significantly decreased., Conclusions: Our results suggest that the CD26/DPP4 level on blood T cells was associated with glucose control status in patients with T2DM.

The gene encoding a β-glucosidase from the archaeon Thermofilum pendens (Tpbgl) was cloned and expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 77.8 kDa and released glucose or mannose from p-nitrophenyl-β-d-glucopyranoside (pNPG), cellobiose, mannobiose, and genistin. Peak Tpbgl activity was detected at 90°C, and 50% activity remained after incubation for 60 min at 95°C. The optimal pH for pNPG hydrolysis was 3.5. When the enzyme was incubated with pNPG in the presence of ethanol and propanol, the glucose moiety was transferred to acceptor alcohols. Tpbgl is the archaeal β-glucosidase from glucoside hydrolase family 3 and found to be most heat stable under extremely acidic conditions (pH 3.5). The kinetic parameters revealed that Tpbgl had the highest catalytic efficiency toward pNPG (kcat/Km = 3.05) with strong substrate affinity for such natural substrates as cellobiose (Km = 0.149) and mannobiose (Km = 0.147). Genistin solubilized in 10-40% DMSO was hydrolyzed to genistein with nearly 99% conversion, indicating that high concentrations of the water-insoluble isoflavone glycoside can be treated by the enzyme. Our results indicate that Tpbgl has great potential in cellulose saccharification and the glucoside hydrolysis of natural compounds., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Thyroid hormone is a potent regulator of metabolic and energy homeostasis implicated in various metabolic diseases. Fibroblast growth factor 21(FGF21) is a systemic metabolic regulator known to modulate various biological functions similar to the actions of thyroid hormone. We investigated the differences in plasma FGF21 concentrations in patients with varying thyroid function. Ninety drug-naïve subjects who underwent thyroid evaluation at Seoul National University Bundang Hospital were enrolled and classified into euthyroid, subclinical hypothyroid, and overtly hypothyroid groups. Biochemical markers and plasma FGF21 levels were measured and analyzed. The mean age of the subjects was 42.6 ± 9.1 years. The mean body mass index (BMI), waist circumference, and fasting glucose concentrations were similar between groups. Overtly hypothyroid subjects exhibited significantly higher concentrations of total cholesterol, triglyceride, and LDL-cholesterol than the other groups (p<0.01). Mean plasma FGF21 concentrations in euthyroid, subclinical hypothyroid and overtly hypothyroid groups were 43.2 ± 39.2 pg/mL, 63.6 ± 73.6 pg/mL, and 101.5 ± 74.9 pg/mL, respectively (p<0.01 between groups). Plasma FGF21 concentrations remained significantly higher in overtly hypothyroid subjects after adjusting for serum triglyceride concentrations (p<0.005). Multivariate analysis revealed a significant positive linear relationship between serum TSH concentrations and plasma FGF21 concentrations (β = 0.192, p = 0.002) and a significant negative linear relationship between free T4 and plasma FGF21 concentrations (β = -0.382, p = 0.037) after adjusting for gender, BMI and serum concentrations of triglycerides and glucose. Plasma FGF21 levels were significantly increased in patients with hypothyroidism independently of BMI, or lipid or glucose metabolism.

Even though the etiology of chronic rejection (CR) is multifactorial, donor specific antibody (DSA) is considered to have a causal effect on CR development. Currently the antibody-mediated mechanisms during CR are poorly understood due to lack of proper animal models and tools. In a clinical setting, we previously demonstrated that induction therapy by lymphocyte depletion, using alemtuzumab (anti-human CD52), is associated with an increased incidence of serum alloantibody, C4d deposition and antibody-mediated rejection in human patients. In this study, the effects of T cell depletion in the development of antibody-mediated rejection were examined using human CD52 transgenic (CD52Tg) mice treated with alemtuzumab. Fully mismatched cardiac allografts were transplanted into alemtuzumab treated CD52Tg mice and showed no acute rejection while untreated recipients acutely rejected their grafts. However, approximately half of long-term recipients showed increased degree of vasculopathy, fibrosis and perivascular C3d depositions at posttransplant day 100. The development of CR correlated with DSA and C3d deposition in the graft. Using novel tracking tools to monitor donor-specific B cells, alloreactive B cells were shown to increase in accordance with DSA detection. The current animal model could provide a means of testing strategies to understand mechanisms and developing therapeutic approaches to prevent chronic rejection., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Leptin regulates energy balance. However, knowledge of the critical intracellular transducers of leptin signaling remains incomplete. We found that Rho-kinase 1 (ROCK1) regulates leptin action on body weight homeostasis by activating JAK2, an initial trigger of leptin receptor signaling. Leptin promoted the physical interaction of JAK2 and ROCK1, thereby increasing phosphorylation of JAK2 and downstream activation of Stat3 and FOXO1. Mice lacking ROCK1 in either pro-opiomelanocortin (POMC) or agouti-related protein neurons, mediators of leptin action, displayed obesity and impaired leptin sensitivity. In addition, deletion of ROCK1 in the arcuate nucleus markedly enhanced food intake, resulting in severe obesity. Notably, ROCK1 was a specific mediator of leptin, but not insulin, regulation of POMC neuronal activity. Our data identify ROCK1 as a key regulator of leptin action on energy homeostasis.

The enzyme phytase has broad biotechnological applications, especially in the reduction of phytate, antinutritional factors that chelate essential minerals, in human and animal food. We investigated the enzymatic properties of β-propeller phytase (BPP) from Bacillus amyloliquefaciens DS11. Thermal refolding analysis demonstrated that BPP can remarkably restore its enzymatic activity in the presence of 5 mM Ca(2+) to 87% of its original activity after heating to 100 °C and subsequent cooling, indicating that the enzyme requires Ca(2+) for appropriate refolding. Furthermore, pH-dependent kinetic studies showed that BPP required excess Ca(2+) for its enzymatic activity as the pH decreased, suggesting that the optimal Ca(2+)-phytate ratio for enzymatic catalysis depends on the pH value of the environment. Finally, we verified the practical application of BPP at two different pH's using soybean meal as a natural source of phytate. As compared to a commercial phytase, BPP efficiently hydrolyzed food phytate over neutral pH ranges.

Ginsenoside Rh2 is one of the most active components of red ginseng, controlling cancer and other metabolic diseases including osteoclast differentiation. However, the molecular mechanism underlying the inhibition of osteoclast differentiation by ginsenoside Rh2 remains poorly understood. In the present study, it was found that ginsenoside Rh2 suppressed osteoclast differentiation from bone marrow macrophages (BMMs) treated with receptor activator of nuclear factor κB ligand (RANKL) without any cytotoxicity. Ginsenoside Rh2 significantly reduced RANKL-induced expression of transcription factors, c-Fos and nuclear factor of activated T-cells (NFATc1), as well as osteoclast markers, TRAP and OSCAR. In defining the signaling pathways, ginsenoside Rh2 was shown to moderately inhibit NF-κB activation and ERK phosphorylation in response to RANKL stimulation in BMM cells without any effect on p38 and c-Jun N-terminal kinase (JNK). Finally, ginsenoside Rh2 blocked osteoporosis in vivo as confirmed by restored bone mineral density (BMD) and other markers associated osteoclast differentiation. Hence, it is suggested that ginsenoside Rh2 could suppress RANKL-induced osteoclast differentiation in vitro and in vivo through the regulation of c-Fos and NFATc1 expressions, not excluding the involvement of NF-κB and ERK. Ginsenoside Rh2 is also suggested to be developed as a therapeutic drug for prevention and treatment of osteoporosis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A role of Rho-associated coiled-coil-containing protein kinase (ROCK)1 in regulating whole-body glucose homeostasis has been reported. However, cell-autonomous effects of ROCK1 on insulin-dependent glucose transport in adipocytes and muscle cells have not been elucidated. To determine the specific role of ROCK1 in glucose transport directly, ROCK1 expression in 3T3-L1 adipocytes and L6 myoblasts was biologically modulated. Here, we show that small interfering RNA-mediated ROCK1 depletion decreased insulin-induced glucose transport in adipocytes and myoblasts, whereas adenovirus-mediated ROCK1 expression increased this in a dose-dependent manner, indicating that ROCK1 is permissive for glucose transport. Inhibition of ROCK1 also impaired glucose transporter 4 translocation in 3T3-L1 adipocytes. Importantly, the ED₅₀ of insulin for adipocyte glucose transport was reduced when ROCK1 was expressed, leading to hypersensitivity to insulin. These effects are dependent on actin cytoskeleton remodeling, because inhibitors of actin polymerization significantly decreased ROCK1's effect to promote insulin-stimulated glucose transport. Unlike ROCK2, ROCK1 binding to insulin receptor substrate (IRS)-1 was not detected by immunoprecipitation, although cell fractionation demonstrated both ROCK isoforms localize with IRS-1 in low-density microsomes. Moreover, insulin's ability to increase IRS-1 tyrosine 612 and serine 632/635 phosphorylation was attenuated by ROCK1 suppression. Replacing IRS-1 serine 632/635 with alanine reduced insulin-stimulated phosphatidylinositol 3-kinase activation and glucose transport in 3T3-L1 adipocytes, indicating that phosphorylation of these serine residues of IRS-1, which are substrates of the ROCK2 isoform in vitro, are crucial for maximal stimulation of glucose transport by insulin. Our studies identify ROCK1 as an important positive regulator of insulin action on glucose transport in adipocytes and muscle cells.

As H. pylori infection progresses, intestinal metaplasia (IM), a key event in gastric carcinogenesis, develops in the stomach. The mechanism by which H. pylori infection causes the trans-differentiation of gastric cells to intestinal-type cells remains an important question. In the current study, we found that RUNX3 is deregulated in all human IM specimens examined by either down regulation or mislocalization; Aberrant localization of a gastric tumor suppressor RUNX3 is observed in most human cases of IM with concurrent H. pylori infection, and RUNX3 is down-regulated in most cases of IM without H. pylori-infection. The cytoplasmic mislocalization of a RUNX3 was associated with H. pylori-induced c-Src activation and RUNX tyrosine phosphorylation. Moreover, gastric epithelial cells of Runx3(-/-) mice expressed the intestinal markers Muc2 and Li-Cadherin, which suggests that the deregulation of Runx3 is a key event in the intestinalization of the gastric epithelium. Collectively, the results of the current study suggest that RUNX3 deregulation is associated with H. pylori-induced pathogenesis and the development of IM., (Copyright © 2011 Wiley Periodicals, Inc.)

In recent years, gold nanoparticles have been increasingly utilized as a promising material for biomedical analysis. We report here for the first time the synthesis of ultrasmall gold nanoparticles with core diameter of 1.2 nm functionalized with hydrazide groups and their use in isolation/enrichment of N-glycosylated peptides. Hydrazide-functionalized gold nanoparticles showed excellent stability in biological samples and exhibited a large capacity for peptide capturing. The captured peptides from tested standard glycoproteins were found to be highly specific as determined by Agilent HPLC chip and quadrupole time-of-flight (Q-TOF) mass spectrometer. The hydrazide-functionalized gold nanoparticles were successfully utilized in the isolation of a real proteome complex, which showed that more than 90% of captured product was glycopeptide. These results demonstrate that the ultrasmall gold nanoparticles can be used for a high-throughput analysis platform of glycoproteins.

Recent genetic screens of fly mutants and molecular analysis have revealed that the Hippo (Hpo) pathway controls both cell proliferation and cell death. Deregulation of its human counterpart (the MST pathway) has been implicated in human cancers. However, how this pathway is linked with the known tumor suppressor network remains to be established. RUNX3 functions as a tumor suppressor of gastric cancer, lung cancer, bladder cancer, and colon cancer. Here, we show that RUNX3 is a principal and evolutionarily conserved component of the MST pathway. SAV1/WW45 facilitates the close association between MST2 and RUNX3. MST2, in turn, stimulates the SAV1-RUNX3 interaction. In addition, we show that siRNA-mediated RUNX3 knockdown abolishes MST/Hpo-mediated cell death. By establishing that RUNX3 is an endpoint effector of the MST pathway and that RUNX3 is capable of inducing cell death in cooperation with MST and SAV1, we define an evolutionarily conserved novel regulatory mechanism loop for tumor suppression in human cancers., (Copyright © 2011 Wiley Periodicals, Inc.)

The mammalian ste20 kinase (MST) signaling pathway plays an important role in the regulation of apoptosis and cell cycle control. We sought to understand the role of MST2 kinase and Salvador homolog 1 (SAV1), a scaffolding protein that functions in the MST pathway, in adipocyte differentiation. MST2 and MST1 stimulated the binding of SAV1 to peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor that plays a key role in adipogenesis. The interaction of endogenous SAV1 and PPARγ was detected in differentiating 3T3-L1 adipocytes. This binding required the kinase activity of MST2 and was mediated by the WW domains of SAV1 and the PPYY motif of PPARγ. Overexpression of MST2 and SAV1 increased PPARγ levels by stabilizing the protein, and the knockdown of SAV1 resulted in a decrease of endogenous PPARγ protein in 3T3-L1 adipocytes. During the differentiation of 3T3-L1 cells into adipocytes, MST2 and SAV1 expression began to increase at 2 days when PPARγ expression also begins to increase. MST2 and SAV1 significantly increased PPARγ transactivation, and SAV1 was shown to be required for the activation of PPARγ by rosiglitazone. Finally, differentiation of 3T3-L1 cells was augmented by MST2 and SAV1 expression and inhibited by knockdown of MST1/2 or SAV1. These results suggest that PPARγ activation by the MST signaling pathway may be a novel regulatory mechanism of adipogenesis.

Background: Traumatic brain injury (TBI) is a leading cause of injury death and long-term disability in the USA. It commonly results from blunt (closed) or penetrating trauma. The majority of civilian TBI is caused by falls or motor vehicle collisions, whereas military TBI mainly results from explosions. Although penetrating injuries are less common than closed injuries in the civilian population, they are far more lethal. Unfortunately, the pathophysiologic differences between penetrating and closed TBI remain poorly understood due to the lack of studies on the subject. Many studies on the prognostic factors of mortality and functional outcome after TBI exclude penetrating brain injuries from their series because they are believed to have a different pathophysiology., Methods: 125 Articles regarding brain injury were reviewed and summarized for this report., Results: Despite the absence of a clear delineation between penetrating and blunt TBI, the current guidelines for penetrating TBI suggest defaulting to management strategies used for closed TBI with limited supportive evidence. Thus, injuries that appear to have different pathophysiologies and outcomes are managed equally and perhaps not optimally., Conclusion: In view of the incomplete understanding of the impact of mechanism of injury on TBI outcomes, as demonstrated in the current review, new research studies are required to improve evidence-based TBI guidelines tailored especially for penetrating injuries.

Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF0356) similar to the enzymes in glycoside hydrolase family 1. This β-glycosidase, designated PFTG (P. furiosus thermostable glycosidase), was cloned and expressed in Escherichia coli. The expressed enzyme was purified by heat treatment and Ni-NTA affinity chromatography. The gene was composed of 1,452 bp encoding 483 amino acids for a protein with a predicted molecular mass of 56,326 Da. The temperature and pH optima were 100°C and 5.0 in sodium citrate buffer, respectively. The substrate specificity of PFTG suggests that it possesses characteristics of both β-galactosidase and β-mannosidase activities. However, through kinetic studies by ITC (Isothermal Titration Colorimetry) which is very sensitive method for enzyme kinetics, PF0356 enzyme revealed the highest catalytic efficiency toward p-nitrophenyl-β-d-mannopyranoside (3.02 k(cat)/K(m)) and mannobiose (4.32 k(cat)/K(m)). The enzyme showed transglycosylation and transgalactosylation activities toward cellobiose, lactose and mannooligosaccharides that could produce GOS (galactooligosaccharides) and MOS (maltooligosaccharides). This novel hyperthermostable β-glycosidase may be useful for food and pharmaceutical applications., (Copyright © 2011 Elsevier B.V. All rights reserved.)

PURPOSE. To solve the shortage of donor corneas, a decellularizing method based on hypertonic saline treatment was introduced, and a favorable outcome was observed in pig-to-rabbit lamellar corneal transplantation. This study was an investigation of the efficacy of pig-to-nonhuman primate lamellar corneal transplantation, using both decellularized and fresh porcine corneas to assess feasibility as a substitute for human corneas. METHODS. Nine Chinese rhesus macaques underwent lamellar corneal transplantation using both decellularized (n = 5) and fresh (n = 4) porcine corneas. Clinically acceptable graft size (7.5 mm in diameter) and minimal immunosuppression based on topical and systemic corticosteroids were applied. Rejection signs, histology of porcine grafts, and serial changes in recipients' blood profile, including memory T-cell subset, anti-α-Gal and donor pig-specific antibodies, and complement were evaluated. Changes in aqueous complement concentration were also assessed at 4 weeks after transplantation. RESULTS. Of the decellularized porcine lamellar grafts, 80% remained transparent for more than 6 months, whereas half of the fresh porcine lamellar grafts developed chronic rejection. Rejected grafts showed extensive cellular infiltration, predominantly CD8(+) T lymphocytes and macrophages. Immunologic profiles of the recipients with rejected grafts showed a significant increase in the concentration of aqueous complement, an enhancement of memory T cells, and an abrupt increase in donor pig-specific antibodies. CONCLUSIONS. The findings suggested that decellularized porcine cornea could be a promising substitute for human corneal allograft. Fresh porcine cornea may be a feasible option for a substitute if combined with more potent immunosuppression or if obtained from transgenic pigs with complement-regulatory proteins.

In this study, alkaline phytase was added to whole-wheat bread and the phytate content and mineral profiles were compared to commercially available acidic phytase. At neutral pH, some phytate (approximately 20%) was degraded by endogenous phytase in wheat flour, while 40% of phytate was hydrolyzed by alkaline phytase DS11 and a 35% reduction was observed with acidic phytase. Most of the enzymatic activity occurred during the proofing stage, and the rate of reaction depended on pH. DS11 phytase effectively degraded the phytate level within a 30 min treatment at pH 7; however, at least 60 min was needed with acidic phytase to achieve the same hydrolysis level. Mineral profiles were also dramatically affected by the phytate reduction. The biggest increase was observed in Fe²⁺ by the phytase treatment. The Fe²⁺ content increased 10-fold at pH 7 and 8-fold at pH 5 with alkaline phytase DS11. Alkaline phytase DS11 was shown to be effective at phytate reduction in whole-wheat bread preparation. Additionally, phytate degradation enhanced the mineral availability of bread., (© 2011 Institute of Food Technologists®)

The study by Dr Peiniger and colleagues in a recent issue of Critical Care indicates that transfusion strategies using an early and more balanced ratio between fresh frozen plasma and red blood cell transfusions provide a survival benefit in patients with acute traumatic coagulopathy requiring massive transfusion within the first 24 hours of hospitalization. However, this topic has never been explored in depth in patients with concomitant severe traumatic brain injury. While the study is retrospective and certainly not a substitute for a well-designed prospective trial, the authors nonetheless should be commended for addressing this issue with their current work. Currently, the optimum fluid resuscitation paradigm for patients with both severe traumatic brain injury and other injuries requiring significant volume resuscitation is not clear.

Background: A newly acquired rhesus macaque was suffering from rapid destruction of the left cheek caused by necrotizing stomatitis., Methods: To restore reconstructive surgery and intensive care with antibiotics, wound protection, wound healing agents, and debridement were applied., Results: Staphylococcus aureus and Enterococcus faecalis were isolated from the culture of the lesion, and the antibiotic susceptibility test revealed methicillin-resistant Staphylococcus aureus infection. Vancomycin and ampicillin-sulbactam effectively treated the bacterial infections, and reconstructive surgery was performed once the infection was cleared. Topical application of recombinant human epidermal growth factor (rhEGF) was useful to treat exposed wound of the noma lesion., Conclusions: Simian noma associated with methicillin-resistant Staphylococcus aureus (MRSA) had not previously been reported in non-human primates. Although noma associated with MRSA is hard to cure because of its rapid and destructive progress, the aggressive therapy used in this study led to the successful resolution of an acute necrotic stomatitis lesion in a rhesus macaque., (© 2011 John Wiley & Sons A/S.)

Background and Objectives: Hyperacute rejection (HAR) is a major obstacle to successful xenotransplantation of vascularized organs. This study was conducted to observe the effect of hemolysis of perfused human whole blood on pig heart function, and determine the major risk factors for preservation of xenoperfused cardiac function using ex-vivo pig to human xenogeneic cardiac perfusion model., Materials and Methods: Harvested pig hearts were perfused with normal human whole blood (group 1), two different types of pre-treated human whole blood (group 2: immunoglobulins were depleted by plasmapheresis, group 3: pre-treated with plasmapheresis, GAS914, cobra venom factor (CVF) and steroid), and normal porcine whole blood as control (group 4) for 3 hours., Results: Duration of heart beat was significantly prolonged in group 2 and group 3. Histological examination showed widespread HAR features but was gradually delayed in groups 2 and 3 compared to group 1. The absolute levels of serum creatine kinase-MB and Troponin I increased gradually, and was lower in group 3. Serum hemoglobin levels were rapidly increased in groups 3 and 4, compared to group 1. Extracellular potassium level increased sharply from the beginning of blood perfusion in groups 1, 2 and 3, compared to group 4., Conclusion: Pretreatment of human whole blood, including immunoglobulin depletion, CVF and steroid reduced and delayed the destruction of pig myocardium by HAR. However, the increased extracellular potassium levels in groups 1, 2 and 3 reflected that these treatments could not prohibit myocardial injury by HAR.

Sphingolipids are membrane components and are involved in cell proliferation, apoptosis and metabolic regulation. In this study we investigated whether de novo sphingolipid biosynthesis in macrophages is regulated by inflammatory stimuli. Lipopolysaccharide (LPS) treatment upregulated Sptlc2, a subunit of serine palmitoyltransferase (SPT), mRNA and protein in Raw264.7 and mouse peritoneal macrophages, but Sptlc1, another subunit of SPT, was not altered. SPT activation by LPS elevated cellular levels of ceramides and sphingomyelin (SM). Pharmacological inhibition of nuclear factor kappa B (NFκB) prevented LPS-induced upregulation of Sptlc2 while transfection of p65 subunit of NFκB upregulated Sptlc2 and increased cellular ceramide levels. In contrast, MAP kinases were not involved in regulation of sphingolipid biosynthesis. Analysis of Sptlc2 promoter and chromatin immunoprecipitation (ChIP) assay showed that NFκB binding sites are located in Sptlc2 promoter region. Our results demonstrate that inflammatory stimuli activate de novo sphingolipid biosynthesis via NFκB and may play a critical role in lipid metabolism in macrophages., (Crown Copyright © 2010. Published by Elsevier Inc. All rights reserved.)

Mammalian MST2 kinase plays an important role in cell proliferation, survival, and apoptosis. In search of interacting proteins of MST2, we found that estrogen receptor α (ERα) co-immunoprecipitates with MST2 and its adaptor protein human Salvador (hSAV). Using reporter assays, we observed that overexpression of MST2 and hSAV leads to ligand-independent activation of ERα in human breast cancer MCF-7 cells, which was attenuated by the knockdown of hSAV. Furthermore, using truncated mutants of hSAV, we observed that the C terminus of hSAV is necessary and sufficient for the induction of ERα transactivation. The expression of hSAV and MST2 results in the phosphorylation of ERα at serine residues 118 and 167 and represses ERα expression. We then investigated the incidence of MST2 and ERα expression with other tumor biomarkers using commercially available tissue microarrays. Among 40 breast cancer samples analyzed, 60% (24 out of 40) expressed MST2. Nineteen among the 40 cases were MST2-positive and ERα-negative, implying a correlation between expressions of MST2 with loss of ERα in breast tumor samples. This study suggests that MST and hSAV act as novel co-regulators of ERα and may play an important role in breast cancer pathogenesis.

Phytate is an antinutritional factor that influences the bioavailability of essential minerals by forming complexes with them and converting them into insoluble salts. To further our understanding of the chemistry of phytate's binding interactions with biologically important metal cations, we determined the stoichiometry, affinity, and thermodynamics of these interactions by isothermal titration calorimetry. The results suggest that phytate has multiple Ca(2+)-binding sites and forms insoluble tricalcium- or tetracalcium-phytate salts over a wide pH range (pH 3.0-9.0). We overexpressed the β-propeller phytase from Hahella chejuensis (HcBPP) that hydrolyzes insoluble Ca(2+)-phytate salts. Structure-based sequence alignments indicated that the active site of HcBPP may contain multiple calcium-binding sites that provide a favorable electrostatic environment for the binding of Ca(2+)-phytate salts. Biochemical and kinetic studies further confirmed that HcBPP preferentially recognizes its substrate and selectively hydrolyzes insoluble Ca(2+)-phytate salts at three phosphate group sites, yielding the final product, myo-inositol 2,4,6-trisphosphate. More importantly, ITC analysis of this final product with several cations revealed that HcBPP efficiently eliminates the ability of phytate to chelate several divalent cations strongly and thereby provides free minerals and phosphate ions as nutrients for the growth of bacteria. Collectively, our results provide significant new insights into the potential application of HcBPP in enhancing the bioavailability and absorption of divalent cations.

RUNX3 is a transcription factor that functions as a tumor suppressor. In some cancers, RUNX3 expression is down-regulated, usually due to promoter hypermethylation. Recently, it was found that RUNX3 can also be inactivated by the mislocalization of the protein in the cytoplasm. The molecular mechanisms controlling this mislocalization are poorly understood. In this study, we found that the overexpression of Src results in the tyrosine phosphorylation and cytoplasmic localization of RUNX3. We also found that the tyrosine residues of endogenous RUNX3 are phosphorylated and that the protein is localized in the cytoplasm in Src-activated cancer cell lines. We further showed that the knockdown of Src by small interfering RNA, or the inhibition of Src kinase activity by a chemical inhibitor, causes the re-localization of RUNX3 to the nucleus. Collectively, our results demonstrate that the tyrosine phosphorylation of RUNX3 by activated Src is associated with the cytoplasmic localization of RUNX3 in gastric and breast cancers.

The p14(ARF)-MDM2-p53 pathway constitutes an effective mechanism for protecting cells from oncogenic stimuli such as activated Ras and Myc. Importantly, Ras activation induces p14(ARF) and often occurs earlier than p53 inactivation during cancer development. Here, we show that RUNX3, a tumor suppressor in various tumors including stomach, bladder, colon, and lung, is stabilized by Ras activation through the p14(ARF)-MDM2 signaling pathway. RUNX3 directly binds MDM2 through its Runt-related DNA-binding domain. MDM2 blocks RUNX3 transcriptional activity by interacting with RUNX3 through an acidic domain adjacent to the p53-binding domain of MDM2 and ubiquitinates RUNX3 on key lysine residues to mediate nuclear export and proteasomal degradation. Our data indicate that the lineage-specific tumor suppressor RUNX3 and the ubiquitous p53 protein are both principal responders of the p14(ARF)-MDM2 cell surveillance pathway that prevents pathologic consequences of abnormal oncogene activation.

Pig blood group antigens may be present on grafted tissue as 16 isoforms, including the major one, A substrance. Seoul National University (SNU) miniature pigs and domestic pigs were used in this study. Polymerase chain reaction (PCR) was performed for the erythrocyte antigen A (EAA) gene, and reverse transcriptase-PCR for pig A transferase fucosyl transferase (FUT) 1 and FUT-2. The hemagglutination test was performed with murine monoclonal anti-A and anti-B antibodies (mAb), and immunohistochemistry with anti-human blood antigen mAb. SNU miniature and domestic pigs showed blood groups A and O. Blood group A SNU miniature pigs expressed either EAA(AA) or EAA(AO) and either S(SS) or S(SO); blood group O miniature pigs expressed EAA(OO) and S(SS) or S(SO), and there was no A(weak). Additionally, blood group A could be divided into blood group A(clotting) and blood group A(not clotting) in hemagglutination tests. Pig A substance was expressed in the lung and kidney in blood group A pigs, but we could not detect pig A substance expression in the lung, kidney, and heart of blood group O pigs or the heart of blood group A pigs. In conclusion, we suggest that blood typing of SNU miniature pigs can be easily performed using immunohistochemistry, PCR, and/or RT-PCR. Molecular-based AO typing described in this study may be useful to select SNU miniature pigs bearing a specific blood group.

Ischemic complications are extremely rare after radical pelvic surgery because of the collateral blood supply in the pelvis. We report a case of complete bladder gangrene 3 weeks after a laparoscopic radical hysterectomy with bilateral hypogastric artery ligation in a 70-year-old woman with cervical cancer.

Background: Sertoli cells (SC) have immunomodulative properties, and chemokine receptor 7 (CCR7) can optimize the systemic immunomodulatory effect by guiding SC from the periphery to the secondary lymphoid organs., Methods: The effect of immortalized neonatal porcine SC (NPSCi) was evaluated by analysis of cytokine levels. Hyporesponsiveness to donor cells was determined by MLC and analysis of splenocyte phenotypes using a murine allogeneic skin graft model. The effect of CCR7-expressing NPSCi (NPSCi-CCR7) combined with cobra venom factor (CVF) was evaluated using a heterotopically transplanted murine allogeneic heart model., Results: Expression of immune cytokines was markedly modulated by NPSCi. The lymphocyte proliferation and splenocyte phenotypes were significantly suppressed by NPSCi-CCR7. Although pre-transplantation of NPSCi or NPSCi-CCR7 did not prolong graft survival of allogeneic cardiac grafts, CVF treatment facilitated pre-transplantation of NPSCi-CCR7 to prolong survival of allogeneic cardiac grafts (25.5 +/- 7.05 vs 9.5 +/- 0.58 days, p < 0.01)., Conclusions: NPSCi may be used as a powerful immunomodulatory tool, and our strategy to traffic NPSCi to lymphoid organs using CCR7 optimizes the systemic immunomodulatory effect in vivo. With the help of initial immunosuppression for humoral mechanisms using CVF, the host immune response against allogeneic cardiac grafts can be effectively ameliorated by immunomodulation of the cellular mechanism with NPSCi-CCR7.

The loci of the Pim and Runx gene families have been identified as targets for viral insertions in CD2-myc mice. Synergistic cooperation between Pim and RUNX was also found in the CD2-Runx2 transgenic mouse lymphoma model. RUNX genes have come to prominence recently because of their roles as essential regulators of cell fate in development. Paradoxically, they appear to function either as tumor-suppressor genes or dominant oncogenes according to the cellular context. However, the molecular mechanism of the ambiguous roles played by this family of transcription factors in cancer has remained largely uninvestigated. Here we demonstrate that Pim-1 phosphorylates four Ser/Thr residues within the Runt domain and stabilizes RUNX3 protein. In addition, Pim-1 markedly altered the cellular localization of RUNX3 from the nucleus to the cytoplasm. Our results demonstrate that the subcellular localization of RUNX3 is altered by phosphorylation. We propose that RUNX family members may behave as oncogenes if mislocalized to a cellular micro-compartment.

The comparison of potent immunomodulating activities of hot-aqueous polysaccharides isolated from Phellinus spp., P. linteus (PL), P. baumii (PB) and P. gilvus (PG), on cellular immunity were investigated. The results showed that all three kinds of Phellinus spp. could stimulate the proliferation of murine splenocytes, and PG has more powerful stimulating activity than other Phellinus spp. Furthermore, PL and PB significantly increased the proliferation of the mixed splenocytes in a concentration-response manner, and the stimulating effect of PL was significantly higher than that of PB at all concentrations used in the present study, but no stimulating effect was found with the addition of PG. The phagocytosis of both peritoneal macrophage and RAW264.7 cells was also increased in the presence of PG, PB or PL, and the stimulating activity of PG was higher than that of PB or PL at all concentrations tested., ((c) 2008 John Wiley & Sons, Ltd.)

Approximately 50,000 traumatic injuries resulting in fractures of the bony spinal column occur annually in the United States. Although some of these lesions are clearly unstable and mandate urgent surgical treatment for stabilization, less severe injuries may be managed initially with bracing and serial imaging to evaluate bony healing and alignment. A proportion of these injuries will require delayed surgical intervention to correct a posttraumatic deformity. In addition, inadequate or ineffective acute spinal stabilization can also result in the progression of delayed spinal deformities. The management of these lesions is frequently complicated by scarring in the body cavities from the inciting trauma or any subsequent surgical interventions, epidural scar formation and spinal cord tethering, solid fusion into the deformed state, medical comorbidities associated with paralysis, and compromised spinal cord function. With these factors in mind, surgical management of these frequently kyphotic deformities can be performed via a posterior approach with osteotomies or a combined anterior approach and posterior procedures.

The adenoviral gene, termed early region 1A (E1A), is crucial for transformation and has been used very effectively as a tool to determine the molecular mechanisms that underlie the basis of cellular transformation. pRb, p107, p130, p300/CBP, p400, TRRAP, and CtBP were identified to be E1A-binding proteins and their roles in cellular transformation have been established. Although the major function of E1A is considered to be the regulation of gene expression that is critical for differentiation and cell cycle exit, one of the most significant questions relating to E1A transformation is how E1A mediates this regulation. RUNX3 is a transcription factor that was first described as a gastric cancer tumor suppressor but is now known to be involved in many different cancers. Exogenous expression of RUNX3 strongly inhibits the growth of cells. Here, we show that the adenovirus oncoprotein E1A interacts with RUNX3 in vitro and in vivo. RUNX3 interacts with the N-terminus (amino acids 2-29) of E1A, which is known to interact with p300/CBP, p400, and TRRAP. E1A interacts directly with the Runt domain of RUNX3 but does not interfere with CBFbeta-RUNX3 interactions. In addition, E1A inhibits the transactivation activity of RUNX3 on the p21(WAF1/CIP1) promoter. Consistent with these observations, the growth inhibition induced by RUNX3 is reduced by E1A. These results demonstrate that E1A specifically binds to RUNX3 and inactivates its transactivation activity. We propose that one of the mechanisms for the oncogenic activity of E1A is the inhibition of RUNX3, similar to that of RB and p300/CBP., ((c) 2008 Wiley-Liss, Inc.)

The understanding of main mechanisms that determine the ability of immune privilege related to Sertoli cells (SCs) will provide clues for promoting a local tolerogenic environment. In this study, we evaluated the property of humoral and cellular immune response modulation provided by porcine SCs. Porcine SCs were resistant to human antibody and complement-mediated formation of the membrane attack complex (38.41+/-2.77% vs. 55.02+/-5.44%, p=0.027) and cell lysis (42.95+/-1.75% vs. 87.99 +/-2.25%, p<0.001) compared to immortalized aortic endothelial cells, suggesting that porcine SCs are able to escape cellular lysis associated with complement activation by producing one or more immunoprotective factors that may be capable of inhibiting membrane attack complex formation. On the other hand, porcine SCs and their culture supernatant suppressed the up-regulation of CD40 expression (p<0.05) on DCs in the presence of LPS stimulation. These novel findings, as we know, suggest that immune modulatory effects of porcine SCs in the presence of other antigen can be obtained from the first step of antigen presentation. These might open optimistic perspectives for the use of porcine SCs in tolerance induction eliminating the need for chronic immunosuppressive drugs.

The emerging future of cerebral surgery will witness the refined evolution of current techniques, as well as the introduction of numerous novel concepts. Clinical practice and basic science research will benefit greatly from their application. The sum of these efforts will result in continued minimalism and improved accuracy and efficiency of neurosurgical diagnostic and therapeutic methodologies.Initially, the refinement of current technologies will further enhance various aspects of cerebral surgery. Advances in computing power and information technology will speed data acquisition, storage, and transfer. Miniaturization of current devices will impact diverse areas, such as modulation of endoscopy and endovascular techniques. The increased penetrance of surgical technologies such as stereotactic radiosurgery, neuronavigation, intraoperative imaging, and implantable electrodes for neurodegenerative disorders and epilepsy will enhance the knowledge and experience in these areas and facilitate refinements and advances in these technologies. Further into the future, technologies that are currently relatively remote to surgical events will fundamentally alter the complexity and scale at which a neurological disease may be treated or investigated. Seemingly futuristic concepts will become ubiquitous in the daily experience of the neurosurgeon. These include diverse fields such as nanotechnology, virtual reality, and robotics. Ultimately, combining advances in multiple fields will yield progress in diverse realms such as brain tumor therapy, neuromodulation for psychiatric diseases, and neuroprosthetics. Operating room equipment and design will benefit from each of the aforementioned advances. In this work, we discuss new developments in three parts. In Part I, concepts in minimalism important for future cerebral surgery are discussed. These include concrete and abstract ideas in miniaturization, as well as recent and future work in microelectromechanical systems and nanotechnology. Part II presents advances in computational sciences and technological fields dependent on these developments. Future breakthroughs in the components of the "computer," including data storage, electrical circuitry, and computing hardware and techniques, are discussed. Additionally, important concepts in the refinement of virtual environments and the brain-machine interface are presented, as their incorporation into cerebral surgery is closely linked to advances in computing and electronics. Finally, Part III offers insights into the future evolution of surgical and nonsurgical diagnostic and therapeutic modalities that are important for the future cerebral surgeon. A number of topics relevant to cerebral surgery are discussed, including the operative environment, imaging technologies, endoscopy, robotics, neuromodulation, stem cell therapy, radiosurgery, and technical methods of restoration of neural function. Cerebral surgery in the near and distant future will reflect the application of these emerging technologies. As this article indicates, the key to maximizing the impact of these advancements in the clinical arena is continued collaboration between scientists and neurosurgeons, as well as the emergence of a neurosurgeon whose scientific grounding and technical focus are far removed from those of his predecessors.

In the following literature review the authors consider the available evidence for the medical management of patients with ankylosing spondylitis (AS), and they critically assess current treatment guidelines. Medical therapy for axial disease in AS emphasizes improvement in patients' pain and overall function. First-line treatments include individualized physical therapy and nonsteroidal antiinflammatory drugs (NSAIDs) in conjunction with gastroprotective therapy. After an adequate trial of therapy with two NSAIDs exceeding 3 months or limited by medication toxicity, the patient may undergo tumor necrosis factor-alpha blockade therapy. Response should occur within 6-12 weeks, and patients must undergo tuberculosis screening. Evidence does not currently support the use of disease modifying antirheumatic drugs, corticosteroids, or radiotherapy in AS.

The pharmacokinetics and dosage regimen of norfloxacin-glycine acetate (NFLXGA) was investigated in pigs after a single intravenous (i.v.) or oral (p.o.) administration at a dosage of 7.2 mg/kg body weight. After both i.v. and p.o. administration, plasma drug concentrations were best fitted to an open two-compartment model with a rapid distribution phase. After i.v. administration of NFLXGA, the distribution (t(1/2alpha)) and elimination half-life (t(1/2beta)) were 0.36 +/- 0.07 h and 7.42 +/- 3.55 h, respectively. The volume of distribution of NFLXGA at steady state (Vd(ss)) was 4.66 +/- 1.39 l/kg. After p.o. administration of NFLXGA, the maximal absorption concentration (C(max)) was 0.43 +/- 0.06 microg/ ml at 1.36 +/- 0.39 h (T(max)). The mean absorption (t(1/2ka)) and elimination half-life (t(1/2beta)) of NFLXGA were 0.78 +/- 0.27 h and 7.13 +/- 1.41 h, respectively. The mean systemic bioavailability (F) after p.o. administration was 31.10 +/- 15.16%. We suggest that the optimal dosage calculated from the pharmacokinetic parameters is 5.01 mg/kg per day i.v. or 16.12 mg/kg per day p.o.

Background: Sertoli cells (SC) are known to have active mechanism for evading humoral immune response and are known to have immune modulatory effects in the presence of other antigens. This has led us to hypothesize that systemic immune modulating effect of SC might be optimized by their residence on peripheral lymph node. This study was designed to evaluate our new strategy to promote preventive or therapeutic effects of SC in systemic immune modulation for organ transplantation., Methods: For this purpose, we created chemokine receptor 7 (CCR7) expressing porcine SC (NPSCi-CCR7) to facilitate their migration into lymphoid organ in vivo and their potential to modulate systemic immune responses was evaluated using mouse allogeneic skin graft model., Results: Directed migration of NPSCi-CCR7 cells from periphery into lymphoid organs was dramatically promoted compared to control NPSCi cells. Also pre-transplantation of NPSCi-CCR7 significantly suppressed lymphocyte proliferation and prolonged the allogeneic skin graft survival., Conclusion: These results suggest that our new strategy to traffic SC to lymphoid organs using CCR7 is very effective and can be extended to traffic other immune modulatory cells or proteins to primary and secondary lymphoid structures to augment their therapeutic potential.

Objective: The pseudarthrosis rate after multisegment anterior cervical fusion is directly related to the number of levels surgically fused. The advent of osteobiological adjuvants offers an opportunity to reduce both the likelihood of failed arthrodesis and the need for posterior instrumentation. Collagen-hydroxyapatite matrix is osteoconductive and has been used with autogenous bone marrow aspirate (BMA) to promote fusion. We report our results of using collagen-hydroxyapatite matrix with BMA for multilevel anterior cervical discectomy and fusion and anterior cervical corpectomy and fusion (ACCF)., Methods: Sixty-six consecutively treated patients underwent a multilevel anterior cervical discectomy and fusion and/or ACCF during a period of 16 months. In all cases, a Smith-Robinson decompression was performed followed by allograft fibula strut grafting filled with collagen-hydroxyapatite matrix and BMA, and anterior semiconstrained cervical plating. A vacuum chamber was used to draw the BMA slowly through the collagen-hydroxyapatite sponges. No patient underwent simultaneous posterior instrumentation. Clinical outcome was determined by an independent observer who evaluated patients on the basis of symptom and neurological examination results. Radiographic fusion was determined by dynamic x-rays and computed tomographic scanning during an 18-month follow-up period., Results: With the inclusion of discectomies performed in ACCF procedures, patients were fused between two and five disc levels (mean, 3.1 levels). Seventeen patients underwent one to four-level corpectomies (mean, two levels). Clinical improvement was observed in 49 patients. Conditions in nine patients remained unchanged, and two patients had radicular palsies. In all, 60 patients were followed and analyzed for radiographic fusion. All but two patients demonstrated successful radiographic fusion., Conclusion: Collagen-hydroxyapatite matrix with BMA can be a safe, effective adjuvant for promoting fusion in multilevel anterior cervical discectomy and fusion and ACCF. Although randomized, controlled studies are necessary to determine whether or not the fusion rates are superior to those obtained from using allograft alone, these results compare favorably to historical data in the literature.

Background: Effective intervention achieved by manipulating cell-mediated xenogeneic immune responses would critically increase the clinical feasibility of xenotransplantation as immediate hyperacute rejections become controllable through genetic modulations of donor organs. Endogenous negative regulatory signals like the programmed death 1 (PD-1)-programmed death ligand 1 (PD-L1) system are candidate targets for the control of cell-mediated xenogeneic immune response., Methods: A porcine PD-L1 molecule was cloned using RACE (rapid amplification of cDNA ends) technology based on the human PD-L1 sequence. The functional effects of cloned porcine PD-L1 were tested on human CD4(+) T cell activation using porcine PD-L1-transfected bystander cells. Cellular proliferation was monitored by [3H] thymidine incorporation, and human T cell apoptosis was measured by flow cytometry., Results: Porcine PD-L1 (GenBank accession number AY837780) was found to have 73.8% sequence homology with human PD-L1 and to contain two immunoglobulin domains in its extracellular region. Moreover, porcine PD-L1 expressed on Chinese hamster ovary (CHO) cells inhibited human CD4(+) T cell proliferation stimulated with anti-CD3 only or anti-CD3 plus anti-CD28. Percentages of apoptotic activated human T cells increased by over 30% in the presence of porcine PD-L1/CHO cells, and the addition of recombinant human PD-1-Fc fusion proteins during human T cell activation reversed the inhibitory effects of porcine PD-L1., Conclusions: Cloned porcine PD-L1 showed high sequence homology with human PD-L1 and a similar molecular structure. Moreover, porcine PD-L1 inhibited human CD4(+) T cell activation in human PD-1-dependent manner, and this involved activated T cell apoptosis. The authors suggest that PD-1-PD-L1 might play an important endogenous immune regulatory role during xenogeneic transplantation, and that the effective application of this system would improve transplanted xenogeneic organ survival.

In the first part of this series, we reviewed the histological, radiographic, and molecular data gathered regarding the brain parenchymal response to radiosurgery and suggested future studies that could enhance our understanding of the topic. With this article, we begin by addressing methods of potentiating the effect of radiosurgery on target lesions of the central nervous system. Much of the work on potentiating the effects of cranial radiation has been performed in the field of whole-brain radiotherapy. Data from Phase III trials evaluating the efficacy of various agents as radiosensitizers or radioenhancers in whole-brain radiotherapy are reviewed, and trials for investigating certain agents as enhancers of radiosurgery are suggested. The roles of gene therapy and nanotechnology in enhancing the therapeutic efficacy of radiosurgery are then addressed. Focus is then shifted to a discussion of strategies of protecting healthy tissue from the potentially deleterious aspects of the brain's response to radiosurgery that were presented in the first article of this series. Finally, comments are made regarding the role of neural progenitor or stem cells in the repair of radiation-induced brain injury after radiosurgery. The importance of both the role of the extracellular matrix and properly directed axonal regrowth leading to appropriate target reinnervation is highlighted.

Sertoli cells (SC) are known to contain immunoprotective properties, which allow them to survive as allografts without the use of immunosuppressive drugs. Experiments were designed to determine which factors are related to prolonged survival of allogeneic SC. Balb/c derived Sertoli (TM4) and colon cancer (CT-26) cell lines were implanted beneath the kidney capsule of non-immunosuppressed C57BL/6 mice and compared their survival as allografts. Compared to TM4 graft, which survived more than 7 days after transplantation, CT-26 showed massive infiltration of polymorphonuclear cells, necrosis and enlargement of draining lymph nodes. Cultured cell lines showed no differences in their expression patterns of FasL, TGF beta1, clusterin and two complement regulatory proteins (CRP, i.e., membrane cofactor protein, MCP; decay accelerating factor, DAF), but protectin (CD59), another member of CRP was expressed only on TM4. These results suggest that CD59 and unknown factors may contribute to the prolonged survival of SC in non-immunoprivileged sites.

Background: An understanding of the main mechanism that determines the ability of immune privilege related to Sertoli cells (SC) will provide clues for promoting a local tolerogenic environment. In this report, we established neonatal porcine SC line and evaluated their characteristics., Methods: SC line was established following the transfection of primary SC (NPSC) from the testis of neonatal pig with plasmid pRNS-1 carrying genes for neomycin resistance and the SV40 large T antigen. Immunohistochemistry and RT-PCR were performed to evaluate the character of immortalized SC lines., Results: Our immortalized SC line (iPS) proliferated stably and had a phenotype similar to NPSC, as indicated by the immunoexpression of follicle stimulating hormone receptor (FSHR), and mRNA expression of androgen receptor (AR), and Wilms' tumor antigen (WT1). Interestingly, NPSC and iPS expressed mRNA of complement regulatory proteins (CRP) such as membrane cofactor protein (CD46), decay accelerating factor (DAF or CD55), and protectin (CD59), but CD59 mRNA expression was negligible in iPS., Conclusion: These results suggest that iPS, immortalized by the introduction of SV40 T, retain their original characteristics, except for the relatively low expression of CD59, and that they may be useful for future in vitro and in vivo studies of immune privilege mechanisms related to SC.

In the present study, we analyzed the antioxidant, antiplatelet aggregation, anti-inflammatory, and tyrosinase inhibitory activities of a variety of solvent extracts of Elaeagnus multiflora Thunb. (Family Elaeagnaceae). Among the solvent extracts of E. multiflora, the ethyl acetate extract (EE) exhibited the highest 1,1-diphenyl-2-picrylhydrazyl radical and xanthine oxidase inhibition activity, as well as the greatest tyrosinase inhibition activity. Only the chloroform extract (CE) inhibited platelet aggregation, and that was a weak effect with 19.29% inhibition at 250 microg/mL, as compared to controls. The CE was also the most potent inhibitor of nitric oxide production among the tested fractions, with almost 100% inhibition at 500 microg/mL. We also detected 2,4-bis(1,1-dimethylethyl)phenol in the CE and EE, via a gas chromatography-mass spectrometry assay. In conclusion, we found that E. multiflora Thunb. has antioxidant and antiplatelet aggregation effects to some extent, and its CE and EE possess potent inhibitory effects on nitric oxide production and tyrosinase activity.

Radiosurgery is now the preferred treatment modality for many intracranial disease processes. Although almost 50 years have passed since it was introduced as a tool to treat neurological disease, investigations into its effects on normal tissues of the central nervous system are still ongoing. The need for these continuing studies must be underscored. A fundamental understanding of the brain parenchymal response to radiosurgery would permit development of strategies that would enhance and potentiate the radiosurgical treatment effects on diseased tissue while mitigating injury to normal structures. To date, most studies on the response of the central nervous system to radiosurgery have been performed on brain tissue in the absence of pathological lesions, such as benign tumors or metastases. Although instructive, these investigations fail to emulate the majority of clinical scenarios that involve radiosurgical treatment of specific lesions surrounded by normal brain parenchyma. This article is the first in a two-part series that addresses the brain parenchyma's response to radiosurgery. This first article analyzes the histological, radiographic, and molecular data gathered regarding the brain parenchymal response to radiosurgery and aims to suggest future studies that could enhance our understanding of the topic. The second article in the series begins by discussing strategies for radiosurgical therapeutic enhancement. It concludes by focusing on strategies for mitigation and repair of radiation-induced brain injury.

Although dendritic cells (DCs) are unrivaled for initiation of immune responses, the immunomodulatory capacity of chemically fixed DC has not been thoroughly evaluated. We monitored the tolerogenic capacity of chemically fixed DCs using allogeneic heart transplantations. Bone marrow progenitors were differentiated into immature DCs which were then chemically fixed and injected intravenously into recipient mice at 14 days before allogeneic heart transplantation. Chemically fixed DCs markedly prolonged graft survival in the major histocompatibility complex (MHC) I/II mismatch cardiac transplantation (B6 --> B10.A; median survival time [MST] 12.5 days vs >70 days). T cells that encountered chemically fixed DCs showed attenuated apoptotic cell death and inactivated phenotypes after allogeneic heterotropic heart transplantation. Furthermore, when DCs from interleukin (IL)-10-/- mice were treated, the in vitro T-cell response was greater than that from IL-12-/- mice. We have suggested that the chemically fixed DCs may mediate peripheral T-cell tolerance, with therapeutic potential for allogeneic transplantation.

Interspecies cloning may be a useful method to help conserve endangered species and to study nuclear-cytoplasm interaction. The present study investigated in vitro development of goral (Naemorhedus goral) intergeneric nuclear transfer embryos produced by fusing goral fibroblasts with enucleated metaphase II (MII) bovine oocytes. After two to five passages, serum-starved or non-starved goral skin fibroblast cells were transferred into enucleated MII bovine oocytes. Couplets were electrically fused and chemically activated, and then cultured in either modified synthetic oviduct fluid (mSOF) or tissue culture medium-199 (TCM-199) supplemented with 10% FBS. Serum starvation of donor cells did not affect the fusion rate and or development to of cells to the two-cell stage, to more than 9-cells, or to morulae, regardless of culture medium. Three blastocysts from 202 fused embryos were obtained when embryos reconstructed with non- serum- starved donor cells were cultured in mSOF. However, no blastocysts were obtained when the embryos reconstructed with serum-starved donor cells were cultured in mSOF. The total cell number of goral intergeneric embryos averaged 130.3 (range 105-180). In conclusion, this study demonstrated that bovine oocytes can support blastocyst development after intergeneric SCNT with goral fibroblasts.

Inositol phosphates are recognized as having diverse and critical roles in biological systems. In this report, kinetic studies and TLC analysis indicate that beta-propeller phytase is a special class of inositol phosphatase that preferentially recognizes a bidentate (P-Ca(2+)-P) formed between Ca(2+) and two adjacent phosphate groups of its natural substrate phytate (InsP(6)). The specific recognition of a bidentate chelation enables the enzyme to sequentially hydrolyze one of the phosphate groups in a bidentate of Ca(2+)-InsP(6) to yield a myo-inositol trisphosphate (InsP(3)) and three phosphates as the final products. A comparative analysis of (1)H- and (13)C NMR spectroscopy with the aid of 2D NMR confirms that the chemical structure of the final product is myo-Ins(2,4,6)P(3). The catalytic properties of the enzyme suggest a potential model for how the enzyme specifically recognizes its substrate Ca(2+)-InsP(6) and produces myo-Ins(2,4,6)P(3) from Ca(2+)-InsP(6). These findings potentially provide evidence for a selective Ca(2+)-InsPs chelation between Ca(2+) and two adjacent phosphate groups of inositol phosphates.

Runx2/Cbfa1/Pebp2aA is a global regulator of osteogenesis and is crucial for regulating the expression of bone-specific genes. Runx2 is a major target of the bone morphogenetic protein (BMP) pathway. Genetic analysis has revealed that Runx2 is degraded through a Smurf-mediated ubiquitination pathway, and its activity is inhibited by HDAC4. Here, we demonstrate the molecular link between Smurf, HDACs and Runx2, in BMP signaling. BMP-2 signaling stimulates p300-mediated Runx2 acetylation, increasing transactivation activity and inhibiting Smurf1-mediated degradation of Runx2. HDAC4 and HDAC5 dea-cetylate Runx2, allowing the protein to undergo Smurf-mediated degradation. Inhibition of HDAC increases Runx2 acetylation, and potentiates BMP-2-stimulated osteoblast differentiation and increases bone formation. These results demonstrate that the level of Runx2 is controlled by a dynamic equilibrium of acetylation, deacetylation, and ubiquitination. These findings have important medical implications because BMPs and Runx2 are of tremendous interest with regard to the development of therapeutic agents against bone diseases.

Background: The immunoprotective nature of the testes has prompted numerous investigations into their supportive roles during allogeneic or xenogeneic cellular grafts. However, the optimal developmental stage of these cells in terms of maximum efficacy for cellular grafts has not been elucidated. In this study, the time-dependent expressions of immune-privilege- and proliferation-related molecules in Sertoli cells were determined., Methods: To investigate the time course of the expression of proteins related to immune privilege and proliferation, immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed using testes of 18 Yorkshire pigs from birth to 20 weeks of age. We included fas ligand (FasL), transforming growth factor beta1 (TGF-beta1) and clusterin as the immune-protective molecules, and follicle stimulating hormone receptor (FSHR), p27Kip1, GATA-4 and Wilm's tumor antigen (WT1) as Sertoli cell proliferation markers. RT-PCR was used to complement histological assessment., Results: The expression of FasL in Sertoli cells gradually increased with age, whereas TGF-beta1 showed the reverse pattern, and clusterin expression showed no age-related difference. p27Kip1 showed a gradual increase in its expression from week 12, but GATA-4 showed earlier postnatal expression from birth to week 12. WT1 expression gradually increased from week 16., Conclusion: We confirmed the age-dependent expression of immune-privilege- and proliferation-related molecules in porcine Sertoli cells. These data may be useful for determining the optimal time for harvesting Sertoli cells with respect to maximal immunoprotection and proliferative activity.

The isoforms of SH2-B, APS, and Lnk form a family of signaling proteins that have been described as activators, mediators, or inhibitors of cytokine and growth factor signaling. We now show that the three alternatively spliced isoforms of human SH2-B readily homodimerize in yeast two-hybrid and cellular transfections assays, and this is mediated specifically by a unique domain in its amino terminus. Consistent with previous reports, we further show that the SH2 domains of SH2-B and APS bind JAK2 at Tyr813. These findings suggested a model in which two molecules of SH2-B or APS homodimerize with their SH2 domains bound to two JAK2 molecules, creating heterotetrameric JAK2-(SH2-B)2-JAK2 or JAK2-(APS)2-JAK2 complexes. We further show that APS and SH2-B isoforms heterodimerize. At lower levels of SH2-B or APS expression, dimerization approximates two JAK2 molecules to induce transactivation. At higher relative concentrations of SH2-B or APS, kinase activation is blocked. SH2-B or APS homodimerization and SH2-B/APS heterodimerization thus provide direct mechanisms for activating and inhibiting JAK2 and other kinases from the inside of the cell and for potentiating or attenuating cytokine and growth factor receptor signaling when ligands are present.