Improving of Catalase Stability Properties by Encapsulation in Alginate/Fe3O4 Magnetic Composite Beads for Enzymatic Removal of H2O2

The aim of this study was enhancing of stability properties of catalase enzyme by encapsulation in alginate/nanomagnetic beads. Amounts of carrier (10–100 mg) and enzyme concentrations (0.25–1.5 mg/mL) were analyzed to optimize immobilization conditions. Also, the optimum temperature (25–50°C), optimum pH (3.0–8.0), kinetic parameters, thermal stability (20–70°C), pH stability (4.0–9.0) operational stability (0–390 min), and reusability were investigated for characterization of the immobilized catalase system. The optimum pH levels of both free and immobilized catalase were 7.0. At the thermal stability studies, the magnetic catalase beads protected 90% activity, while free catalase maintained only 10% activity at 70°C. The thermal profile of magnetic catalase beads was spread over a large area. Similarly, this system indicated the improving of the pH stability. The reusability, which is especially important for industrial applications, was also determined. Thus, the activity analysis was done 50 times in succession. Catalase encapsulated magnetic alginate beads protected 83% activity after 50 cycles.     

Formation of an Antral Follicle-Like Structure by Bovine Cumulus-Oocyte Complexes Embedded with Fragmin/Protamine Microparticles

Fragmin/protamine microparticles (F/P MPs) approximately 0.5-1 µM in diameter were prepared by the simple mixing of fragmin with protamine. This study investigated the effects of F/P MP-containing collagen gels as a hormone carrier on the formation of antral follicle-like structures and on the development of growing bovine oocytes. The supplementation of F/P MPs in collagen gels contributed to the beneficial effects of follicle stimulating hormone (FSH) on the formation and size of antral follicle-like structures. The F/P MPs may serve as potential hormone carriers for the growth of cultured bovine oocytes from early antral follicles.     

SIGN-R1, a C-type lectin,enhances apoptotic cell clearance through the complement deposition pathway by interacting with C1q in the spleen

Complements, such as C1q and C3, and macrophages in the splenic marginal zone (MZMs) play pivotal roles in the efficient uptake and processing of circulating apoptotic cells. SIGN-R1, a C-type lectin that is highly expressed in a subpopulation of MZMs, regulates the complement fixation pathway by interacting with C1q, to fight blood-borne Streptococcus pneumoniae. Therefore, we examined whether the SIGN-R1-mediated classical complement pathway plays a role in apoptotic cell clearance and immune tolerance. SIGN-R1 first-bound apoptotic cells and this binding was significantly enhanced in the presence of C1q. SIGN-R1–C1q complex then immediately mediated C3 deposition on circulating apoptotic cells in the MZ, leading to the efficient clearance of them. SIGN-R1-mediated C3 deposition was completely abolished in the spleen of SIGN-R1 knockout (KO) mice. Given that SIGN-R1 is not expressed in the liver, we were struck by the finding that C3-deposited apoptotic cells were still found in the liver of wild-type mice, and dramatically reduced in the SIGN-R1 KO liver. In particular, SIGN-R1 deficiency caused delayed clearance of apoptotic cells and aberrant secretion of cytokines, such as TNF-α, IL-6, and TGF-β in the spleen as well as in the liver. In addition, anti-double- and single-stranded DNA antibody level was significantly increased in SIGN-R1-depleted mice compared with control mice. These findings suggest a novel mechanism of apoptotic cell clearance which is initiated by SIGN-R1 in the MZ and identify an integrated role of SIGN-R1 in the systemic clearance of apoptotic cells, linking the recognition of apoptotic cells, the opsonization of complements, and the induction of immune tolerance.     

Human HOXA5 homeodomain enhances protein transduction and its application to vascular inflammation

Cellular protein delivery is an emerging technique by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an Escherichia coli expression vector including human specific gene sequences for protein cellular delivery. The plasmid was generated by ligation the nucleotides 770–817 of the homeobox A5 mRNA sequence which was matched with protein transduction domain (PTD) of homeodomain protein A5 (HOXA5) into pET expression vector. The cellular uptake of HOXA5-PTD-EGFP was detected in 1 min and its transduction reached a maximum at 1 h within cell lysates. The cellular uptake of HOXA5-EGFP at 37 °C was greater than in 4 °C. For study for the functional role of human HOXA5-PTD, we purified HOXA5-APE1/Ref-1 and applied it on monocyte adhesion. Pretreatment with HOXA5-APE1/Ref-1 (100 nM) inhibited TNF-α-induced monocyte adhesion to endothelial cells, compared with HOXA5-EGFP. Taken together, our data suggested that human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins.     

Protective potential of resveratrol against oxidative stress and apoptosis in Batten disease lymphoblast cells

Batten disease (BD) is the most common form of a group of disorders called neuronal ceroid lipofuscinosis, which are caused by a CLN3 gene mutation. A variety of pathogenic lysosomal storage disorder mechanisms have been suggested such as oxidative stress, endoplasmic reticulum (ER) stress, and altered protein trafficking. Resveratrol, a stilbenoid found in red grape skin, is a potent antioxidant chemical. Recent studies have suggested that resveratrol may have a curative effect in many neurodegenerative diseases. Therefore, we investigated the activities of resveratrol at the levels of oxidative and ER stress and apoptosis factors using normal and BD lymphoblast cells. We report that the BD lymphoblast cells contained low-levels of superoxide dismutase-1 (SOD-1) due to the long-term stress of reactive oxygen species. However, when we treated the cells with resveratrol, SOD-1 increased to levels observed in normal cells. Furthermore, we investigated the expression of glucose-regulated protein 78 as an ER stress marker. BD cells underwent ER stress, but resveratrol treatment resolved the ER stress in a dose-dependent manner. We further demonstrated that the levels of apoptosis markers such as apoptosis induce factor, cytochrome c, and cleavage of poly (ADP)-ribose polymerase decreased following resveratrol treatment. Thus, we propose that resveratrol may have beneficial effects in patients with BD.     

RASSF1A suppresses the activated K-Ras-induced oxidative DNA damage

Dendritic cells are the most potent antigen presenting cells responsible for the development of immune responses in cancer. However, it is known that the function of dendritic cells in tumor-bearing hosts is severely compromised. Our previous studies demonstrated that the defects in dendritic cell function are due, to a large extent, to the accumulation of high amounts of lipids – predominantly triglycerides – in a substantial proportion of dendritic cells in tumor-bearing mice and patients with cancer. The dendritic cells accumulation of lipids is likely associated with their up-regulation of a scavenger receptor A. This receptor is primarily responsible for uptake of modified lipids. Here, by using different versions of liquid chromatography–mass spectrometry, we identified several molecular species of oxygenated lipids in plasma of tumor-bearing animals that may be responsible for their uptake and accumulation by dendritic cells via scavenger receptor A-dependent pathway – the effect that may be associated with the loss of dendritic cell’s immune surveillance function in cancer.     

Anatomical and nutlet differentiation between <Emphasis Type="Italic">Teucrium montanum</Emphasis> and <Emphasis Type="Italic">T. polium</Emphasis> from Turkey

Teucrium montanum L. and T. polium L. are the two closest Teucrium L. species from sect. Polium (Mill.) Schreb in Turkey. In addition, they are sympatric for some part of their range in Turkey. In this study, comparative anatomical and micromorphological studies of the two species are carried out. They have been investigated by their leaf and stem anatomical features, as well as nutlet micromorphological characteristics. The results of anatomical studies show that the anatomical characters of both taxa are observed to be similar to the general features of the family Lamiaceae anatomy, except for lacking rich collenchyma at the corners. Both taxa are similar in stem anatomy and their leaves exhibit xeromorphy. However, trichome morphology on the stems and the leaves appear to have a taxonomic value in segregation of the two taxa. Light and scanning electron microscope studies on the nutlets also show that nutlet shapes, measures and surface micromorphologies are different in the two species. Whereas nutlet surfaces are bireticulate in both species, the nutlets are larger and primary sculpturing is more distinct in T. polium than in T. montanum. Moreover, the nutlets are oblong to oblong-ovoid and larger in T. polium, but ovoid and smaller in T. montanum.     

Denitrosylation of S-nitrosylated OGT is triggered in LPS-stimulated innate immune response

Rad is a member of a subclass of small GTP-binding proteins, the RGK family. In the present study we investigated the role of Rad protein in regulating cardiomyocyte viability. DNA fragmentation and TUNEL assays demonstrated that Rad promoted rat neonatal cardiomyocyte apoptosis. Rad silencing fully blocked serum deprivation induced apoptosis, indicating Rad is necessary for trigger cardiomyocyte apoptosis. Rad overexpression caused a dramatic decrease of the anti-apoptotic molecule Bcl-x_L, whereas Bcl-x_L overexpression protected cardiomyocytes against Rad-induced apoptosis. Rad-triggered apoptosis was mediated by the activation of p38 MAPK. The p38 blocker SB203580 effectively protected cardiomyocytes against Rad-evoked apoptosis.     

Comparative analysis of the role of small G proteins in cell migration and cell death: cytoprotective and promigratory effects of RalA

Small G protein superfamily consists of more than 150 members, and is classified into six families: the Ras, Rho, Rab, Arf, Ran, and RGK families. They regulate a wide variety of cell functions such as cell proliferation/differentiation, cytoskeletal reorganization, vesicle trafficking, nucleocytoplasmic transport and microtubule organization. The small G proteins have also been shown to regulate cell death/survival and cell shape. In this study, we compared the role of representative members of the six families of small G proteins in cell migration and cell death/survival, two cellular phenotypes that are associated with inflammation, tumorigenesis, and metastasis. Our results show that small G proteins of the six families differentially regulate cell death and cell cycle distribution. In particular, our results indicate that Rho family of small G proteins is antiapoptotic. Ras, Rho, and Ran families promoted cell migration. There was no significant correlation between the cell death- and cell migration-regulating activities of the small G proteins. Nevertheless, RalA was not only cytoprotective against multiple chemotherapeutic drugs, but also promigratory inducing stress fiber formation, which was accompanied by the activation of Akt and Erk pathways. Our study provides a framework for further systematic investigation of small G proteins in the perspectives of cell death/survival and motility in inflammation and cancer.     

Increase in CIP2A expression is associated with doxorubicin resistance

The cancerous inhibitor of protein phosphatase 2A (CIP2A) increases the migration and metastasis of various cancer cells. Overexpression of CIP2A has been shown to increase the proliferation of MDA-MB-231 cells. We thus assessed whether CIP2A expression is associated with sensitivity to doxorubicin. MDA-MB-231 cells showed an increase in CIP2A expression after treatment with doxorubicin, while MCF-7 cells showed a decrease in CIP2A expression. The overexpression of CIP2A in MCF-7 cells overcame the inhibition of cell proliferation in response to doxorubicin treatment. CIP2A expression was not affected by wild-type or mutant p53. However, mutant p53 blocked doxorubicin-mediated CIP2A down-regulation in HCT116 cells. As a regulation mechanism of doxorubicin-mediated CIP2A expression, we showed that phosphorylated Akt was involved in the suppression of CIP2A expression.