Decoy receptor 3 suppresses TLR2-mediated B cell activation by targeting NF-κB

Decoy receptor 3 (DcR3) is a soluble protein in the TNFR superfamily. Its known ligands include Fas ligand, homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, TNF-like molecule 1A, and heparan sulfate proteoglycans. DcR3 has been reported to modulate the functions of T cells, dendritic cells, and macrophages; however, its role in regulating B cell activation is largely unknown. In this study, we found that the DcR3.Fc fusion protein bound to human and mouse B cells and suppressed the activation of B cells. DcR3.Fc attenuated Staphylococcus aureus, IgM-, Pam(3)CSK(4)-, and LPS-mediated B cell proliferation but did not affect cytokine-induced B cell growth. In the presence of these mitogens, DcR3.Fc did not induce B cell apoptosis, suggesting that DcR3 may inhibit the signal(s) important for B cell activation. Because the combination of Fas.Fc, LT-βR.Fc (homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes receptor), and DR3.Fc (TNF-like molecule 1A receptor) did not suppress B cell proliferation and because the biological effect of DcR3.Fc on B cells was not blocked by heparin, we hypothesize that a novel ligand(s) of DcR3 mediates its inhibitory activity on B cells. Moreover, we found that TLR2-stimulated NF-κB p65 activation and NF-κB-driven luciferase activity were attenuated by DcR3.Fc. The TLR2-induced cytokine production by B cells was consistently reduced by DcR3. These results imply that DcR3 may regulate B cell activation by suppressing the activation of NF-κB.     

Ha-ras overexpression mediated cell apoptosis in the presence of 5-fluorouracil

By using a mouse NIH3T3 derivate designed 7-4 harboring the inducible Ha-ras oncogene, we demonstrated the close relationship between Ha-ras expression level and sensitization of 5-flurouracil (5-FU)-treated cells. Further studies revealed that the cells susceptible to 5-FU treatment died of apoptosis, which was demonstrated by caspase-3 activation, loss of mitochondria membrane potential (MMP), and DNA fragmentation. The 7-4 cells coexpressing dominant negative Ras (Ras(Asn17)), dominant negative Raf-1 (Raf-1(CB4)), Bcl-2, or active form of phosphatidylinositol 3-kinase (PI3K) became resistant to 5-FU, and apoptosis was prevented. In contrast, the cells coexpressing dominant negative Rac 1 (Rac1(Asn17)) or dominant negative Rho A (RhoA(Asn19)) showed no change of sensitivity to 5-FU. These results indicate that Ras, Bcl-2, as well as Raf-1 and PI3K pathways play pivotal roles in 5-FU-induced apoptosis under Ha-ras-overexpressed condition. Aberrant levels of cyclin E and p21(Cip/WAF-1) expression as well as Cdc 2 phosphorylation at Tyrosine 15 suggest that perturbation of G1/S and G2/M transitions in cell cycle might be responsible for 5-FU triggered apoptosis. Sensitization of Ha-ras-related cells to 5-FU was also demonstrated in human bladder cancer cells. Through understanding the mechanism of 5-FU induced apoptosis in tumor cells, a new direction toward the treatment of Ha-ras oncogene-related cancers with 5-FU at more optimal dosages is possible and combinational therapy with other drugs that suppress PI3K and Bcl-2 activities can also be considered.     

Cdh1 Regulates Cell Cycle through Modulating the Claspin/Chk1 and the Rb/E2F1 Pathways

APC/Cdh1 is a major cell cycle regulator and its function has been implicated in DNA damage repair; however, its exact role remains unclear. Using affinity purification coupled with mass spectrometry, we identified Claspin as a novel Cdh1-interacting protein and further demonstrated that Claspin is a novel Cdh1 ubiquitin substrate. As a result, inactivation of Cdh1 leads to activation of the Claspin/Chk1 pathway. Previously, we demonstrated that Rb interacts with Cdh1 to influence its ability to degrade Skp2. Here, we report that Cdh1 reciprocally regulates the Rb pathway through competing with E2F1 to bind the hypophosphorylated form of Rb. Although inactivation of Cdh1 in HeLa cells, with defective p53/Rb pathways, led to premature S phase entry, acute depletion of Cdh1 in primary human fibroblasts resulted in premature senescence. Acute loss of many other major tumor suppressors, including PTEN and VHL, also induces premature senescence in a p53- or Rb-dependent manner. Similarly, we showed that inactivation of the p53/Rb pathways by overexpression of SV40 LT-antigen partially reversed Cdh1 depletion–induced growth arrest. Therefore, loss of Cdh1 is only beneficial to cells with abnormal p53 and Rb pathways, which helps explain why Cdh1 loss is not frequently found in many tumors.     

Production and Purification of d-Aminoacylase from Alcaligenes denitrificans and Taxonomic Study of the Strain

A d-aminoacylase-producing microorganism, strain DA181, isolated from soil was identified as Alcaligenes denitrificans subsp. denitrificans. This strain produced about 29,300 units (micromoles of product formed per hour) of d-aminoacylase and 2,300 units of l-aminoacylase per gram of cells (wet weight) when cultivated in a medium containing 1% N-acetyl-dl-leucine as the carbon source. The d-aminoacylase was purified 345-fold. The specific activity of the purified enzyme was 108,600 units per mg of protein when N-acetyl-d-methionine was used as a substrate. The apparent molecular weight was 58,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-Acetyl-d-methionine was the favored substrate, followed by N-acetyl-d-phenylalanine. This enzyme had a high stereospecificity, and its hydrolysis of N-acetyl-l-amino acids was almost negligible.     

Focused Ultrasound-Induced Blood–Brain Barrier Opening to Enhance Temozolomide Delivery for Glioblastoma Treatment: A Preclinical Study

The purpose of this study is to assess the preclinical therapeutic efficacy of magnetic resonance imaging (MRI)-monitored focused ultrasound (FUS)-induced blood-brain barrier (BBB) disruption to enhance Temozolomide (TMZ) delivery for improving Glioblastoma Multiforme (GBM) treatment. MRI-monitored FUS with microbubbles was used to transcranially disrupt the BBB in brains of Fisher rats implanted with 9L glioma cells. FUS-BBB opening was spectrophotometrically determined by leakage of dyes into the brain, and TMZ was quantitated in cerebrospinal fluid (CSF) and plasma by LC-MS\MS. The effects of treatment on tumor progression (by MRI), animal survival and brain tissue histology were investigated. Results demonstrated that FUS-BBB opening increased the local accumulation of dyes in brain parenchyma by 3.8-/2.1-fold in normal/tumor tissues. Compared to TMZ alone, combined FUS treatment increased the TMZ CSF/plasma ratio from 22.7% to 38.6%, reduced the 7-day tumor progression ratio from 24.03 to 5.06, and extended the median survival from 20 to 23 days. In conclusion, this study provided preclinical evidence that FUS BBB-opening increased the local concentration of TMZ to improve the control of tumor progression and animal survival, suggesting its clinical potential for improving current brain tumor treatment.     

Effects of butylated hydroxyanisole,butylated hydroxytoluene and tertiary butylhydroquinone on growth and luteoskyrin production byPenicillium islandicum

Butylated hydroxyanisole (BHA), Butylated hydroxytoluene (BHT) and tertiary butylhydroquinone (TBHQ) alone in cultural media were tested for the inhibition of growth and luteoskyrin production by two toxigenic strains ofPenicillium islandicum UST-11 andP. islandicum HLT-6. In potato dextrose agar, the concentrations of BHA and TBHQ from 0.2 mg/disc, BHT from 5.0 mg/disc did affect the growth of both tested strains, but the initial concentrations of these antioxidants to reduced luteoskyrin production by UST-11 strain were BHA 0.5 mg/disc, BHT 1.0 mg/disc and TBHQ 0.4 mg/disc, while for HLT-6, BHA 0.4 mg/disc, BHT and TBHQ were 0.2 mg/disc, respectively. In grainy and powdery rice media, the effects of BHA, BHT and TBHQ on luteoskyrin production byP. islandicum UST-11 and HLT-6 were clearly demonstrated. The efficiency of the inhibitory effect was not only closely related to the concentration of antioxidants, but also completely inhibited the luteoskyrin production at a concentration of 200 mg/kg or higher. Also, the antioxidants at a concentration higher than 20 mg/kg reduced significantly the growth and luteoskyrin production by both strains ofP. islandicum.     

Sorting nexin 17 prevents lysosomal degradation of β1 integrins by binding to the β1-integrin tail

Integrin functions are controlled by regulating their affinity for ligand, and by the efficient recycling of intact integrins through endosomes. Here we demonstrate that the Kindlin-binding site in the β1-integrin cytoplasmic domain serves as a molecular switch enabling the sequential binding of two FERM-domain-containing proteins in different cellular compartments. When β1 integrins are at the plasma membrane, Kindlins control ligand-binding affinity. However, when they are internalized, Kindlins dissociate from integrins and sorting nexin 17 (SNX17) is recruited to free β1-integrin tails in early endosomes to prevent β1-integrin degradation, leading to their recycling back to the cell surface. Our results identify SNX17 as a β1-integrin-tail-binding protein that interacts with the free Kindlin-binding site in endosomes to stabilize β1 integrins, resulting in their recycling to the cell surface where they can be reused.     

Characterization of alpha-actinin from Acanthamoeba

Characterization of a protein from Acanthamoeba that was originally called gelation protein [T.D. Pollard, J. Biol. Chem. 256:7666-7670, 1981] has shown that it resembles the actin filament cross-linking protein, alpha-actinin, found in other cells. It comprises about 1.5% of the total amoeba protein and can be purified by chromatography with a yield of 13%. The native protein has a molecular weight of 180,000 and consists of two polypeptides of 90,000 Da. The Stokes' radius is 8.5 nm, the intrinsic viscosity is 0.35 dl/dm, and the extinction coefficient at 280 mm is 1.8 X 10(5)M-1 X cm-1. Electron micrographs of shadowed specimens show that the molecule is a rod 48 nm long and 7 nm wide with globular domains at both ends and in the middle of the shaft. On gel electrophoresis in sodium dodecylsulfate the pure protein can run as bands with apparent molecular weights of 60,000, 90,000, 95,000, or 134,000 depending on the method of sample preparation. Rabbit antibodies to electrophoretically purified Acanthamoeba alpha-actinin polypeptides react with all of these electrophoretic variants in samples of purified protein and cell extracts. By indirect fluorescent antibody staining of fixed amoebas, alpha-actinin is distributed throughout the cytoplasmic matrix and concentrated in the hyaline cytoplasm of the cortex. The protein cross-links actin filaments in the presence and absence of Ca++. It inhibits slightly the time course of the spontaneous polymerization of actin monomers but has no effect on the critical concentration for actin polymerization even though it increases the apparent rate of elongation to a small extent. Like some other cross-linking proteins, amoeba alpha-actinin inhibits the actin-activated ATPase of muscle myosin subfragment-1. Although Acanthamoeba alpha-actinin resembles the alpha-actinin from other cells in shape and ability to cross-link actin filaments, antibodies to amoeba and smooth muscle alpha-actinins do not cross react and there are substantial differences in the amino acid compositions and molecular dimensions.