Involvement of IL-10 and Bcl-2 in resistance against an asbestos-induced apoptosis of T cells

To analyze the possibility that immunological alteration in asbestos-related diseases (ARDs) such as asbestosis (ASB) and malignant mesothelioma (MM) may affect the progression of cancers, a human adult T cell leukemia virus-immortalized T cell line (MT-2Org) was continuously exposed to 10 μg/ml of chrysotile-B (CB), an asbestos. After at least 8 months of exposure, the rate of apoptosis in the cells became very low and the resultant subline was designated MT-2Rst. The MT-2Rst cells were characterized by (i) enhanced expression of bcl-2, with regain of apoptosis-sensitivity by reduction of bcl-2 by siRNA, (ii) excess IL-10 secretion and expression, and (iii) activation of STAT3 that was inhibited by PP2, a specific inhibitor of Src family kinases. These results suggested that the contact between cells and asbestos may affect the human immune system and trigger a cascade of biological events such as activation of Src family kinases, enhancement of IL-10 expression, STAT3 activation and Bcl-2 overexpression. This speculation was partially confirmed by the detection of elevated bcl-2 expression levels in CD4 + peripheral blood T cells from patients with MM compared with those from patients with ASB or healthy donors. Further studies will be required to verify the role of T cells with enhanced bcl-2 expression in tumor progression induced by asbestos exposure.     

Genetic evidence for functional dependency of p18Ink4c on Cdk4

The INK4 family of cyclin-dependent kinase (CDK) inhibitors negatively regulates cyclin D-dependent CDK4 and CDK6 and induces the growth-suppressive function of Rb family proteins. Mutations in the Cdk4 gene conferring INK4 resistance are associated with familial and sporadic melanoma in humans and result in a wide spectrum of tumors in mice, suggesting that INK4 is a major regulator of CDK4. Mice lacking the Cdk4 gene exhibit various defects in many organs associated with hypocellularity, whereas loss of the p18(Ink4c) gene results in widespread hyperplasia and organomegaly. To genetically test the notion that the function of INK4 is dependent on CDK4, we generated p18; Cdk4 double-mutant mice and examined the organs and tissues which developed abnormalities when either gene is deleted. We show here that, in all organs we have examined, including pituitary, testis, pancreas, kidney, and adrenal gland, hyperproliferative phenotypes associated with p18 loss were canceled. The double-mutant mice exhibited phenotypes very close to or indistinguishable from that of Cdk4 single-mutant mice. Mice lacking p27(Kip1) develop widespread hyperplasia and organomegaly similar to those developed by p18-deficient mice. The p27; Cdk4 double-mutant mice, however, displayed phenotypes intermediate between those of p27 and Cdk4 single-mutant mice. These results provide genetic evidence that in mice p18(Ink4c) and p27(Kip1) mediate the transduction of different cell growth and proliferation signals to CDK4 and that p18(Ink4c) is functionally dependent on CDK4.     

Structure of the N-terminal domain of PEX1 AAA-ATPase. Characterization of a putative adaptor-binding domain

Peroxisomes are responsible for several pathways in primary metabolism, including beta-oxidation and lipid biosynthesis. PEX1 and PEX6 are hexameric AAA-type ATPases, both of which are indispensable in targeting over 50 peroxisomal resident proteins from the cytosol to the peroxisomes. Although the tandem AAA-ATPase domains in the central region of PEX1 and PEX6 are highly similar, the N-terminal sequences are unique. To better understand the distinct molecular function of these two proteins, we analyzed the unique N-terminal domain (NTD) of PEX1. Extensive computational analysis revealed weak similarity (<10% identity) of PEX1 NTD to the N-terminal domains of other membrane-related type II AAA-ATPases, such as VCP (p97) and NSF. We have determined the crystal structure of mouse PEX1 NTD at 2.05-A resolution, which clearly demonstrated that the domain belongs to the double-psi-barrel fold family found in the other AAA-ATPases. The N-domains of both VCP and NSF are structural neighbors of PEX1 NTD with a 2.7- and 2.1-A root mean square deviation of backbone atoms, respectively. Our findings suggest that the supradomain architecture, which is composed of a single N-terminal domain followed by tandem AAA domains, is a common feature of organellar membrane-associating AAA-ATPases. We propose that PEX1 functions as a protein unfoldase in peroxisomal biogenesis, using its N-terminal putative adaptor-binding domain.     

Discovery of novel (4-piperidinyl)-piperazines as potent and orally active acetyl-CoA carboxylase 1/2 non-selective inhibitors: F-Boc and triF-Boc groups are acid-stable bioisosteres for the Boc group

Novel (4-piperidinyl)-piperazine derivatives were synthesized and evaluated as ACC1/2 non-selective inhibitors. Optimization of the substituents on the nitrogen of the piperidine ring led to the identification of the fluorine substituted tert-butoxycarbonyl group. Advanced analog, 1,1,1-trifluoro-2-methylpropan-2-yl 4-{4-[(2-amino-6-methyl-1-benzothiophen-3-yl)carbonyl]piperazin-1-yl}piperidine-1-carboxylate (12c) showed potent inhibitory activities in enzyme-assay and cell-based assays. Compound 12c also exhibited reduction of hepatic de novo fatty acid synthesis in rats after oral administration.     

Systemic CD8+ T Cell-Mediated Tumoricidal Effects by Intratumoral Treatment of Oncolytic Herpes Simplex Virus with the Agonistic Monoclonal Antibody for Murine Glucocorticoid-Induced Tumor Necrosis Factor Receptor

Oncolytic virotherapy combined with immunomodulators is a novel noninvasive strategy for cancer treatment. In this study, we examined the tumoricidal effects of oncolytic HF10, a naturally occurring mutant of herpes simplex virus type-1, combined with an agonistic DTA-1 monoclonal antibody specific for the glucocorticoid-induced tumor necrosis factor receptor. Two murine tumor models were used to evaluate the therapeutic efficacies of HF10 virotherapy combined with DTA-1. The kinetics and immunological mechanisms of DTA-1 in HF10 infection were examined using flow cytometry and immunohistochemistry. Intratumoral administration of HF10 in combination with DTA-1 at a low dose resulted in a more vigorous attenuation of growth of the untreated contralateral as well as the treated tumors than treatment with either HF10 or DTA-1 alone. An accumulation of CD8⁺ T cells, including tumor- and herpes simplex virus type-1-specific populations, and a decrease in the number of CD4⁺ Foxp3⁺ T regulatory cells were seen in both HF10- and DTA-1-treated tumors. Studies using Fc-digested DTA-1 and Fcγ receptor knockout mice demonstrated the direct participation of DTA-1 in regulatory T cell depletion by antibody-dependent cellular cytotoxicity primarily via macrophages. These results indicated the potential therapeutic efficacy of a glucocorticoid-induced tumor necrosis factor receptor-specific monoclonal antibody in oncolytic virotherapy at local tumor sites.     

Novel metabolic pathway for salicylate biodegradation via phenol in yeast <Emphasis Type="Italic">Trichosporon moniliiforme</Emphasis>

A novel metabolic pathway was found in the yeast Trichosporon moniliiforme WU-0401 for salicylate degradation via phenol as the key intermediate. When 20 mM salicylate was used as the sole carbon source for the growth of strain WU-0401, phenol was detected as a distinct metabolite in the culture broth. Analysis of the products derived from salicylate or phenol through reactions with resting cells and a cell-free extract of strain WU-0401 indicated that salicylate is initially decarboxylated to phenol and then oxidized to catechol, followed by aromatic ring cleavage to form cis-cis muconate.     

Intranasal administration of Lactobacillus rhamnosus GG protects mice from H1N1 influenza virus infection by regulating respiratory immune responses

Aims: To investigate whether intranasal Lactobacillus administration protects host animals from influenza virus (IFV) infection by enhancing respiratory immune responses in a mouse model. Methods and Results: After 3 days of intranasal exposure to Lactobacillus rhamnosus GG (LGG), BALB/c mice were infected with IFV A/PR/8/34 (H1N1). Mice treated with LGG showed a lower frequency of accumulated symptoms and a higher survival rate than control mice (P < 0·05). The YAC‐1 cell‐killing activity of lung cells isolated from mice treated with LGG was significantly greater than those isolated from control mice (P < 0·01). Intranasal administration of LGG significantly increased mRNA expression of interleukin (IL)‐1β, tumour necrosis factor (TNF) and monocyte chemotactic protein (MCP)‐1 (P < 0·01). Conclusions: These results suggest that intranasal administration of LGG protects the host animal from IFV infection by enhancing respiratory cell‐mediated immune responses following up‐regulation of lung natural killer (NK) cell activation. Significance and Impact of Study: We have demonstrated that probiotics might protect host animals from viral infection by stimulating immune responses in the respiratory tract.     

Prosaposin Overexpression following Kainic Acid-Induced Neurotoxicity

Because excessive glutamate release is believed to play a pivotal role in numerous neuropathological disorders, such as ischemia or seizure, we aimed to investigate whether intrinsic prosaposin (PS), a neuroprotective factor when supplied exogenously in vivo or in vitro, is up-regulated after the excitotoxicity induced by kainic acid (KA), a glutamate analog. In the present study, PS immunoreactivity and its mRNA expression in the hippocampal and cortical neurons showed significant increases on day 3 after KA injection, and high PS levels were maintained even after 3 weeks. The increase in PS, but not saposins, detected by immunoblot analysis suggests that the increase in PS-like immunoreactivity after KA injection was not due to an increase in saposins as lysosomal enzymes after neuronal damage, but rather to an increase in PS as a neurotrophic factor to improve neuronal survival. Furthermore, several neurons with slender nuclei inside/outside of the pyramidal layer showed more intense PS mRNA expression than other pyramidal neurons. Based on the results from double immunostaining using anti-PS and anti-GABA antibodies, these neurons were shown to be GABAergic interneurons in the extra- and intra-pyramidal layers. In the cerebral cortex, several large neurons in the V layer showed very intense PS mRNA expression 3 days after KA injection. The choroid plexus showed intense PS mRNA expression even in the normal rat, and the intensity increased significantly after KA injection. The present study indicates that inhibitory interneurons as well as stimulated hippocampal pyramidal and cortical neurons synthesize PS for neuronal survival, and the choroid plexus is highly activated to synthesize PS, which may prevent neurons from excitotoxic neuronal damage. To the best of our knowledge, this is the first study that demonstrates axonal transport and increased production of neurotrophic factor PS after KA injection.