Synthesis and properties of bifunctional chloroalkyl nitrosamines with an intercalating moiety

Three N-nitroso-N-(arylcarbonyloxymethyl)-3-chloropropylamines were synthesized, and their chemical and biological properties were studied. All arylcarboxylates intercalated with double-stranded DNA, and their mutagenicity and DNA cross-linking activity were affected by their ring structure. The DNA interstrand cross-link formation increased dose dependently after treatment with the acridine analog. The anthraquinone analog showed the highest bacterial mutagenicity among the three nitrosamines in Salmonella typhimurium TA100, while in Salmonella typhimurium TA92, which can detect cross-linking agents, the acridine analog showed the highest mutagenicity. This agreed with the result of a cross-linking assay. These results suggest that the three-ring aromatic moiety gives DNA-intercalating ability to cross-linkable chloropropyl nitrosamine, and the acridine analog is considered as a possible new antitumor lead compound.     

Regulation mode of evaporative cooling underlying a strategy of the heat-tolerant FOK rat for enduring ambient heat

Compared with other rat strains, the inbred FOK rat is extremely heat tolerant. This increased heat tolerance is due largely to the animal's enhanced saliva spreading abilities. The aims of the present study were to 1) quantify the heat tolerance capacity of FOK rats and 2) determine the regulatory mode of the enhanced salivary cooling in these animals. Various strains of rats were acutely exposed to heat. In the heat-intolerant strains, saliva spreading was insufficient and the core temperature (Tc) rose rapidly. In contrast, FOK rats maintained an elevated Tc plateau (39.5 +/- 0.7 degrees C) for 5-6 h over a wide range of ambient temperatures (Ta) (37.5-42.5 degrees C). In hot environments the FOK rats secreted copious amounts of saliva and spread it over more than the entire ventral body surface. FOK rats had a low Tc threshold for salivation, and the salivation rate increased linearly in proportion to the Tc deviation from the threshold. No strain difference or temperature effect was observed in the saliva secretion rate from in vitro submandibular glands perfused by sufficient doses of ACh. These results suggest that 1) the ability of FOK rats to maintain a moderate steady-state hyperthermia (39.5 +/- 0.7 degrees C) over a wide Ta range is enabled by a lowered threshold Tc for salivation and functional negative-feedback control of saliva secretion and 2) strain differences in ability to endure heat stress are mainly attributable to changes in the thermoregulatory control system rather than altered secretory abilities of the salivary glands.     

Nicotine enhances neovascularization and promotes tumor growth

Solid tumors require vascularization for their growth. Bone marrow-derived endothelial progenitor cells participate in tumor angiogenesis. Here, we show that nicotine markedly accelerated growth of colon cancer cells inoculated subcutaneously in mice but had no effect on proliferation of carcinoma cells in vitro. We found that the tumor growth was associated with increased vascularization of the tumor and that bone marrow-derived cells contributed to the formation of the new blood vessels. Our findings show that nicotine promotes tumor growth, at least in part, by stimulating tumor-associated neovascularization.     

AIP is a mitochondrial import mediator that binds to both import receptor Tom20 and preproteins

Most mitochondrial preproteins are maintained in a loosely folded import-competent conformation by cytosolic chaperones, and are imported into mitochondria by translocator complexes containing a preprotein receptor, termed translocase of the outer membrane of mitochondria (Tom) 20. Using two-hybrid screening, we identified arylhydrocarbon receptor-interacting protein (AIP), an FK506-binding protein homologue, interacting with Tom20. The extreme COOH-terminal acidic segment of Tom20 was required for interaction with tetratricopeptide repeats of AIP. An in vitro import assay indicated that AIP prevents preornithine transcarbamylase from the loss of import competency. In cultured cells, overexpression of AIP enhanced preornithine transcarbamylase import, and depletion of AIP by RNA interference impaired the import. An in vitro binding assay revealed that AIP specifically binds to mitochondrial preproteins. Formation of a ternary complex of Tom20, AIP, and preprotein was observed. Hsc70 was also found to bind to AIP. An aggregation suppression assay indicated that AIP has a chaperone-like activity to prevent substrate proteins from aggregation. These results suggest that AIP functions as a cytosolic factor that mediates preprotein import into mitochondria.     

Akt is an endogenous inhibitor toward tumor necrosis factor-related apoptosis inducing ligand-mediated apoptosis in rheumatoid synovial cells

Akt is known to be activated in the rheumatoid synovial tissues. We examined here functional role of Akt during tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in rheumatoid synovial cells. Rheumatoid synovial cells in vitro were rapidly committed to apoptosis in response to TRAIL in mitochondria-dependent manner whereas Akt and extracellular signal-regulated kinase (ERK) were also phosphorylated. TRAIL-mediated apoptosis in synovial cells was significantly increased through inactivation of Akt by LY294002, however, that process was not so changed by adding ERK inhibitor, PD98059. Platelet-derived growth factor (PDGF) clearly phosphorylated both Akt and ERK in synovial cells, and PDGF pretreatment markedly suppressed TRAIL-mediated synovial cell apoptosis. The use of not PD98059 but LY294002 abrogated PDGF-mediated inhibitory effect toward TRAIL-induced apoptosis in synovial cells. The above protective effect of Akt was confirmed by the use of short interfering RNA (siRNA)-directed inhibition of Akt. Our data suggest that Akt is an endogenous inhibitor during TRAIL-mediated synovial cell apoptotic pathway, which may explain that synovial cells in situ of the rheumatoid synovial tissues are resistant toward apoptotic cell death in spite of death receptor expression.     

A G protein-coupled receptor responsive to bile acids

So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5'-O-3-thio-triphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids.     

Abnormal activation of glial cells in the brains of prion protein-deficient mice ectopically expressing prion protein-like protein, PrPLP/Dpl

BACKGROUND: Some lines of mice homozygous for a disrupted prion protein gene (Prnp), including Ngsk Prnp(0/0) mice, exhibit Purkinje cell degeneration as a consequence of the ectopic overexpression of the downstream gene for prion protein-like protein (PrPLP/Dpl) in the brain, but others, such as Zrch I Prnp(0/0) mice, show neither the neurodegeneration nor the expression of PrPLP/Dpl. In the present study, we found that Ngsk Prnp(0/0), but not Zrch I Prnp(0/0) mice, developed gliosis involving both astrocytes and microglia in the brain. MATERIALS AND METHODS: The brains from wild-type (Prnp(+/+)), Ngsk Prnp(0/0), Zrch I Prnp(0/0), and reconstituted Ngsk Prnp(0/0) mice carrying a mouse PrP transgene, designated Tg(P) Ngsk Prnp(0/0) mice, were subjected into Northern blotting and in situ hybridization using probes of glial fibrillary acidic protein (GFAP) and lysozyme M (LM) specific for astrocytes and microglia, respectively. Immunohistochemistry was also performed on the brain sections using anti-GFAP and anti-F4/80 antibodies. RESULTS: Northern blotting demonstrated upregulated expression of the genes for GFAP and LM in the brains of Ngsk Prnp(0/0), but not in Zrch I Prnp(0/0) mice. A transgene for normal mouse PrP(C) successfully rescued Ngsk Prnp(0/0) mice from the glial activation. In situ hybridization and immunohistochemistry revealed activated astrocytes and microglia mainly in the white matter of both the forebrains and cerebella. In contrast, there was no evidence of neuronal injury except for the Purkinje cell degeneration. Moreover, the glial cell activation was notable well before the onset of the Purkinje cell degeneration. CONCLUSIONS: These findings strongly suggest that ectopic PrPLP/Dpl in the absence of PrP(C) is actively involved in the glial-cell activation in the brain.     

Stimulation of the secretion of pro-inflammatory cytokines by Bifidobacterium strains

To characterize the ability of bifidobacteria to affect the production of macrophage-derived cytokines, a murine macrophage-like cell line, J774.1, was cultured in the presence of 27 strains of heat-inactivated bifidobacteria. Bifidobacterium adolescentis and B. longum, known as adult-type bifidobacteria, induced significantly more pro-inflammatory cytokine secretion, IL-12 and TNF-alpha, by J774.1 cells, than did the infant-type bifidobacteria, B. bifidum, B. breve, and B. infantis (P<0.01). In contrast, B. adolescentis did not stimulate the production of anti-inflammatory IL-10 from J774.1 cells as the other tested bacteria did. The results suggest that the adult-type bifidobacteria, especially B. adolescentis, may be more potent to amplify but less able to down-regulate the inflammatory response.