Shigenobu Tone

IMMOBILIZATION STRESS REDUCED THE EXPRESSION OF NEUROTROPHINS AND THEIR RECEPTORS IN THE RAT BRAIN

Exposure to stressful events and elevated level of stress hormones are associated with impaired spatial memory and neuronal damage in the hippocampus. These neurons are considered to be maintained by neurotrophins such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) and trk family of neurotrophin receptors. Male Wistar rats (6 weeks old) were exposed to immobilization stress for 8 h and their brains were processed for in situ hybridization histochemistry. Exposure to long-lasting immobilization stress reduced mRNA levels for neurotrophins and their high affinity receptors in the brain, especially in the hippocampus. Our results provide, some new information that may be relevant to the pathogenesis of stress-induced disturbances of memory and learning.

Lipopolysaccharide induction of indoleamine 2,3‐dioxygenase is mediated dominantly by an IFN‐γ‐independent mechanism

Indoleamine 2,3‐dioxygenase (IDO) is a rate‐limiting enzyme in the L‐tryptophan‐kynurenine pathway, which converts an essential amino acid, L‐tryptophan, to N‐formylkynurenine. It has been speculated that IFN‐γ is a dominant IDO inducer in vivo. The present study used IFN‐γ or TNF‐α gene‐disrupted mice and IFN‐γ antibody‐treated mice to demonstrate that lipopolysaccharide (LPS)‐induced systemic IDO is largely dependent on TNF‐α rather than IFN‐γ. IFN‐γ‐independent IDO induction was also demonstrated in vitro with LPS‐stimulated monocytic THP‐1 cells. These findings clearly indicate that there is an IFN‐γ‐independent mechanism of IDO induction in addition to the IFN‐γ‐dependent mechanism.

Expression of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase in early concepti

Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan degradation in the placenta has been implicated in the prevention of the allogeneic fetus rejection [Munn, Zhou, Attwood, Bondarev, Conway, Marshall, Brown, and Mellor (1998) Science 281, 1191-1193]. To determine how IDO is associated with the development of the fetus and placenta, the time course of IDO expression (tryptophan-degrading activity, IDO protein and IDO mRNA) in the embryonic and extra-embryonic tissues as well as maternal tissues of mice was examined. A high tryptophan-degrading activity was detected in early concepti on days 6.5 and 7.5, whereas IDO protein and its mRNA were not expressed during early gestation, but appeared 2-3 days later, lasted for about 3 days and declined rapidly thereafter. The expression of IDO basically coincided with the formation of the placenta. On the contrary, the early tryptophan-degrading activity was due to gene expression of tryptophan 2,3-dioxygenase (TDO), as shown by Northern and Western analysis. These findings indicate that IDO is transiently expressed in the placenta but that the expression does not last until birth, and that the IDO expression is preceded by expression of another tryptophan-degrading enzyme, TDO, in the maternal and/or embryonic tissues in early concepti.

Inhibition of Cytochrome c Release in Fas-mediated Signaling Pathway in Transgenic Mice Induced to Express Hepatitis C Viral Proteins

Persistent hepatitis C virus (HCV) infection often progresses to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Numerous viruses have been reported to escape from apoptotic mechanism to maintain persistent infection. In the present study, we characterized the effect of HCV proteins on the Fas signal using HCV transgenic mice, which expressed core, E1, E2, and NS2 proteins, regulated by the Cre/loxP switching system. The transgene expression of HCV transgenic mice caused resistance to Fas antibody stimulated lethality. Apoptotic cell death in the liver of HCV protein expressing mice was significantly reduced compared with nonexpressing mice. Histopathological analysis and DNA fragmentation analysis revealed that the HCV proteins suppressed Fas-mediated apoptotic cell death. To identify the target pathway of HCV proteins, we characterized caspase activity. The activation of caspase-9 and -3/7 but not caspase-8 was inhibited by HCV proteins. Cytochromec release from mitochondria was inhibited in HCV protein expressing mice. These results indicated that the expression of HCV proteins may directly or indirectly inhibit Fas-mediated apoptosis and death in mice by repressing the release of cytochrome cfrom mitochondria, thereby suppressing caspase-9 and -3/7 activation. These results suggest that HCV may cause persistent infection, as a result of suppression of Fas-mediated cell death.

Immunoactivative role of indoleamine 2,3-dioxygenase in human hepatocellular carcinoma.

Background:  Indoleamine 2,3‐dioxygenase (IDO) is a tryptophan catabolic enzyme. Recent studies have focused on the immunoregulatory role of IDO in mononuclear cells. The role of IDO in hepatocellular carcinoma (HCC) cell lines and HCC patients was examined.

Detection of DNA cleavage in apoptotic cells.

At least two discrete deoxyribonuclease activities can be detected during apoptotic death, one that generates 30- to 500-kilobase pair (kbp) domain-sized fragments and another that mediates internucleosomal DNA degradation. The latter nuclease has been identified as the caspase-activated deoxyribonuclease (CAD)/CPAN, a unique enzyme that is normally inhibited by the regulatory subunit ICAD (inhibitor of CAD)/DFF45 (DNA fragmentation factor). In this chapter, techniques widely used to detect DNA cleavage in apoptotic cells, including pulsed-field gel electrophoresis, conventional agarose gel electrophoresis, and terminal transferase-mediated dUTP nick end-labeling (TUNEL), are briefly reviewed. In addition, the use of ICAD to inhibit apoptosis-associated nuclease activity is illustrated. When properly applied, these techniques are widely applicable to the characterization of apoptotic cells.

Quantitative and spatial differences in the expression of tryptophan-metabolizing enzymes in mouse epididymis

Previous reports have suggested that indoleamine 2,3-dioxygenase (IDO) activity is particularly important in mouse epididymis tissue. We show here, using reverse transcription/polymerase chain reaction assays, Northern assays, Western blotting experiments, and immunohistochemistry that IDO is indeed highly expressed in mouse epididymis, and that IDO mRNA distribution and protein location are precisely regionalized within the organ and within sub-territories of the proximal part of the epididymal duct, the so-called caput epididymidis. Within the caput epididymidis, both the principal and the apical cells have been shown to express IDO. On the contrary, tryptophan dioxygenase (TDO), a sister enzyme of IDO, is weakly and uniformly expressed in mouse epididymis and, in contrast to IDO, is also expressed in testis. In the epididymis, TDO protein expression has been found in a totally different cell type in the smooth muscle layer surrounding the epididymal tubules. Finally, IDO is not secreted into the epididymal lumen, whereas the testis-expressed TDO is present on the head of spermatozoa retrieved from the cauda epididymidis. On the basis of the various functions that have been associated with IDO/TDO, we discuss the putative impacts of IDO/TDO expression on the physiology of mammalian epididymis and spermatozoa.

The Hsp70 homolog gene, Hsc70t, is expressed under translational control during mouse spermiogenesis

Hsc70t is a member of the Hsp70 family of genes and is constitutively expressed after meiosis in mouse spermatogenesis. Immunohistochemistry and in situ hybridization techniques were used to examine the precise localization of the Hsc70t product during the various stages of spermatogenesis. A rabbit antiserum raised against the mouse Hsc70t‐lacZ fusion protein detected the Hsc70t protein in the late spermatid‐enriched fraction after two‐dimensional Western blot analyses. On histological sections, the protein appears in the cytoplasm of spermatids as they progress from step 9 to the final step of spermatogenesis. An antisense RNA probe generated from the 3′ untranslated region of Hsc70t cDNA detected Hsc70t mRNA in late round spermatids from step 7 onward with the signal disappearing in spermatids at step 15. Thus, Hsc70t mRNA first appears after meiosis in haploid cells but is not translated effectively until these cells progress to the transcriptionally inactive stage which coincides with chromatin condensation. These results establish that the synthesis of Hsc70t protein is under strict translational control. Mol. Reprod. Dev. 52:383–391, 1999.

Heat-induced apoptosis and p53 in cultured mammalian cells

Heat-induced apoptosis was studied in M10 and MOLT-4 cells by determining nuclear morphological changes, decrease in cell size, DNA degradation into fragments of about 30 kbp, and the appearance of internucleosomal DNA fragments (DNA ladders). Morphological changes in the nucleus were detected within 30 min after heat-treatment at 44 degrees C in M10 cells, but much later (> 5 h) in MOLT-4 cells. In M10 cells, 30 kbp-DNA fragments were observed even at the end of the heat-treatment and decreased 10 min later, while the DNA ladder increased at 10-30 min after heat treatment. DNA fragments of 30 kbp appeared in MOLT-4 cells at 1 h after the heat-treatment and apparently accumulated for up to 24 h. Heat-treatment increased p53 protein in MOLT-4 cells but not in M10 cells. Analysis of the DNA sequence of the p53 gene revealed that M10 cells have a heterozygous mutation in codon 173 of exon 5. These results suggest that apoptosis is induced by hyperthermia in a cell-line dependent manner, that the formation of 30 kbp-DNA fragments is a very early event in apoptosis, that DNA fragmentation into a DNA ladder occurs via the 30 kbp fragments, and that apoptosis in heat-treated M10 cells may be independent of p53.

Indoleamine 2,3-dioxygenase is necessary for cytolytic activity of natural killer cells.

Indoleamine 2,3‐dioxygenase (IDO) is a tryptophan catabolic enzyme that is widely distributed in various tissues. In peripheral blood mononuclear cells (PBMCs), production of IDO by macrophages or dendritic cells has been reported to inhibit T‐cell activation and proliferation. In the present study, we have determined that other phenotypes of PBMCs also express IDO. In cultures of PBMCs, IDO was found predominantly in monocyte by immunohistochemistry. Reverse transcriptase polymerase chain reaction analysis showed that IDO mRNA was expressed in T lymphocytes, B lymphocytes and natural killer (NK) cells and that expression was increased upon activation with interferon‐γ. The cytotoxicity of NK cells against K562 and HepG2 cells was reduced by IDO inhibitor. These results suggest that IDO in NK cells is essential for NK cells to generate killing activity against cancer cells.