Effects of fasting on thermoregulatory processes and the daily oscillations in rats

To investigate the mechanism involved in the reduction of body core temperature (T(core)) during fasting in rats, which is selective in the light phase, we measured T(core), surface temperature, and oxygen consumption rate in fed control animals and in fasted animals on day 3 of fasting and day 4 of recovery at an ambient temperature (T(a)) of 23 degrees C by biotelemetry, infrared thermography, and indirect calorimetry, respectively. On the fasting day, 1) T(core) in the light phase decreased (P < 0.05) from the control; however, T(core) in the dark phase was unchanged, 2) tail temperature fell from the control (P < 0.05, from 30.7 +/- 0.1 to 23.9 +/- 0.1 degrees C in the dark phase and from 29.4 +/- 0.1 to 25.2 +/- 0.2 degrees C in the light phase), 3) oxygen consumption rate decreased from the control (P < 0.05, from 24.37 +/- 1.06 to 16.24 +/- 0.69 ml. min(-1). kg body wt(-0.75) in the dark phase and from 18.91 +/- 0.64 to 14.00 +/- 0.41 ml. min(-1). kg body wt(-0.75) in the light phase). All these values returned to the control levels on the recovery day. The results suggest that, in the fasting condition, T(core) in the dark phase was maintained by suppression of the heat loss mechanism, despite the reduction of metabolic heat production. In contrast, the response was weakened in the light phase, decreasing T(core) greatly. Moreover, the change in the regulation of tail blood flow was a likely mechanism to suppress heat loss.     

Structure-activity study of L-amino acid-based N-type calcium channel blockers

Synthesis and structure-activity relationship (SAR) study of L-amino acid-based N-type calcium channel blockers are described. The compounds synthesized were evaluated for inhibitory activity against both N-type and L-type calcium channels focusing on selectivity to reduce cardiovascular side effects due to blocking of L-type calcium channels. In the course of screening of our compound library, N-(t-butoxycarbonyl)-L-aspartic acid derivative 1a was identified as an initial lead compound for a new series of N-type calcium channel blockers, which inhibited calcium influx into IMR-32 human neuroblastoma cells with an IC(50) of 3.4 microM. Compound 1a also exhibited blockade of N-type calcium channel current in electrophysiological experiment using IMR-32 cells (34% inhibition at 10 microM, n=3). As a consequence of conversion of amino acid residue of 1a, compound 12a, that include N-(t-butoxycarbonyl)-L-cysteine, was found to be a potent N-type calcium channel blocker with an IC(50) of 0.61 microM. Thus, L-cysteine was selected as a potential structural motif for further modification. Optimization of C- and N-terminals of L-cysteine using S-cyclohexylmethyl-L-cysteine as a central scaffold led to potent and selective N-type calcium channel blocker 21f, which showed improved inhibitory potency (IC(50) 0.12 microM) and 12-fold selectivity for N-type calcium channels over L-type channels.     

Intratracheal gene transfer of decorin reduces subpleural fibroproliferation induced by bleomycin

Decorin, a small leucin-rich proteoglycan, is a negative regulator of transforming growth factor-beta, but the antifibrotic effect of decorin gene transfer has not been examined in a mouse model of usual interstitial pneumonia (UIP). We constructed a replication-defective recombinant adenovirus harboring human decorin gene (AdCMV.DC) and administered 1 x l0(9) plaque-forming units of AdCMV.DC intratracheally or intravenously to C57BL/6 mice with intraperitoneal injection of bleomycin, which induces a subpleural fibroproliferation, mimicking UIP, by day 28. Only intratracheal administration of AdCMV.DC increased decorin mRNA expression in the lung and decreased the hydroxyproline content augmented in bleomycin-induced pulmonary fibrosis (1.13 +/- 0.02 to 0.96 +/- 0.02, P = 0.006). In contrast, intravenous administration of AdCMV.DC increased the decorin expression only in the liver, but not in the lung, and without reducing lung fibrosis. These results indicate that adenoviral decorin gene transfer is effective only by direct administration to fibrosing lungs.     

Targeting apoptotic tumor cells to Fc gamma R provides efficient and versatile vaccination against tumors by dendritic cells

Dendritic cells (DCs) loaded with tumor-associated Ags (TAAs) act as potent adjuvant that initiates antitumor immune responses in vivo. However, TAA-based DC vaccination requires prior identification of TAAs. Apoptotic tumor cells (ATCs) can be an excellent source for DC loading because their potential uncharacterized Ags would be efficiently presented to T cells without any prior characterization and isolation of these Ags. However, ATCs alone are considered to be inefficient for activating antitumor immunity, possibly because of their inability to induce DC maturation. In this study, the aim was to enhance antitumor immune response by taking advantage of ATCs that have been opsonized with IgG (ATC-immune complexes, ATC-ICs) so as to target them to FcR for IgG (FcgammaRs) on DCs. It was found that when compared with ATCs, ATC-ICs were efficiently internalized by DCs via FcgammaRs, and this process induced maturation of DCs, which was more efficient than that of ATCs. Importantly, ATC-IC loading was shown to be more efficient than ATCs alone in its capacity for inducing antitumor immunity in vivo, in terms of cytotoxic T cell induction and tumor rejection. These results show that using ATC-ICs may overcome the limitations and may enhance the immune response of current ATC-based DC vaccination therapy.     

Proteasome activator PA28gamma-dependent nuclear retention and degradation of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein plays an important role in the formation of the viral nucleocapsid and a regulatory protein involved in hepatocarcinogenesis. In this study, we have identified proteasome activator PA28gamma (11S regulator gamma) as an HCV core binding protein by using yeast two-hybrid system. This interaction was demonstrated not only in cell culture but also in the livers of HCV core transgenic mice. These findings are extended to human HCV infection by the observation of this interaction in liver specimens from a patient with chronic HCV infection. Neither the interaction of HCV core protein with other PA28 subtypes nor that of PA28gamma with other Flavivirus core proteins was detected. Deletion of the PA28gamma-binding region from the HCV core protein or knockout of the PA28gamma gene led to the export of the HCV core protein from the nucleus to the cytoplasm. Overexpression of PA28gamma enhanced the proteolysis of the HCV core protein. Thus, the nuclear retention and stability of the HCV core protein is regulated via a PA28gamma-dependent pathway through which HCV pathogenesis may be exerted.     

Human leukocyte-derived arginine aminopeptidase. The third member of the oxytocinase subfamily of aminopeptidases

In this study we report the cloning and characterization of a novel human aminopeptidase, which we designate leukocyte-derived arginine aminopeptidase (L-RAP). The sequence encodes a 960-amino acid protein with significant homology to placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase. The predicted L-RAP contains the HEXXH(X)18E zinc-binding motif, which is characteristic of the M1 family of zinc metallopeptidases. Phylogenetic analysis indicates that L-RAP forms a distinct subfamily with placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in the M1 family. Immunocytochemical analysis indicates that L-RAP is located in the lumenal side of the endoplasmic reticulum. Among various synthetic substrates tested, L-RAP revealed a preference for arginine, establishing that the enzyme is a novel arginine aminopeptidase with restricted substrate specificity. In addition to natural hormones such as angiotensin III and kallidin, L-RAP cleaved various N-terminal extended precursors to major histocompatibility complex class I-presented antigenic peptides. Like other proteins involved in antigen presentation, L-RAP is induced by interferon-gamma. These results indicate that L-RAP is a novel aminopeptidase that can trim the N-terminal extended precursors to antigenic peptides in the endoplasmic reticulum.     

Cytopathologic and clinicopathologic features of ovarian hepatoid carcinoma. A case report

BACKGROUND: Hepatoid carcinoma is a rare ovarian tumor and is thought to be a different histopathologic subtype from hepatoid-type yolk sac tumor based upon its pathologic features. However, the cytopathologic characteristics of ovarian hepatoid carcinoma (OHC) have not been reported previously. We report the clinicopathologic and cytopathologic features and immunoreactivity of a case of OHC. CASE: A 36-year-old woman presented to our department with lower abdominal pain. A left ovarian tumor was found on pelvic examination, magnetic resonance imaging and computed tomography. The tumor was diagnosed as a hepatoid carcinoma of the left ovary based upon the histopathology of the surgically resected specimen. Cytopathologic specimens from a tumor touch preparation of the tumor exhibited pleomorphic tumor cells with abundant cytoplasm. The nuclei contained rough, granular chromatin and large, prominent nucleoli. Several tumor cells were multinucleated. Tumor cells were immunoreactive for alpha-fetoprotein (AFP). Hematoxylin and eosin staining revealed that the tumor cells were in a sinusoidal pattern resembling hepatocellular carcinoma without any glandular formation. The tumor cells were negative for human chorionic gonadotropin while positive for AFP, alpha-1-antitripsin, CA-125 and carcinoembryonic antigen. CONCLUSION: Cytopathologic examination is of considerable aid in the diagnosis of OHC since cytopathologic preparations highlight the characteristic cell pleomorphism.     

Lipid membrane with low proton permeability

This work reports the production of a liposomal formulation, having a lipidic membrane with known chemical composition and a low proton permeability, as confirmed by physicochemical characterization of the maintenance of a transmembranic pH gradient. These liposomes consist of DSPC, DSPE-PEG, DSPG and cholesterol, with low internal pH. To verify the low proton permeability of these liposomal bilayers, a study of proton migration according to the fluorescence quenching of 9-aminoacridine (9AA), as well as CPT-11 encapsulation, were used to monitor the acidification of the intravesicular space. Both experiments showed that this liposomal formulation is able to maintain a transmembranic proton gradient.     

Coupling between cyclooxygenases and prostaglandin F(2alpha) synthase. Detection of an inducible,glutathione-activated,membrane-bound prostaglandin F(2alpha)-synthetic activity

Distinct functional coupling between cyclooxygenases (COXs) and specific terminal prostanoid synthases leads to phase-specific production of particular prostaglandins (PGs). In this study, we examined the coupling between COX isozymes and PGF synthase (PGFS). Co-transfection of COXs with PGFS-I belonging to the aldo-keto reductase family into HEK293 cells resulted in increased production of PGF(2alpha) only when a high concentration of exogenous arachidonic acid (AA) was supplied. However, this enzyme failed to produce PGF(2alpha) from endogenous AA, even though significant increase in PGF(2alpha) production occurred in cells transfected with COX-2 alone. This poor COX/PGFS-I coupling was likely to arise from their distinct subcellular localization. Measurement of PGF(2alpha)-synthetic enzyme activity in homogenates of several cells revealed another type of PGFS activity that was membrane-bound, glutathione (GSH)-activated, and stimulus-inducible. In vivo, membrane-bound PGFS activity was elevated in the lung of lipopolysaccharide-treated mice. Taken together, our results suggest the presence of a novel, membrane-associated form of PGFS that is stimulus-inducible and is likely to be preferentially coupled with COX-2.