In vivo effects of a recombinant molt-inhibiting hormone on molt interval and hemolymph ecdysteroid level in the kuruma prawn, Marsupenaeus japonicus

In order to determine the function of molt-inhibiting hormone (MIH) in vivo, we examined the effects of injecting of a recombinant MIH on the molt interval and hemolymph ecdysteroid level in the kuruma prawn, Marsupenaeus japonicus. The injection of recombinant MIH significantly prolonged the molt interval (9.0 +/-0.4 days in the control group, 9.5+/-0.5 days in the 2500 ng/g-body weight/injection-group, mean+/-SD), and significantly decreased the hemolymph ecdysteroid level (ratio of levels between after and before injection: 1.94+/-1.09 in the control and 1.28+/-0.39 in the 3000 ng/g-body weight/injection-group, mean+/-SD). These results conclusively show the inhibitory effects of MIH on molting in vivo.     

Dose-dependent effects of L-carnosine on the renal sympathetic nerve and blood pressure in urethane-anesthetized rats

The physiological function of L-carnosine (beta-alanyl-L-histidine) synthesized in mammalian muscles has been unclear. Previously, we observed that intravenous (i.v.) injection of L-carnosine suppressed renal sympathetic nerve activity (RSNA) in urethane-anesthetized rats, and L-carnosine administered via the diet inhibited the elevation of blood pressure (BP) in deoxycorticosterone acetate salt hypertensive rats. To identify the mechanism, we examined effects of i.v. or intralateral cerebral ventricular (l.c.v.) injection of various doses of L-carnosine on RSNA and BP in urethane-anesthetized rats. Lower doses (1 microg i.v.; 0.01 microg l.c.v.) of L-carnosine significantly suppressed RSNA and BP, whereas higher doses (100 microg i.v.; 10 microg l.c.v.) elevated RSNA and BP. Furthermore, we examined effects of antagonists of histaminergic (H1 and H3) receptors on L-carnosine-induced effects. When peripherally and centrally given, thioperamide, an H3 receptor antagonist, blocked RSNA and BP decreases induced by the lower doses of peripheral L-carnosine, whereas diphenhydramine, an H1 receptor antagonist, inhibited increases induced by the higher doses of peripheral L-carnosine. Moreover, bilateral lesions of the hypothalamic suprachiasmatic nucleus eliminated both effects on RSNA and BP induced by the lower (1 microg) and higher (100 microg) doses of peripheral L-carnosine. These findings suggest that low-dose L-carnosine suppresses and high-dose L-carnosine stimulates RSNA and BP, that the suprachiasmatic nucleus and histaminergic nerve are involved in the activities, and that L-carnosine acts in the brain and possibly other organs.     

Pituitary adenylate cyclase-activating polypeptide (PACAP)-like immunoreactivity in the brain of a teleost, Uranoscopus japonicus: immunohistochemical relationship between PACAP and adenohypophysial hormones

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) could play a role in stimulating pituitary hormone release in fish brain. In this study, we used immunochemical techniques to examine the histological and quantitative distribution of PACAP in the central nervous system (CNS) of a teleost, the stargazer, Uranoscopus japonicus. In addition, high performance liquid chromatographic (HPLC) analysis was performed to characterize the form of PACAP present, while the relationship between PACAP and adenohypophysial hormones was also determined immunohistochemically. PACAP-like immunoreactive (LI) neuronal cell bodies and fibers were found not only in the hypothalamo-pituitary region but also in the midbrain and hindbrain regions. PACAP-LI fibers were identified in the neurohypophysis in close proximity to pituitary cells containing immunoreactive hormones such as somatolactin, the N-terminal peptide of proopiomelanocortin, and N-acetyl endorphin. The concentration of immunoreactive PACAP in whole brain tissue was approximately 300 pmol/g wet weight. The average concentrations of immunoreactive PACAP in regions of the telencephalon, diencephalon, tectum, cerebellum, and rhombencephalon were 217.53, 510.26, 83.30, 148.64, and 364.62 pmol/g, respectively. In reverse-phase HPLC experiments, the predominant form of immunoreactive PACAP eluted closely with synthetic stargazer PACAP38, while PACAP27-like immunoreactivity was negligible. These results suggest that PACAP38 is the predominant PACAP form in the stargazer CNS, and that PACAP acts not only as a hypophysiotropic factor for adenohypophysial hormone release but also as a neurotransmitter and neuromodulator in the CNS.     

Use of a cDNA microarray to study immunity against viral hemorrhagic septicemia (VHS) in Japanese flounder (Paralichthys olivaceus) following DNA vaccination

Japanese flounder, Paralichthys olivaceus juveniles were vaccinated against viral hemorrhagic septicemia (VHS) by intramuscular injection of 10 microg of a plasmid DNA vector which encodes the viral hemorrhagic septicemia virus (VHSV) glycoprotein (G) gene under the control of the cytomegalovirus promoter. Experimental challenge of two viral doses (1 x 10(2) TCID50 and 1 x 10(3) TCID50) one month post-vaccination revealed that the G gene was able to induce protective immunity against VHS and this lasted until 21 days after the challenge. The VHSV G-protein gene DNA vaccine had a high protective efficiency, giving relative percentage survival (RPS) values of at least 93%. The defense mechanisms activated by the DNA vaccine were further elucidated by microarray analysis. Non-specific immune response genes such as NK, Kupffer cell receptor, MIP1-alpha and Mx1 protein gene were observed to be up-regulated by the VHSV G-protein DNA vaccine at 1 and 3 days post-immunization. Also, specific immune-related genes including the CD20 receptor, CD8 alpha chain, CD40 and B lymphocyte cell adhesion molecule were also up-regulated during that time. We observed significant up-regulation of some immune-related genes that are necessary for antiviral defense. Significant up- and/or down-regulation of unknown genes was also observed upon DNA vaccination. Our results confirm previous reports that the VHSV G gene elicits strong humoral and cellular immune responses which may play a pivotal role in protecting the fish during virus infections.     

A crucial role for antigen-presenting cells and alloantigen expression in graft-versus-leukemia responses

Graft-versus-leukemia (GVL) response after allogeneic bone marrow transplantation (BMT) represents one of the most potent forms of immunotherapy against malignant diseases. Antigen-presenting cells (APCs) are crucial for the induction of graft-versus-host disease (GVHD), the most serious complication of allogeneic BMT, but their role in GVL responses is unclear. Using a series of clinically relevant mouse GVL tumor models, we found that APCs and alloantigen expression on tumors are crucial for GVL. Moreover, APCs of host origin predominated in GVL responses although donor APCs contributed as the acuity of tumor burden decreased.     

Regulation of substrate specificity of plant alpha-mannosidase by cobalt ion: in vitro hydrolysis of high-mannose type N-glycans by Co2+-activated Ginkgo alpha-mannosidase

In our previous study (Woo, K. K., et al., Biosci. Biotechnol. Biochem., 68, 2547-2556 (2004), we purified an alpha-mannosidase from Ginkgo biloba seeds; it was activated by cobalt ions and highly active towards high-mannose type free N-glycans occurring in plant cells. In the present study, we have found that the substrate specificity of Ginkgo alpha-mannosidase is significantly regulated by cobalt ions. When pyridylamino derivative of Man9GlcNAc2 (M9A) was incubated with Ginkgo alpha-mannosidase in the absence of cobalt ions, Man5GlcNAc2-PA (M5A) having no alpha1-2 mannosyl residue was obtained as a major product. On the other hand, when Man9GlcNAc2-PA was incubated with alpha-mannosidase in the presence of Co2+ (1 mM), Man3-1GlcNAc2-PA were obtained as major products releasing alpha1-3/6 mannosyl residues in addition to alpha1-2 mannosyl residues. The structures of the products (Man8-5GlcNAc2-PA) derived from M9A by enzyme digestion in the absence of cobalt ions were the same as those in the presence of cobalt ions. These results clearly suggest that the trimming pathway from M9A to M5A is not affected by the addition of cobalt ions, but that hydrolytic activity towards alpha1-3/6 mannosyl linkages is stimulated by Co2+. Structural analysis of the products also showed clearly that Ginkgo alpha-mannosidase can produce truncated high-mannose type N-glycans, found in developing or growing plant cells, suggesting that alpha-mannosidase might be involved in the degradation of high-mannose type free N-glycans.     

Glycoform analysis of Japanese cedar pollen allergen, Cry j 1

In our previous study (Y. Kimura et al., Biosci. Biotechnol. Biochem., 69, 137-144 (2005)), we found that plant complex type N-glycans harboring Lewis a epitope are linked to the mountain cedar pollen allergen Jun a 1. Jun a 1 is a glycoprotein highly homologous with Japanese cedar pollen glycoallergen, Cry j 1. Although it has been found that some plant complex type N-glycans are linked to Cry j 1, the occurrence of Lewis a epitope in the N-glycan moiety has not been proved yet. Hence, we reinvestigated the glycoform of the pollen allergen to find whether the Lewis a epitope(s) occur in the N-glycan moiety of Cry j 1. From the cedar pollen glycoallergen, the N-glycans were liberated by hydrazinolysis and the resulting sugar chains were N-acetylated and then coupled with 2-aminopyridine. Three pyridylaminated sugar chains were purified by reversed-phase HPLC and size-fractionation HPLC. The structures were analyzed by a combination of exo- and endo-glycosidase digestions, sugar chain mapping, and electrospray ionization mass spectrometry (ESI-MS). Structural analysis clearly indicated that Lewis a epitope (Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-), instead of the Galbeta1-4(Fucalpha1-6)GlcNAc, occurs in the N-glycans of Cry j 1.     

The testicular fatty acid binding protein PERF15 regulates the fate of germ cells in PERF15 transgenic mice

The quality control of sperm is critical for efficient reproduction. In germ cells, cell death involves different processes to those in somatic cells, and in many cases, the trigger to induce cell death in deficient germ cells is still unclear. It is known that the fatty acid composition of sperm is related to fertility. Composition of the fatty acid of germ cells changes dynamically during spermatogenesis, and fatty acid binding protein (FABP) may be involved in these changes. In this study, we developed transgenic mice with a testicular germ-cell-specific FABP (PERF15) transgene, whose expression was controlled by the Cre-LoxP site-specific recombination system. We also developed transgenic mice with the Cre gene under the control of the spermatocyte specific Pgk2 promoter. In double transgenic mice, following Cre-mediated recombination of the PERF15 containing transgene, PERF15 was strongly overexpressed. Its overexpression induced multinucleate symplasts to form, indicating programmed germ cell death occurred at the elongated spermatid stage. As a result, sperm harboring the transgene were significantly decreased, but the surviving sperm demonstrated higher fertility than natural sperm. Therefore, we conclude that PERF15 associate with the direction of germ cell fates and preserve the quality of sperm.     

Influence of length on cytotoxicity of multi-walled carbon nanotubes against human acute monocytic leukemia cell line THP-1 in vitro and subcutaneous tissue of rats in vivo

Carbon nanotubes (CNTs) are single- or multi-cylindrical graphene structures that possess diameters of a few nanometers, while the length can be up to a few micrometers. These could have unusual toxicological properties, in that they share intermediate morphological characteristics of both fibers and nanoparticles. To date, no detailed study has been carried out to determine the effect of length on CNT cytotoxicity. In this paper, we investigated the activation of the human acute monocytic leukemia cell line THP-1 in vitro and the response in subcutaneous tissue in vivo to CNTs of different lengths. We used 220 nm and 825 nm-long CNT samples for testing, referred to as "220-CNTs" and "825-CNTs", respectively. 220-CNTs and 825-CNTs induced human monocytes in vitro, although the activity was significantly lower than that of microbial lipopeptide and lipopolysaccharide, and no activity appeared following variation in the length of CNTs. On the other hand, the degree of inflammatory response in subcutaneous tissue in rats around the 220-CNTs was slight in comparison with that around the 825-CNTs. These results indicated that the degree of inflammation around 825-CNTs was stronger than that around 220-CNTs since macrophages could envelop 220-CNTs more readily than 825-CNTs. However, no severe inflammatory response such as necrosis, degeneration or neutrophil infiltration in vivo was observed around both CNTs examined throughout the experimental period.