Production and characterization of murine monoclonal anti-human DNase II antibodies,and their use for immunoaffinity purification of DNase II from human liver and urine

Four murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion (SRED) and DNA-cast polyacrylamide gel electrophoresis (DNA-cast PAGE) were very useful for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining of the 32-kDa enzyme band, which is one of the three non-identical subunits of human DNase II molecule separated by sodium dodecyl sulfate (SDS)-PAGE followed by blotting on a transfer membrane. A formyl-cellulofine resin conjugated with each antibody specifically adsorbed and efficiently desorbed the active DNase II enzyme. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.     

Matrix metalloproteinases regulate mesonephric cell migration in developing XY gonads which correlates with the inhibition of tissue inhibitor of metalloproteinase-3 by Sry

In the mouse, the sex determining gene Sry, on the Y chromosome, controls testis differentiation during embryogenesis. Following Sry expression, indifferent XY gonads increase their size relative to XX gonads and form cord-like structures with the adjacent mesonephros, providing XY gonad somatic cells. This mesonephric cell migration is known to depend on Sry, but the molecular mechanism of mesonephric cell migration remains unknown. In this study, it was shown that cells expressing Sry induced proliferation of mesonephric cells migrating into male gonads, and inhibited expression of the tissue inhibitor of metalloproteinases (TIMP)-3 gene, which is the endogenous inhibitor of matrix metalloproteinases (MMP). In addition, the mesonephric cell migration was blocked by a chemically synthesized inhibitor of MMP in a gonad/mesonephros organ co-culture system with enhanced green fluorescent protein transgenic embryos. The findings indicate that MMP may play a critical role in mesonephric cell migration, and the function of MMP may be regulated by a Sry-TIMP-3 cascade. These findings are an important clue for the elucidation of testicular formation in developing gonads.     

Chemoprevention of precursors to colon cancer by dehydroepiandrosterone (DHEA)

Although dehydroepiandrosterone (DHEA) is recognized as one of the major adrenal androgens, its precise physiological role in the human endocrine system remains to be elucidated. In particular, the effect of DHEA on carcinogenesis has not been fully characterized. We undertook this study to determine whether DHEA has a chemopreventative effect on the precursors of colon cancer in a murine model of azoxymethane (AOM)-induced aberrant crypt foci (ACF). The number of ACF was significantly decreased in mice treated with 0.4% (p < 0.001) and 0.8% DHEA (p < 0.001), but there were no significant differences between DHEA-treated and control mice in terms of the ACF size, 3-catenin expression or level of dysplasia. This is the first study of colon cancer carcinogenesis demonstrating that DHEA treatment can decrease the number of ACF without apparently modifying their malignant potential. These data strongly suggest that DHEA might be a potential chemopreventative agent against human colon cancer.     

Effect of heat shock preconditioning on NF-kappaB/I-kappaB pathway during I/R injury of the rat liver

Hepatic ischemia-reperfusion (I/R) injury continues to be a fatal complication after liver surgery. Heat shock (HS) preconditioning is an effective strategy for protecting the liver from I/R injury, but its exact mechanism is still unclear. Because the activation of nuclear factor-kappaB (NF-kappaB) is an important event in the hepatic I/R-induced inflammatory response, the effect of HS preconditioning on the pathway for NF-kappaB activation was investigated. In the control group, NF-kappaB was activated 60 min after reperfusion, but this activation was suppressed in the HS group. Messenger RNA expressions of proinflammatory mediators during reperfusion were also reduced with HS preconditioning. Concomitant with NF-kappaB activation, NF-kappaB inhibitor I-kappaB proteins were degraded in the control group, but this degradation was suppressed in the HS group. This study shows that HS preconditioning protected the liver from I/R injury by suppressing the activation of NF-kappaB and the subsequent expression of proinflammatory mediators through the stabilization of I-kappaB proteins.     

Status of Bone Allografting in Japan – Nation-Wide Survey of Bone Grafting Performed from 1995 through 1999

We report the status of bone allografting in Japan on the basis of the information obtained through questionnaires performed by the Japanese Orthopaedic Association (JOA). JOA performed a nation-wide survey in 2000, in order to clarify the current status of musculoskeletal tissue grafting in the orthopaedic practices in Japan. Conducted period was for 5 years from 1995 to 1999. As the results of this survey, it had been clarified that 92,984 bone graftings, which included autografts, allografts and synthetic bone substitutes, were performed during conducted 5 years. While the allografts were used only in 3,212 cases (3%), autograftings were performed in 64,193 cases (69%), synthetic bone substitutes were used in 25,576 cases (28%) in this series. The proportion of the number of operations for use bone substitutes increased every year, whereas that autografting decreased. The proportion of the number of allografting remained almost unaltered. Of the 706 institutions which answered to have experiences of tissue grafting, only 193 (27%) performed allograft.Since Kitasato University Hospital Bone Bank was developed in 1971, we have applied to clinical while doing basic research for preserved bone allograft. When extensive bone graft is required, allograft is very useful. In Japan, however, allograft is not performed widely. The foundation of regional bone banks is expected to resolve this problem. Since excision of bone preparations from cadaver donors is not common, bone allografts are not supplied sufficiently at present. It is needed to develop a network connecting bone banks in Japan. The enlightenment activities to the ordinary people and medical institutions will also be required.     

M-LP,Mpv17-like protein,has a peroxisomal membrane targeting signal comprising a transmembrane domain and a positively charged loop and up-regulates expression of the manganese superoxide dismutase gene

M-LP (Mpv17-like protein) has been identified as a new protein that has high sequence homology with Mpv17 protein, a peroxisomal membrane protein involved in the development of early onset glomerulosclerosis. In this study, we verified the peroxisomal localization of M-LP by performing dual-color confocal analysis of COS-7 cells cotransfected with green fluorescent protein-tagged M-LP and DsRED2-PTS1, a red fluorescent peroxisomal marker. To characterize the peroxisomal membrane targeting signal, we examined the intracellular localizations of several green fluorescent protein-tagged deletion mutants and demonstrated that, of the three transmembrane segments predicted, the first near the NH(2) terminus and NH(2)-terminal half of the following loop region, which is abundant in positively charged amino acids, were necessary and sufficient for peroxisomal targeting. To elucidate the function of M-LP, we examined the activities of several enzymes involved in reactive oxygen species metabolism in COS-7 cells and found that transfection with M-LP increased the superoxide dismutase activity significantly. Quantitative real-time PCR analysis revealed that the manganese SOD (SOD2) mRNA level of COS-7 cells transfected with M-LP was elevated. These results indicate that M-LP participates in reactive oxygen species metabolism.     

The lymphocytic cholinergic system and its contribution to the regulation of immune activity

Lymphocytes express most of the cholinergic components found in the nervous system, including acetylcholine (ACh), choline acetyltransferase (ChAT), high affinity choline transporter, muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), and acetylcholinesterase. Stimulation of T and B cells with ACh or another mAChR agonist elicits intracellular Ca2+ signaling, up-regulation of c-fos expression, increased nitric oxide synthesis and IL-2-induced signal transduction, probably via M3 and M5 mAChR-mediated pathways. Acute stimulation of nAChRs with ACh or nicotine causes rapid and transient Ca2+ signaling in T and B cells, probably via alpha7 nAChR subunit-mediated pathways. Chronic nicotine stimulation, by contrast, down-regulates nAChR expression and suppresses T cell activity. Activation of T cells with phytohemagglutinin or antibodies against cell surface molecules enhances lymphocytic cholinergic transmission by activating expression of ChAT and M5 mAChR, which is suggestive of local cholinergic regulation of immune system activity. This idea is supported by the facts that lymphocytic cholinergic activity reflects well the changes in immune system function seen in animal models of immune deficiency and immune acceleration. Collectively, these data provide a compelling picture in which lymphocytes constitute a cholinergic system that is independent of cholinergic nerves, and which is involved in the regulation of immune function.     

Pralidoxime iodide (2-pAM) penetrates across the blood-brain barrier

The in vivo rat brain microdialysis technique with HPLC/UV was used to determine the blood-brain barrier (BBB) penetration of pralidoxime iodide (2-PAM), which is a component of the current nerve agent antidote therapy. After intravenous dosage of 2-PAM (10, 50, 100 mg/kg), 2-PAM appeared dose-dependently in the dialysate; the striatal extracellular/blood concentration ratio at 1 h after 50 mg/kg dosage was 0.093 ± 0.053 (mean ± SEM). This finding offered conclusive evidence of the BBB penetration of 2-PAM. We also examined whether the BBB penetration of 2-PAM was mediated by a certain specific transporter, such as a neutral or basic amino acid transport system. Although it was unclear, the neural uptake of 2-PAM was Na⁺ dependent. The mean BBB penetration by 2-PAM was approximately 10%, indicating the intravenous administration of 2-PAM might be to a degree effective to reactivation of the blocked cholinesterase in the brain.