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The experimental asthma caused by IgE antibody in rats was inhibited by HSR-6071 (6-(1-pyrrolidinyl)-N-(1H-tetrazol-5-yl)-2-pyrazinecarboxamide) (0.01-0.1 mg kg−1 i.v.) in a dose-dependent manner. The inhibitory activity of HSR-6071 was more potent than those of disodium cromoglycate and ketotifen, and equipotent with amlexanox. The bronchoconstriction mediated by IgE or IgG antibody in guinea-pigs was also prevented by HSR-6071 (0.3, 1 and 3 mg kg−1 i.v.), amlexanox (3, 10 and 30 mg kg−1 i.v.) and ketotifen (0.1 mg kg−1 i.v.) but not by disodium cromoglycate (10 mg kg−1 i.v.). HSR-6071 was more potent than amlexanox, but less potent than ketotifen. HSR-6071 suppressed antigen-induced histamine and SRS-A release from minced lung tissues of guinea-pigs sensitized passively with rabbit anti-EA serum and was a more potent inhibitor of the release of SRS-A than of histamine. On the other hand, histamine- or acetylcholine-induced bronchoconstriction in guinea-pigs was scarcely affected by HSR-6071 at doses sufficient to inhibit the experimental asthma, but LTD4-induced bronchoconstriction was dramatically inhibited. These results indicate that the inhibitory action on experimental allergic asthma of HSR-6071 may be due to suppression of antigen-induced histamine and SRS-A release from lung tissues and to antagonism of SRS-A action. In addition, HSR-6071 inhibited cyclic AMP phosphodiesterase activity and produced relaxation of the guinea-pig isolated trachea. These pharmacological actions may contribute to the production of the anti-allergic action of HSR-6071.

Dermatitis caused by contact with tentacles of jellyfish was studied on 25 volunteers. Two tentacles cut from a living jellyfish, Carybdea rastonii, were applied on each of the forearms and skin reactions were observed. All volunteers complained of severe pain, which lasted from 10 min to 8 hrs. Erythema and wheal appeared within 3 to 4 min and enlarged for 15 to 20 min. Erythema subsided within 24 hrs to 3 days in all but two individuals. Seven to 13 days after the application, linear erythema and papulo-vesicular lesions with pruritus were observed on the forearms of 15 out of 25 volunteers tested. These flare-up lesions lasted for one week leaving slight pigmentation. Histological findings from the flare-up lesions corresponded to those of allergic contact dermatitis. The lymphocyte response to the jellyfish venom in the subjects who had recurring lesions was greater than that in either the subjects with no recurring lesions or the control group, who was never exposed to jellyfish.

Various pyrazine derivatives were synthesized and their antiallergic activity was examined. The inhibitory activity on allergic histamine release of the compounds bearing a 5-tetrazolyl group was more potent than that of the corresponding carboxyl derivatives. The introduction of -CONH- or -NHCO- between the pyrazine ring and the 5-tetrazolyl group as a spacer greatly enhanced the activity. N-(1H-Tetrazol-5-yl)-2-pyrazinecarboxamide (I-3) was estimated to exhibit nearly the same potency as disodium cromoglycate (DSCG). The structure-activity relationship among various derivatives modified by introducing some substituents onto the 3-, 5- or 6-position of the pyrazine ring of I-3 was investigated. The activity remained unchanged or was reduced when such substituents as methyl, chloro, methoxy, methylamino and dimethylamino were introduced at the 3- or 5-position. In contrast, 6-substitution with various alkylamino groups more or less increased the activity. Among them, the 6-dimethylamino (I-17c) and 6-(1-pyrrolidinyl) (I-34) derivative were proved to be most potent. The IC50 values (concentration which produces 50% inhibition of the allergic histamine release) of I-17c and I-34 were determined to be 4.7 x 10(-10) and 4.6 x 10(-10) M, respectively. These two compounds produced a potent inhibitory activity on passive cutaneous anaphylaxis (PCA) in rat, not only by the intravenous route (ED50 = 0.0096 mg/kg for both compounds) but also by the oral route (ED50 = 0.19 and 0.18 mg/kg, respectively). On the other hand, when the pyrazine ring of some representative compounds was replaced with a pyridine ring, the inhibitory activity on histamine release was significantly reduced.

The effect of structural modifications of 6-substituted N-(1H-tetrazol-5-yl)-2-pyrazinecarboxamides on their anti-allergic activity was analyzed quantitatively by means of the Hansch-Fujita method. The activity of these compounds was correlated with hydrophobic (pi) and steric (molecular refractivity and STERIMOL B1) effects of the 6-substituent on the pyrazine ring. The 6-substituents with a length greater than n-propylamino possess an extra effect enhancing the activity. Moreover, the activity increased progressively from 6-non-amino via alkylamino- to dialkylamino-substituted compounds, other factors being equal. This could be attributable to an electronic effect of substituents. Electron-donating small and yet symmetric substituents with high hydrophobicity longer than n-propylamino seemed to be favorable to the activity. By compromising these contradictory requirements, small dialkylamino (including cyclic amino) groups were decided to be the most favorable substituents. This analysis was in agreement with the observation that the most effective compounds were the 6-dimethylamino (I-27) and 6-(1-pyrrolidinyl) (I-34) derivatives.

This study employs three nuclear genes (PHYA, LFY, and GAI1) to reconstruct the phylogenetic and biogeographic history of Magnoliaceae. A total of 104 samples representing 86 taxa from all sections and most subsections were sequenced. Twelve major groups are well supported to be monophyletic within Magnoliaceae and these groups are largely consistent with the recent taxonomic revision at the sectional and subsectional levels. However, relationships at deeper nodes of the subfamily Magnolioideae remain not well resolved. A relaxed clock relying on uncorrelated rates suggests that the complicated divergent evolution of Magnolioideae began around the early Eocene (54.57 mya), concordant with paleoclimatic and fossil evidence. Intercontinental disjunctions of Magnoliaceae in the Northern Hemisphere appear to have originated during at least two geologic periods. Some occurred after the middle Miocene, represented by two well-recognized temperate lineages disjunct between eastern Asia and eastern North America. The others may have occurred no later than the Oligocene, with ancient separations between or within tropical and temperate lineages.

SUMMARY Microsporidian Encephalitozoon cuniculi has a unique organelle called a polar tube (PT), the extrusion of which is absolutely required to invade a host cell. We recently detected anti-E. cuniculi PT immunoglobulin (Ig) M antibodies in sera from many healthy individuals. The present one-dimensional (1-D) immunoblot analysis predominantly detected a band at 52 kDa in all of the examined human sera with anti-PT IgM. The use of mouse monoclonal antibody confirmed that the 52-kDa band detected in 1-D immunoblots was an antigen derived from the PT, which represents a glycoprotein nature. In addition, from changes in the immunoreactivity of the 52-kDa band before and after treatment with NaOH, we determined that the 24 human serum samples with anti-PT IgM activities could be roughly grouped into three types: (i) sera containing antibodies against only a saccharic determinant (n = 3); (ii) sera containing antibodies against only a proteinic determinant (n = 11); and (iii) sera showing dual recognition of saccharic and proteinic determinants (n = 10). Further two-dimensional (2-D) immunoblot analysis followed by proteomic analysis confirmed that human sera with anti-PT IgM reacted with E. cuniculi polar tube protein 1 (PTP1). Such circulating IgM antibodies may be important in the first line of defence against E. cuniculi infection.

The physiological properties of endothelin-1 in ocular choroidal melanocytes are not well-known, although endothelin-1 increases the intracellular calcium concentration through endothelin-B receptors in skin melanocytes. We studied the effect of endothelin-1 on choroidal melanocytes in rabbit ocular suprachoroidal tissue. Fura-2 microfluorophotometry indicated that endothelin-1 and endothelin-B receptor selective agonists, endothelin-3 and BQ3020, increased the intracellular calcium concentration of the tissue. Endothelin-B receptor selective antagonist BQ788 blunted the response. Immuno-electron microscopy showed that endothelin-B receptor-like immunoreactivity was located only on melanocytes. These results indicate that endothelin-1 increases intracellular calcium concentration in rabbit suprachoroidal tissue via endothelin-B receptors on melanocytes.

Nitric oxide (NO) has been suggested to play a key role in the pathogenesis of pulmonary hypertension (PH). To determine which mechanism exists to affect NO production, we examined the concentration of endogenous nitric oxide synthase (NOS) inhibitors and their catabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) activity and protein expression (DDAH1 and DDAH2) in pulmonary artery endothelial cells (PAECs) of rats given monocrotaline (MCT). We also measured NOS and arginase activities and NOS protein expression. Twenty-four days after MCT administration, PH and right ventricle (RV) hypertrophy were established. Endothelium-dependent, but not endothelium-independent, relaxation and cGMP production were significantly impaired in pulmonary artery specimens of MCT group. The constitutive NOS activity and protein expression in PAECs were significantly reduced in MCT group, whereas the arginase, which shares l-arginine as a common substrate with NOS, activity was significantly enhanced in PAECs of MCT group. The contents of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA), were increased in PAECs of MCT group. The DDAH activity and DDAH1, but not DDAH2, protein expression were significantly reduced in PAECs of MCT group. These results suggest that the impairment of cGMP production as a marker of NO production is possibly due to the blunted endothelial NOS activity resulting from the downregulation of endothelial NOS protein, accumulation of endogenous NOS inhibitors, and accelerated arginase activity in PAECs of PH rats. The decreased overall DDAH activity accompanied by the downregulation of DDAH1 would bring about the accumulation of endogenous NOS inhibitors.

Recombinant human serum albumin (HSA) including the synthetic iron(II)-porphyrin (FeP), albumin-heme (HSA-FeP), is a unique O2-carrying plasma hemoprotein as a red blood cell substitute. We have investigated the possible influence of HSA-FeP on the complement system and platelet activation in vitro. The amounts of the serum complement titer CH50 and terminal complement complex SC5b-9 of human blood serum, incubated with HSA-FeP (10, 20, and 40 vol %), were almost the same as those of the corresponding samples with HSA. The effect of HSA-FeP on the platelet reactivity has been demonstrated by conformational changes in the membrane glycoprotein IIb/IIIa and surface expression of an α-granule membrane protein P-selectin. Platelet activation in response to the ADP-stimulation was not influenced by the presence of HSA-FeP. It can be concluded that the albumin-heme solution does not facilitate the immunological reaction and platelet activation. Moreover, a 20% exchange transfusion with HSA-FeP into anesthetized rats has been performed to evaluate the circulation and blood parameters for 6 h. Time course changes in all parameters showed features identical to the control group (without infusion) and HSA group. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007

Encephalitozoon cuniculi , a spore-forming obligate intracellular parasitic pathogen belonging to the phylum Microsporidia, has a unique and highly specialized organelle called the polar tube. Using an enzyme immunostaining assay in which germinated E. cuniculi spores were coated onto plastic surfaces, we tested healthy and human immunodeficiency virus (HIV)-infected individuals in Japan for anti-polar tube antibodies of each immunoglobulin (Ig) class. Anti-polar tube IgG was detected in just 4 of 380 healthy individuals; no anti-polar tube IgA was detected in any individuals; however, unexpectedly, anti-polar tube IgM antibodies were detected in 138 individuals (36%). When the healthy individuals were grouped by age, the highest rate of positivity to anti-polar tube IgM antibodies was seen in individuals aged 20 years old or younger. Fifty-nine percent (24/41) of the individuals aged 20 years or younger were anti-polar tube IgM antibody positive. This rate tended to decrease among individuals in older age groups. However, no anti-polar tube IgM antibodies were detected in 21 HIV-infected persons who were younger than 30 years of age and who had CD4 cell levels below 250/μl. These seroepidemiological results clearly indicate that circulating anti-polar tube IgM antibodies that are capable of strongly reacting with filaments extruded from geminated spores exist and suggest that such antibodies may play a part in protective immunity.

Based on colonoscopy findings, we made a preoperative diagnosis of primary mucinous cystadenocarcinoma of the appendix with features of a submucosal tumor (SMT) in the ascending colon. A 59-year-old woman who presented with right lower quadrant abdominal pain underwent colonoscopy, which revealed an SMT with three nodules covered with mucus in the ascending colon. Examination of colonoscopic biopsy specimens indicated “very” well-differentiated adenocarcinoma with mucus lakes. Abdominal computed tomography showed irregular wall thickness from the cecum to the ascending colon. The adjacent appendix had an enhanced wall and unclear border against the ascending colon. Thus, we performed right hemicolectomy, with good results. Histopathological examination revealed mucinous cystadenocarcinoma of the appendix, invading the ascending colon with fistula formation. Appendiceal tumors can manifest with a variety of colonoscopic features, and curative surgical resection should be attempted even if there is fistula formation.

In a preliminary experiment, we found that lavender essential oil relaxes vascular smooth muscle. Thus, the present experiments were designed to investigate the relaxation mechanism of linalyl acetate as the major ingredient of lavender essential oil in rabbit carotid artery specimens. Linalyl acetate produced sustained and progressive relaxation during the contraction caused by phenylephrine. The relaxation effect of linalyl acetate at a concentration near the EC50 was partially but significantly attenuated by nitroarginine as an inhibitor of nitric oxide synthase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one as an inhibitor of guanylyl cyclase, or by the denudation of endothelial cells. In specimens without endothelium, the phenylephrine-induced contraction and phosphorylation of myosin light chain (MLC) were significantly attenuated after the pretreatment with linalyl acetate. The relaxation caused by linalyl acetate in the endothelium-denuded specimens was clearly inhibited by calyculin A as an inhibitor of MLC phosphatase, although not by ML-9 as an inhibitor of MLC kinase. Furthermore, suppression of the phenylephrine-induced contraction and MLC phosphorylation with linalyl acetate was canceled by the pretreatment with calyculin A. These results suggest that linalyl acetate relaxes the vascular smooth muscle through partially activation of nitric oxide/cyclic guanosine monophosphate pathway, and partially MLC dephosphorylation via activating MLC phosphatase.

This study was designed to investigate the role of arginase in regulating myometrial contractions during gestation in the rat. Arginase activity in the myometrium was significantly decreased during the 7th-21st day of gestation, with the lowest value on the 14th day. However, the enzyme activity became significantly higher at term gestation (22nd day) than that in the non-pregnant myometrium. Arginase I protein was undetectable in the non-pregnant myometrium, at 7th and 14th day of gestation and after delivery. A slight positive signal for arginase I was detectable at 21st day of gestation. However, the protein was clearly up-regulated at term gestation (22nd day), although arginase II protein was down-regulated during gestation, with the lowest value on the 14th day. Gestational changes in arginase activity negatively correlated with those in cyclic GMP production, whereas the changes positively correlated with those in endogenous nitric oxide synthase (NOS) inhibitors and endothelin-1 (ET-1) contents. Myometrial arginase activity was inhibited by N(G)-hydroxy-L-arginine as an intermediate of NO production from L-arginine in a concentration-dependent manner. Both basal and stimulated guanylyl cyclase activities were enhanced at mid- and reduced at term gestation and after delivery, thereby partly increasing cyclic GMP production at mid- and partly decreasing the nucleotide production at term gestation and after delivery. These results suggest that the decreased arginase activity at mid-gestation possibly results from the down-regulation of arginase II protein. Whereas, the enhanced overall arginase activity at term gestation seems to be because of the induced functional arginase I in concert with the attenuated arginase II expression. The enhanced arginase activity at term gestation would be implicated in increasing myometrial contractions mediated by the increased ET-1. The increased peptide production at term gestation is possibly because of the reduced cyclic GMP production resulting from enhanced arginase activity, accumulated endogenous NOS inhibitors and attenuated guanylyl cyclase activity.

BACKGROUND The aim of this study was to find a simple and feasible method for ex vivo expansion of human cytomegalovirus (HCMV)-specific cytotoxic T cells from peripheral blood mononuclear cells (PBMNCs) without the aid of exogenous antigen-presenting cells (APCs) such as cultured dendritic cells. STUDY DESIGN AND METHODS PBMNCs from three HLA-A*2402-seropositive donors were stimulated with HCMV pp65(341-350) peptide on Day 1 and then cultured with interleukin-2 and allogeneic feeder cells for 3 to 4 weeks. HCMV peptide-specific T cells were purified with HLA-A*2402/pp65(341-350) tetramer on Days 12 to 13 and harvested on Days 23 to 27. RESULTS The initial numbers of PBMNCs were 2 x 10(7), 1.5 x 10(7), and 2.5 x 10(7) and the increases in HCMV peptide-specific T cells were 3.5 x 10(4)-, 2.0 x 10(3)-, and 1.1 x 10(3)-fold, respectively. The estimated final numbers of tetramer-positive cells were 9.1 x 10(7), 9.0 x 10(6), and 5.3 x 10(6), respectively. The purities of the tetramer-positive cell population in culture were 72.6, 75.0, and 80.9 percent, respectively. The cells killed peptide-pulsed B-lympoblastoid cell lines and secreted interferon-gamma in a HLA-restricted manner. They did not have natural killer cell activity or lymphokine activated killer cell activity. Most of them had an effector-memory phenotype. They did not express killer inhibitory receptors. CONCLUSION This method makes it possible to obtain more than 1 x 10(7) HCMV-specific T cells from approximately 2 x 10(7) to 5 x 10(7) PBMNCs without exogenous APCs such as cultured dendritic cells.

Present experiments were designed to investigate the effects of ovariectomy (OVX) and estrogen replacement (ER) on neointimal formation after balloon injury of the rat carotid artery. Young adult female rats were divided into 3 groups of sham operation (control), ovariectomy, and ovariectomy plus estrogen replacement. Estrogen replacement was initiated by implanting a sustained release pellet containing water-soluble 17beta-estradiol 1 week after the ovariectomy. Carotid arteries were harvested 2 weeks after the balloon injury for determinations. The balloon injury caused intimal hyperplasia, which was accompanied by the impaired endothelium-dependent relaxation and cyclic GMP production, and accumulation of asymmetric dimethylarginine (ADMA) as an endogenous NOS inhibitor. Bilateral ovariectomy accelerated the intimal hyperplasia. The acceleration was accompanied by the enhanced impairment of NO production, attenuated reendothelialization, and enhanced accumulation of ADMA. The estrogen replacement improved the accelerated intimal hyperplasia with concomitant improvement of the impaired NO production and accumulated asymmetric dimethylarginine, and facilitated reendothelialization. These results suggests that the enhanced impairment of NO production, which possibly results from the accumulated asymmetric dimethylarginine and lack of reendothelialization, may contribute to the acceleration of intimal hyperplasia by ovariectomy and that estrogen replacement effectively improves the intimal hyperplasia by restoring the impaired NO production through reducing endogenous NOS inhibitor and facilitating reendothelialization.

l -Arginine is the common substrate for arginase and nitric oxide synthase (NOS). Arginase converts l -arginine to urea and ornithine, which is the principal precursor for production of polyamines required for cell proliferation. Human placenta expresses endothelial NOS (eNOS) in syncytiotrophoblasts, but the expression of arginase has not been fully elucidated. Our aim was to investigate the expression and distribution patterns of arginase-I (A-I) and arginase-II (A-II) in human placental villi in the first trimester and at term using immunohistochemistry, RT-PCR and Western blot analysis. The arginase enzyme activity in placental villi was also measured. Immunohistochemistry showed different distribution patterns of the arginase isoforms during gestation: A-I was observed only in cytotrophoblasts, while A-II was observed in both cytotrophoblasts and syncytiotrophoblasts. RT-PCR and Western blot analysis showed expression of A-I and A-II in the first trimester and at term in human placental villi. Expression of A-II and arginase activity was greater in the first trimester than at term. Differentiation of cytotrophoblasts into syncytiotrophoblasts may be associated with l -arginine metabolism through modulation of l -arginine availability for eNOS and A-I. And elevated arginase activity in the early gestational period may be responsible for proliferation of trophoblasts by increasing polyamines production. These results suggest that the l -arginine–ornithine–polyamine and l -arginine–nitric oxide pathways play a role in placental growth and development.

The cellular receptor for hepatitis B virus (HBV) infection has not yet been identified. The purpose of this study was to address the possibility of participation by desialylated HBV and the asialoglycoprotein receptor (ASGP-R) exclusively expressed on liver parenchymal cells, in infection. Assays for viral binding and entry were performed by culturing a hepatoblastoma cell line, HepG2, and HBV particles derived from the HBV carrier in the presence or absence of neuraminidase (NA). Viral binding and entry were clearly enhanced in the presence of NA, and the enhancement of the binding could be blocked by asialo-fetuin and ethylenediamine-tetraacetic acid (EDTA). In addition, covalently closed circular (CCC)-DNA, as a marker of infectivity, was detected in the presence of NA, but not in its absence. The optimal concentration of NA raised infectivity more than 1000 times. We concluded that this method makes it feasible to evaluate the infectivity of HBV in vitro and that ASGP-R may be a specific HBV receptor once viral particles are desialylated.

We analyzed mechanisms decreasing hind-limb perfusion pressure (PP) with blackcurrant concentrate (BC) in the rat. The decrease in PP with BC was abolished by endothelial removal, nitroarginine plus tetraethylammonium, nitroarginine plus catalase or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one as an inhibitor of guanylyl cyclase and potassium channel(s), and accompanied by the increased cyclic GMP level. Partial but significant inhibition caused by KCl was observed during contraction. Authentic H2O2 decreased the PP in a sensitive manner to catalase and tetraethylammonium. The decrease in PP with BC in the presence of nitroarginine was significantly attenuated by diverse potassium channel blockers. Two delphinidins of 4 anthocyanins purified from BC definitely decreased the PP through similar mechanisms to BC. These results suggest that the decreased PP with BC is possibly mediated by endothelial NO and H2O2, and partially through the activation of diverse potassium channels, and that 2 delphinidins play a major role as active components of BC.

Little is known about L-arginine catabolism following ischemia in the bladder. We examined the changes in nitric oxide synthase, arginase and ornithine decarboxylase activity, polyamine biosynthesis and the ability to produce nitric oxide following ischemia of the rabbit bladder.Bladder ischemia was created by ligation of a unilateral bladder artery. At various time points, that is 1, 4, 8, 24, 48 and 72 hours following ligation, the bladders were excised and harvested for determinations.Constitutive nitric oxide synthase, inducible nitric oxide synthase arginase and ornithine decarboxylase activities increased with time, peaking at 48 hours without significant differences between the ligated and nonligated sides in the whole layer. Arginase and ornithine decarboxylase increased mainly in the muscularis following ischemia. Also, putrescine in the muscularis was significantly higher than in the mucosa 48 hours following ischemia. Baseline nitrite/nitrate production in the whole detrusor on the ligated side at 24 hours was significantly lower than that in the normal detrusor. However, nor-hydroxyarginine as an arginase inhibitor and L-arginine increased nitrite/nitrate production in the ischemic detrusor without changing in the normal detrusor. This increasing effect of nor-hydroxyarginine was abolished by nitroarginine methylester as a nitric oxide synthase inhibitor.Enzymes related to L-arginine catabolism were involved in the early events of ischemic bladder. Arginase may have 2 independent roles, that is 1) activation of arginase/ornithine decarboxylase/polyamines pathways in the muscle injury and remodeling following ischemia, and 2) endogenous negative regulation of nitric oxide production by limiting the L-arginine substrate for nitric oxide synthase.

Hemoglobin vesicles (HbV) are artificial oxygen carriers that encapsulate a concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane. The oxygen transporting ability of HbV in vivo has been demonstrated by the transfusion of HbV into hemorrhagic shock rodent models. However, the compatibility of HbV with human blood cells must be evaluated. Preincubation of platelets with concentrations of 20% or 40% HbV had no effect on the binding of PAC-1, a monoclonal antibody that detects activation-dependent conformational changes in alphaIIbbeta3 on platelets, or the surface expression of CD62P in whole blood. ADP-induced increases in PAC-1 binding were significantly enhanced by exposing the platelets to concentrations of either 20% or 40% HbV, whereas the ADP-induced increases in CD62P expression were not affected by HbV treatment at either concentration. Preincubation of platelet-rich plasma (PRP) with HbV minimally reduced the spontaneous release of TXB2 and RANTES, but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with ADP. Similarly, preincubation of PRP with HbV minimally reduced the spontaneous release of RANTES but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with collagen, although collagen-induced serotonin release tended to decrease with HbV pretreatment. These data suggest that the exposure of human platelets to high concentrations of HbV (up to 40%) in vitro did not cause platelet activation and did not adversely affect the formation and secretion of prothrombotic substances or proinflammatory substances triggered by platelet agonists, although one of the earliest events in ADP-induced platelet activation was slightly potentiated by HbV pretreatment at the doses tested. Taken together, these results imply that HbV, at concentrations of up to 40%, do not have any aberrant interactions with either unstimulated or agonist-induced platelets.

Background Anti-CD36 isoantibody in blood recipients is reported to cause refractoriness to platelet (PLT) transfusions and posttransfusion purpura-like syndrome. There are few reports, however, about the effects of passively transfused blood products containing this isoantibody on recipients. Case report A 67-year-old Japanese woman underwent brain surgery. On the 6th postoperative day, the patient experienced tightness of the chest and nausea after receiving a transfusion of fresh frozen plasma (FFP). When she manifested hypotension, the transfusion was discontinued. No cutaneous manifestation was observed. The patient's condition gradually improved soon after the administration of steroids. Results Her pretransfusion PLT count was 17.1 x 10(4) per microL. It decreased to 1.9 x 10(4) per microL 12 hours after transfusion and recovered to 15.4 x 10(4) per microL 8 days after transfusion. The donor of the FFP had a Type I CD36 deficiency. Flow cytometric analysis identified anti-CD36 isoantibody in the FFP. The cross-match between the patient's PLTs and the FFP was positive. The FFP induced the aggregation of PLTs derived from healthy adults. Conclusion This is the first reported case of life-threatening adverse effects and thrombocytopenia caused by passively transfused anti-CD36 isoantibody. The possibility of passive infusion of this antibody should be considered in the evaluation of life-threatening transfusion reactions followed by thrombocytopenia.

Background and Objectives Antibodies to CD36 (anti-CD36) are clinically important. As some platelet immunoglobulins produced by transfusion or pregnancy have been shown to induce platelet activation and to play roles in non-haemolytic transfusion reactions (NHTRs), we investigated the in vitro response of platelets to plasma containing anti-CD36. Materials and Methods Plasma containing anti-CD36, implicated in the development of NHTRs and subsequent thrombocytopenia, was incubated with CD36-positive platelets. Plasma-induced platelet activation was examined by evaluating platelet aggregation and RANTES (regulated on activation, normal, T-cell expressed, and presumably secreted) release. Results Platelet activation was induced by plasma alone in four out of 20 CD36-positive subjects. In seven subjects, platelet activation was synergistically induced by the combination of epinephrine priming and the plasma. The platelets of the nine remaining subjects failed to respond to the plasma. Platelet activation induced by either the plasma alone or by synergy with epinephrine required the involvement of FcγRIIa. The different responsiveness of the platelets was partially associated with the surface levels of CD36 and FcγRIIa, but not with FcγRIIa polymorphisms. Conclusions Plasma containing anti-CD36, implicated in the development of NHTRs, exhibited a platelet-activating capability. Additionally, platelets from healthy human subjects exhibited a considerable degree of heterogeneity in their responsiveness to this plasma. The heterogeneity of these responses may determine the occurrence of anti-CD36-related NHTRs.

In order to specify phylogenetic positions of the genera of Tofieldiaceae (Tofieldia, Triantha, Pleea, Harperocallis, Isidrogalvia), and to suggest reasonable circumscription of the family and genera of Tofieldiaceae, we determined DNA sequences of matK and rbcL for each genus of the family, and analyzed them phylogenetically with the 45 families and 113 genera of the monocots other than Tofieldiaceae, whose matK and rbcL sequences have already been reported. We found that Tofieldia, Triantha, Pleea, and Harperocallis form the same clade, which receives 100 % bootstrap support. This clade can be regarded as corresponding to Tofieldiaceae, and is embedded in the clade of Alismatales (98 %). On the other hand, Isidrogalvia is not included in this Tofieldiaceae clade, and positioned as sister to Narthecium (100 %), embedded in the clade of Nartheciaceae (Dioscoreales) (100 %). In the Tofieldiaceae, Pleea first diverges from the remaining three genera (100 %), and then, Harperocallis diverges from the Tofieldia-Triantha complex (100 %). In the Tofieldia-Triantha complex, five Tofieldia species form the same clade (100 %), and two Triantha species form another clade (100 %). Thus, Isidrogalvia should be transferred from Tofieldiaceae to Nartheciaceae. As Isidrogalvia, as well as the Nartheciaceae, have the carpels that are fully connate into a single style, Isidrogalvia fits the Nartheciaceae well with respect to carpel connation. After this transfer, the Tofieldiaceae correspond mainly to plants with almost free carpels and three styles. Pleea is better treated as an independent genus than included in Tofieldia. Triantha can be treated either as an independent genus or as congeneric with Tofieldia.

Osmotic diuretics are therapeutic agents used to reduce endolymphatic hydrops. However, glycerol-induced change in endolymph volume is followed by a rebound phenomenon. In this study, we investigated the rebound phenomenon occurring with isosorbide, an osmotic diuretic used as a therapeutic agent for Ménière’s disease in Japan. Forty guinea pigs underwent surgical obliteration of the endolymphatic sac. Thirty received isosorbide orally 1 month after surgery. These animals were sacrificed 3, 6, or 12 h after isosorbide intake. The remaining 10 animals served as controls. Quantitative assessment of changes in the endolymphatic space was performed light-microscopically. Isosorbide reduced cochlear endolymph volume, with a peak reduction 6 h after intake. Thereafter, no prominent rebound phenomenon was noted. Clinically, since isosorbide is orally administered every 8 h, rebound phenomenon need not be considered in the treatment with isosorbide.

We investigated the possible role of the endogenous nitric oxide (NO) synthase (NOS) inhibitors N-monomethyl-L-arginine (L-NMMA) and asymmetrical N, N-dimethyl-L-arginine (ADMA) in inhibiting urethral relaxation following estrogen supplementation in ovariectomized rabbits.A total of 16 mature Japanese White female rabbits were divided into 2 groups. In the control group rabbits were sacrificed 2 weeks after bilateral ovariectomy. In the estrogen group estradiol was administered subcutaneously for 2 weeks with the aid of sustained release pellet from 2 weeks after ovariectomy until sacrifice. Isolated urethra was cut into transverse strips for functional study and processed to determine endogenous NOS inhibitors, NOS activity, dimethylarginine dimethylaminohydrolase (DDAH) activity as a metabolizing enzyme of endogenous NOS inhibitors and cyclic guanosine monophosphate production.Electrical field stimulation produced NO mediated and neurogenic relaxation of the urethral strip in the presence of guanethidine and atropine under contraction with phenylephrine. Relaxation was significantly decreased in the estrogen group and accompanied by decreased cyclic guanosine monophosphate production. Sodium nitroprusside induced relaxation was not different between the 2 groups. The content of L-NMMA plus ADMA in the urethra was significantly increased in the estrogen group. Ca dependent NOS activity in the urethra remained unaffected. DDAH activity was significantly lower in the estrogen group.Estrogen supplementation leads to decreased NO mediated and neurogenic urethral relaxation through the accumulation of L-NMMA and ADMA in the urethra. The accumulation of NOS inhibitors is possibly brought about by impaired DDAH activity.

Delphinidin-3-rutinoside (D3R) is the major anthocyanin component in blackcurrant (Ribes nigrum L.) fruits. We investigated the relaxation mechanism of D3R in bovine ciliary smooth muscle (CM). D3R at a concentration of 10(-5) m produced a sustained and progressive relaxation during the contraction induced by endothelin (ET)-1 in the bovine CM specimens. After the pre-treatment with D3R, the anthocyanin exerted an inhibitory effect on the ET-1-induced contraction with a concomitant increase in cyclic GMP production and decreased phosphorylation ratio of myosin light chain (RLC). The inhibitory effect of D3R was significantly attenuated in the presence of either N(G)-nitro-L-arginine (NOARG) as a nitric oxide synthase (NOS) inhibitor, carboxy-PTIO as a NO scavenger, ODQ as an inhibitor of guanylyl cyclase, or BQ788 as a selective ET(B) receptor antagonist. The atteuation with NOARG was reversed by the addition of excess L-arginine. However, iberiotoxin as a Ca2+-activated K+ channel inhibitor, propranolol as a beta-adrenoceptor antagonist, and indomethacin as a cyclooxygenase inhibitor failed to modify the inhibitory effect of D3R. Scatchard plot analysis revealed that the [125I]-ET-1 binding site constituted a single population with Kd of 54.5+/-4.6 nm and maximum binding site (B(max)) of 168.4+/-25.4 fmol/mg protein in the ciliary epithelium (CE), and Kd of 141.7+/-18.0 nm and B(max) of 357.7+/-35.8 fmol/mg protein in CM. [125I]-ET-1 binding was completely displaced by BQ788 with K(i) values of 56.7+/-10.8 pm in CE and 93.4+/-23.3 pm in CM. Meanwhile, partial displacement (approximately 40%) was observed by BQ123 as a selective ET(A) receptor antagonist in both preparations. ET(B) receptor was predominant subtype in CE and CM, whereas kinetics of the binding was different in two preparations. These results suggest that D3R possibly stimulates ET(B) receptors to produce/release NO, and results in an inhibition of myosin RLC phosphorylation and/or acceleration of dephosphorylation, thereby causing relaxation and producing an inhibitory effect on the ET-1-induced contraction in the bovine CM.

The intercellular space in the stria vascularis (intrastrial space) is a closed space and isolated from both the endolymph and the perilymph in normal tissue. Loop diuretics such as bumetanide and furosemide cause an acute enlargement of the intrastrial space in association with a decline in the endocochlear potential. It is known that bumetanide inhibits the Na+-K+-2Cl- cotransporter, which is expressed abundantly in the basolateral membrane of marginal cells. We studied ionic mechanisms underlying the bumetanide-induced enlargement of the intrastrial space using perilymphatic perfusion in guinea pigs. Perilymphatic perfusion with artificial perilymph containing 100 microM bumetanide caused marked enlargement of the intrastrial space, as reported previously. Removal of K+ from the perilymph did not affect the bumetanide-induced enlargement, whereas removal of Na+ from the perilymph inhibited it almost completely. Perilymph containing 1 mM amiloride also inhibited the enlargement of the intrastrial space almost completely. These results indicate that perilymphatic Na+, but not K+, and amiloride-sensitive pathways are essential to the bumetanide-induced enlargement of the intrastrial space. Two possible pathways could yield these results. Na+ in the perilymph could enter the endolymph via Reissner's membrane or the basilar membrane; Na+ in the endolymph would then be taken up by marginal cells via the apical membrane and secreted into the intrastrial space by Na+-K+-ATPase in the basolateral membrane of them. Another, less likely possibility is that Na+ in the perilymph is transported into basal cells or fibrocytes in the spiral ligament, then into intermediate cells via gap junctions, and finally secreted into the intrastrial space via Na+-K+-ATPase of intermediate cells.

Bacterial lipopolysaccharide (LPS), the major structural component of the outer wall of Gram-negative bacteria, is a potent activator of macrophages. Activated macrophages produce a variety of inflammatory cytokines. Excessive production of cytokines in response to LPS is regarded as the cause of septic shock. On the other hand, macrophages exposed to suboptimal doses of LPS are rendered tolerant to subsequent exposure to LPS and manifest a profoundly altered response to LPS. Increasing evidence suggests that monocytic cells from patients with sepsis and septic shock survivors have characteristics of LPS tolerance. Thus, an understanding of the molecular mechanisms underlying activation and deactivation of macrophages in response to LPS is important for the development of therapeutics for septic shock and the treatment of septic shock survivors. Over the past several years, significant progress has been made in identifying and characterizing several key molecules and signal pathways involved in the regulation of macrophage functions by LPS. In this paper, we summarize the current findings of the functions of the LPS receptor complex, which is composed of CD14, Toll-like receptor 4 (TLR4), and myeloid differentiation protein-2 (MD-2), and the signal pathways of this LPS receptor complex with regard to both activation and deactivation of macrophages by LPS. In addition, recent therapeutic approaches for septic shock targeting the LPS receptor complex are described.

Trimenia moorei (Oliv.) Philipson is an andromonoecious liane with >0·40 of the total flower buds maturing as bisexual flowers. Male and bisexual flowers are strongly scented with pollen, anther sacs and receptacle scars testing positively for volatile emissions. Scent analyses detect over 20 components. The major fatty acid derivative is 8-heptadecene, and 2-phenylethanol dominates the benzenoids. While hover-flies in the genera Melangyna and Triglyphus contact the stigma with their probosces, the stigma secretes no free-flowing, edible fluids. Copious pollen is the only edible reward consumed by hover-flies (Syprhidae), sawflies (Pergidae) and bees in the families Apidae, Colletidae and Halictidae. All these insects carried pollen of T. moorei on their heads, legs and thoraces and female bees in the genera Apis, Exoneura, Leioproctus and Lasioglossum stored pollen on their hind legs. Pollen traps also indicate that pollen is shed directly into the air, permitting wind pollination. When bisexual flower buds are bagged (isolated from insect foragers) on the liane then subjected to a series of hand-pollination experiments after perianth segments open, the structural analyses of pollen–carpel interactions indicate that T. moorei has a trichome-rich dry-type stigma with an early-acting self-incompatibility (SI) system. Bicellular pollen grains deposited on stigmas belonging to the same plant germinate but fail to penetrate intercellular spaces, while grains deposited following cross-pollination reach the ovule within 24 h. Fluorescence analyses of 76 carpels collected at random from unbagged (open-pollinated) flowers on five plants indicates that at least 64 % of carpels are cross-pollinated in situ. Trimenia moorei is the first species within the ANITA group, and second within reilictual-basal angiosperm lineages, to exhibit stigmatic SI in combination with dry-type stigma and bicellular pollen, a condition once considered to be atypical for angiosperms as a whole but now known to be present in numerous taxa.

RANTES, one of the PLT-derived biologic response modifiers, accumulates in PLT concentrates (PCs) during storage and may play a causative role in nonhemolytic transfusion reactions (NHTRs) after PC transfusion.To investigate the association of RANTES with NHTRs, the biologic activity of RANTES in the supernatant of stored PC at the intravascular concentration expected after PC transfusion was assessed by examining chemotaxis and histamine release in human basophils. In addition, the levels of RANTES in PCs involved in NHTRs were compared with those in PCs causing no transfusion reactions.The supernatant of PC diluted to contain 1 nM RANTES significantly increased the migration of and release of histamine from basophils. Neutralizing antibody to RANTES suppressed the PC-triggered migration, but not histamine release. The levels of RANTES in PCs involved in NHTRs after PC transfusion were comparable to those in PCs that did not cause any transfusion reactions.RANTES that accumulated in PCs during storage was biologically active in a basophil chemotaxis assay at the intravascular concentration expected after PC transfusion. However, the NHTRs after PC transfusion were not simply related to the RANTES level in PCs.

Thrombopoietin (TPO) is a hematopoietic cytokine that regulates megakaryocytosis and thrombocytosis by binding to its receptor (c-Mpl). The signaling pathways downstream of c-Mpl include the Ras/Raf/MAP kinase and JAK/STAT pathway and are transduced into the regulation of immediate early-, early- and delayed-response genes. How these genes couple c-Mpl activation to the biochemical machinery of cell growth and cell cycle progression in hematopoietic cells is still unclear. UT-7/TPO is a recently characterized TPO-dependent cell line. Using RNA fingerprinting with arbitrarily primed PCR (RAP-PCR) to identify the TPO-regulated genes in this cell line, we found that the mRNA expression of nucleolin was upregulated in the UT-7/TPO cells in response to TPO. Concomitantly, the TPO-stimulated cells expressed an increased amount of full length nucleolin as determined by immunoblot analysis. The TPO-induced upregulation of nucleolin mRNA was not inhibited by the MEK1/2 inhibitor PD98059, suggesting that ERK/MAPK activation is not necessary for elevation of nucleolin gene expression in response to TPO in UT-7/TPO. Nucleolin is a multifunctional nucleolar protein thought to be involved in many cellular processes, including ribosome biogenesis, the processing of ribosomal RNA (rRNA), mRNA stability, transcriptional regulation, and cell proliferation. Thus, these results indicate that the upregulation of nucleolin mRNA and protein may be important for the TPO-induced effects of hematopoietic cells.

Loop diuretics such as bumetanide and furosemide cause an acute enlargement of the intrastrial space of the stria vascularis, with an associated decline in the endocochlear DC potential (EP). The aim of this study was to determine the role played by the Na+-K+-ATPase in the bumetanide-induced enlargement of the intrastrial space, and to examine the importance of the balance between the activities of the Na+-K+-2Cl- cotransporter and the Na+-K+-ATPase to the physiological function of the stria vascularis.Albino guinea pigs were used in experiments involving perilymphatic perfusion, EP measurement and electron microscopy. The effects of bumetanide on the stria vascularis were examined following inhibition of the Na+-K+-ATPase by ouabain. Ouabain was administered to the perfusate and, when the EP reached 0 mV, both ouabain and bumetanide were administered.Although there was no enlargement of the intrastrial space, vacuoles were apparent in marginal cells. The vacuolar change in marginal cells was similar to that caused by ouabain alone.This study indicates that the enlargement of the intrastrial space requires not only the blockade of the Na+-K+-2Cl- cotransporter but also normal activity of the Na+-K+-ATPase, and suggests that the bumetanide-induced enlargement of the intrastrial space resulted from the imbalance between the activities of the Na+-K+-2Cl- cotransporter and the Na+-K+-ATPase.

Endothelin (ET)-1, which had been discovered as the most potent vasoconstrictive peptide, plays active roles in the various biological responses. However, it is controversial how is ET-1 involved in the vascular remodeling process. Therefore, present experiments were performed to investigate the role of ET-1 for the initiation/progression of intimal hyperplasia. Intimal hyperplasia was caused with the time to peak of 4 to 6 weeks after endothelial removal of the rabbit carotid artery and accompanied by increased ET-1 content as well as ET receptor density within the hyperplastic vessel wall. ET-1 receptors could be classified into three subtypes of ETA, ETB and tentative nonETA/nonETB. TUNEL- and Ki-67-positive cells were detectable with the time to peak of 1 to 2 weeks, whereas all positive cells disappeared within 6 weeks. ATZ1993 as a mixed type antagonist for ET-A, ET-B and nonETA/nonETB receptor subtypes inhibited the intimal hyperplasia, of which inhibition was accompanied by increased TUNEL- and p53-positive cells and decreased Bcl-2-positive cells. ET-1 accelerated the [3H]-thymidine incorporation by cultured vascular smooth muscle cells and, on the other hand, reduced TUNEL-positive cells, which was caused by the serum deprivation. The reduction of TUNEL-positive cells with ET-1 was blocked by ATZ1993 or BQ123 as an antagonist for ETA receptor, but unaffected by BQ788 as an antagonist for ETB receptor. These results strongly suggest that ET-1 plays crucial roles as a mitogen and an inhibitory factor of apoptosis in the vascular remodeling process after endothelial removal.

We investigated the different distributions of nitric oxide synthase (NOS) and soluble guanylate cyclase activities in the detrusor and proximal urethra in the rabbit.The detrusor and proximal urethra were excised, the mucosa and muscularis were separated and each layer was homogenized to measure the activities of NOS as well as baseline and sodium nitroprusside (SNP) activated soluble guanylate cyclase. Isometric tension experiments were performed in detrusor and urethral strips with and without mucosa.In the detrusor NOS and sodium nitroprusside activated soluble guanylate cyclase activities in the mucosa were much higher than in the muscularis. While in the proximal urethra NOS activity in the 2 layers was similar, SNP activated soluble guanylate cyclase activity in the muscularis was 2.8 times higher than in the mucosa. Removing the mucosa reduced nitric oxide (NO) mediated neurogenic relaxation in the proximal urethra, while no relaxation responses could be elicited by electrical field stimulation with or without mucosa in the detrusor. On the other hand, removing the mucosa did not affect SNP or 8-bromo-cyclic guanosine monophosphate induced relaxation in the detrusor and proximal urethra.In the detrusor NOS and soluble guanylate cyclase activities were mainly detected in the mucosa, while in the urethra these activities were distributed throughout the mucosa and muscularis. Also, the mucosa in the urethra but not in the detrusor contributed to electrical field stimulation induced, NO mediated relaxation. These findings confirm previous studies demonstrating that NO has a different role in the detrusor than in the proximal urethra.

BACKGROUND: An ex vivo culture system was previously established for stem cell expansion using human marrow stromal cells and serum-free medium. However, the stromal cells were prepared using long-term culture medium containing horse serum and FCS, which may transmit infectious diseases of xenogeneic origin. In this study, therefore, a method was established to prepare stromal cells using an AB serum-based medium. In the case that serum from a transplant recipient or PBPC donor is available, additional infectious diseases would not be transmitted. STUDY DESIGN AND METHODS: Cord blood CD34+ cells were cultured with thrombopoietin, stem cell factor, and flt3/flk2 ligand on a monolayer of human marrow primary stromal cells prepared using long-term culture medium or AB serum-based medium. After 2 weeks, clonogenic progenitor activity and SCID mouse-reconstituting cell activity were assayed. mRNA expression of cytokines and Notch ligand by stromal cells was also examined. RESULTS: There were no remarkable differences in expansion-supporting activity and mRNA expression between stromal cells established by the two methods. CONCLUSION: An ex vivo expansion system completely based on AB serum has been established.

Objective: A change in the summating potential (SP) obtained from a patient with posterior fossa meningioma associated with surgical removal of the tumor was reported and possible mechanisms underlying the change are discussed. Methods: This paper is a retrospective case review. A tertiary care referral center was the setting for the study. The authors present a 46-year-old female patient with posterior fossa meningioma, which was removed surgically. Pure tone audiometry and electrocochleography (ECoG) were performed before and after surgical removal of the tumor. Results: Pure tone audiogram showed normal hearing levels before surgery, and there were no apparent postoperative changes in hearing levels. ECoG showed a normal compound action potential (AP) level and an elevated SP/AP ratio (0.53) before surgery. After the tumor removal, the SP/AP ratio became 0.16, which was a normal level in our department. Conclusions: The increased SP/AP ratio was normalized after surgery. Possible mechanisms underlying the increase in SP/AP ratio are an inhibition of the olivocochlear bundle function and endolymphatic hydrops.

We examined whether endogenous inhibitors of nitric oxide (NO) synthesis are involved in the impaired cavernosal relaxation with ischemia in rabbits. Two weeks after cavernosal ischemia caused by partial vessel occlusion, endothelium-dependent and electrical field stimulation (EFS)-induced neurogenic NO-mediated relaxations, but not sodium nitroprusside (SNP)-induced relaxation, were significantly impaired in the isolated corpus cavernosum. The Ca2+-dependent NO synthase (NOS) activity and the basal and stimulated cGMP productions with carbachol or EFS were significantly decreased after ischemia. Supplementation of excessl-arginine partially recovered both of the impaired relaxations. The contents of NG-monomethyl-l-arginine (l-NMMA) and asymmetric NG, NG-dimethyl-l-arginine (ADMA) but not l-arginine and symmetric NG, N′G-dimethyl-l-arginine (SDMA) were increased in the cavernosal tissues after ischemia. Authentic l-NMMA and ADMA but not SDMA concentration dependently inhibited both relaxations without affecting the relaxation produced by SNP in the control. Excess l-arginine abolished the inhibition with l-NMMA and ADMA. These results suggest that the impaired NO-mediated cavernosal relaxations after ischemia are closely related to the decreased NOS activity and the increased accumulation of l-NMMA and ADMA.

Background In vitro maintenance and expansion of human hematopoietic stem cells is crucial for many clinical applications, and investigators have been using xenogeneic, especially munne, stromal cells for stem-cell expansion. In addition, many such culture systems utilize FCS-containing medium or serum-free medium that contains human- or animal-derived proteins. However, the possible transmission of infectious diseases has led to a debate about the safety of the delivery of grafts expanded in culture using cells and proteins of allogeneic or xenogeneic origin. Using primary human BM stromal cells, we have established an AB serum-based co-culture system to expand human primitive progenitors and transplantable stem cells. Methods Cord blood CD34 + cells were cultured on a monolayer of human BM-derived primary stromal cells with thrombopoietin (TPO), stem-cell factor (SCF) andflt3/flk2 ligand (FL) in the presence of either FCS or AB serum. One to three weeks later, cells were examined for total cells, CD34 + cells, CD34 + CD38 − cells, and clonogenic progenitors. SCID mouse reconstituting cell (SRC) activity was also studied. Results Three weeks of culture with TPO, SCF, and FL supported more than a 250-fold expansion of CD34 + cells, CD34 + CD38 − cells and CFU-C, regardless of the kind of serum used. SRC assay revealed that transplantable stem cells were moderately expanded as well. Discussion This ex vivo expansion system should prove valuable in clinical settings in which stromal cells and serum are available from recipients or stem-cell donors.

This study investigated whether activated protein C (APC) improves the cerebral vasospasm in an experimental subarachnoid hemorrhage that was produced by the intracisternal injection of autologous blood. Male rabbits were divided into the following four groups: APC 0.1-and 0.5-mg groups, in which 0.1 and 0.5 mg APC were injected into the cisterna magna, respectively; a placebo group, in which saline was injected instead of APC; and a sham operation group that did not get injections of autologous blood, APC, and saline. On day 2, amount of clot in the basal cistern was significantly (p < 0.01) decreased in the APC 0.5-mg group. Percent diameter of the basilar artery on day 2 to that before injecting the blood was angiographically determined as 97.1 +/- 3.8% in the APC 0.5-mg group, which was significantly (p < 0.001) greater than the corresponding value in the placebo group (74.8 +/- 3.4%). The impaired endothelium-dependent relaxation following subarachnoid hemorrhage was normalized in the APC 0.5-mg group (p < 0.0001). These results suggest that APC would improve cerebral vasospasm following subarachnoid hemorrhage, possibly by decreasing the amount of subarachnoid clot and normalizing the impaired nitric oxide production/release.

A new lignan 1 was isolated together with the five known lignans 2-6 and four sesquiterpenes 7-10 from the seeds of Magnolia praecocissima. The structure of 1 was elucidated by analysis of spectroscopic data and chemical reaction. Furthermore, the absolute configurations of 1, 2, and 3 were determined by the modified Mosher's method.

To examine the possibility of adoptive cellular immunotherapy such as donor lymphocyte infusion using ex vivo expanded cord blood (CBL) lymphocytes, the potential expansion ability of CBL lymphocytes and the function of expanded CBL lymphocytes were evaluated.Mononuclear cell fractions derived from CBL or peripheral blood (PBL) were placed in anti-CD3 monoclonal antibody-coated flasks and cultured in the presence of recombinant human interleukin-2 for 4 days. Cells then were transferred to noncoated flasks and cultured for another 2 weeks. On day 14, polyclonality, cell surface markers, killer activity, and intracellular cytokine profiles were evaluated.Cells were polyclonally expanded. The differences in cumulative fold expansion on day 14 between CBL [1174 +/- 637 (292-1939), n = 6] and PBL [1247 +/- 568 (517-2328), n = 9] were not significant (p = 0.95). Phenotypic patterns of both expanded CBL and PBL were similar. CD4/CD8 ratio of expanded CBL appeared to remain greater than 1 on day 8. In contrast, that of expanded PBL became less than 1. In both cases, approximately 20% of cells had the CD3(+)CD8(+)CD56(+) phenotype. At an effector to target ratio (E/T) of 40:1, the natural killer activity of expanded CBL (64.5% +/- 10.8%, n = 9) was significantly higher than that of expanded PBL (48.3% +/- 16.8%, n = 9) (p0.01, Mann-Whitney U-test). However, there was no significant difference in lymphokine-activated killer activity between expanded CBL (45.3% +/- 25.2%, n = 7) and expanded PBL (67.2% +/- 12.3%, n = 7). Interferon-gamma-producing cells were dominant in both cases.It was feasible to achieve approximately 1000-fold expansion of CBL, and the phenotype and function of expanded CBLs were essentially equivalent to those of expanded PBL. This suggested that ex vivo expanded CBL may be applied to adoptive cellular immunotherapy such as donor lymphocyte infusion.

Protein arginine N-methyltransferases (PRMTs) catalyse the methylation of guanidinonitrogen(s) of arginine to produce NG-monomethyl-L-arginine (L-NMMA), asymmetric NG,NG-dimethyl-L-arginine (ADMA) and symmetric NG,NG-dimethyl-L-arginine (SDMA), which are subsequently released into the cytoplasm following proteolysis. Free intracellular L-NMMA and ADMA, but not SDMA, are inhibitors of all three isoforms of nitric oxide synthases (nNOS, eNOS and iNOS). L-NMMA and ADMA, but not SDMA, are actively metabolized by dimethylarginine dimethylaminohydrolase (DDAH) to L-citrulline and methylamine (and dimethylamine). Free methylarginines are detectable in cell cytosol, plasma and tissues. Elevated ADMA has been detected in the plasma of patients or experimental animals with hypercholesterolemia, renal failure, atherosclerosis, hypertension, thrombotic microangiopathy, peripheral arterial occlusive disease and in the regenerated endothelial cells after angioplasty. Moreover, in the non-cardiovascular field, ADMA was increased in the urethral tissue following ischemia and in the plasma of patients with schizophrenia and multiple sclerosis. Altered biosynthesis of NO has been implicated in the pathogenesis of these diseases, and it is possible to consider that the accumulation of endogenous L-NMMA and ADMA underlies the impaired NO generation and increased O2- production. We described herein the biosynthesis, transmembrane transport, metabolic pathway and possible pathophysiological roles of endogenous methylarginines.