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Axl and Mer are members of the TAM (Tyro3-Axl-Mer) family of receptor tyrosine kinases. Previously, we reported that enzyme-mediated inhibition of Mer by an Axl/Mer dual inhibitor led to retinal toxicity in mice, whereas selective Axl inhibition by compound 1 did not. On the other hand, compound 1 showed low membrane permeability. Here, we designed and synthesized a novel series of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine derivatives and evaluated their Axl and Mer inhibitory activities, leading to identification of ER-001259851-000 as a potent and selective Axl inhibitor with drug-likeness and a promising pharmacokinetic profile in mice.

Axl and Mer are a members of the TAM (Tyro3-Axl-Mer) family of receptor tyrosine kinases, which, when activated, can promote tumor cell survival, proliferation, migration, invasion, angiogenesis, and tumor-host interactions. Chronic inhibition of Mer leads to retinal toxicity in mice. Therefore, successful development of an Axl targeting agent requires ensuring that it is safe for prolonged treatment. Here, to clarify whether enzyme inhibition of Mer by a small molecule leads to retinal toxicity in mice, we designed and synthesized Axl/Mer inhibitors and Axl-selective inhibitors. We identified an Axl/Mer dual inhibitor 28a, which showed retinal toxicity at a dose of 100 mg/kg in mice. Subsequent derivatization of a pyridine derivative led to the discovery of a pyrimidine derivative, 33g, which selectively inhibited the activity of Axl over Mer without retinal toxicity at a dose of 100 mg/kg in mice. Additionally, the compound displayed in vivo anti-tumor effects without influencing body weight in a Ba/F3-Axl isogenic subcutaneous model.

Effects of liposomal particles on immune function have not been adequately investigated. Earlier reports indicate that intravenous injection of rats with pegylated liposomes comprising chemically defined specific lipids produces myeloid derived suppressor-cell (MDSC)-like cells in the spleen. After liposome injection, we sought a cell surface marker expressed specifically on splenic macrophages. Then we assessed the immunosuppressive activity of macrophages positive for the marker. Furthermore, we investigated whether immunosuppression induction is an immunopharmacological action specific to this pegylated liposome, or not. After using a microarray system to screen genes enhanced by this liposome, we evaluated cell surface expression of gene products using flow cytometry. Liposomes of several kinds, each comprising one type of phospholipid, were prepared and evaluated for their ability to induce T-cell suppression. Microarray analysis indicated enhanced B7-H3 expression. Flow cytometry revealed that the B7-H3 molecule was expressed on splenic macrophages after liposome injection. B7-H3+ macrophages were positive for iNOS. Removing B7-H3+ cells restored T-cell proliferation. Similarly to this liposome, various liposomes with different long chain fatty acids induced T-cell suppression when accumulated in the spleen. Immunosuppressive cells induced by this pegylated liposome closely resemble MDSCs, especially B7-H3+ MDSCs. Immunosuppression induction is not a phenomenon specific to this liposome. Accumulation of long chain fatty acid in macrophages by internalization of liposomal nanoparticles might be related to macrophage acquisition of immunosuppressive activity in vivo.

BACKGROUND AND OBJECTIVES There are few reports on renal dysfunction in the remote period after biventricular repair, and biomarkers for early detection of renal dysfunction are not well understood. We examined whether early fluctuation of biomarkers of renal function occurs in the remote period after biventricular repair in patients with congenital heart disease (CHD). METHODS Fourteen patients with CHD after biventricular repair were included. The examination values obtained by cardiac catheterization test and renal function indices based on blood and urine sampling were compared. RESULTS The median estimated glomerular filtration rate (eGFR) of creatinine was 113 mL/min/1.73 m², and the median eGFR of cystatin C was 117 mL/min/1.73 m². A urine albumin-to-creatinine ratio (UACR) ≥10 mg/gCr was considered a risk factor for cardiovascular disease in 6 (43%) patients. There was a significant difference in right ventricular ejection fraction and deviation in right ventricular end-diastolic volume from the normal value between the 2 groups divided by UACR. Cyanosis before biventricular repair was noted in 2 (25%) patients with UACR

Antibody-mediated phagocytosis of platelets using a flow cytometric monocyte-based phagocytosis assay (FMPA) has been shown to predict the outcome of platelet transfusion. The easy adherence between platelets and monocytes even in the absence of an antibody is regarded as one of limitations of the FMPA. To improve the FMPA for prediction of transfusion outcome, we used the pH-sensitive dye pHrodo succinimidyl ester (pHrodo-SE), which has weak fluorescence at neutral pH and has increased fluorescence intensity in low pH conditions such as in lysomes. Platelets stained with pHrodo-SE were sensitized with an HLA class I monoclonal antibody (w6/32 clone) or anti-HLA class I containing antisera. The platelets were incubated with monocyte-enriched mononuclear cells. Phagocytic activity was assessed by the percentage of monocytes that phagocytosed platelets. Sensitization of platelets with w6/32 significantly increased platelet phagocytosis by monocytes in dose- and time-dependent manners. Anti-HLA class I antibody-containing sera caused platelet phagocytosis in a cognate antigen-antibody-dependent manner. There was a significant correlation (r=0.69, p

Background Neonatal diabetes mellitus (NDM) is a monogenic insulin-dependent diabetes that develops within 6 months of age. The progression of hyperglycemia until diagnosis is unknown. Glycemic control indicators at diagnosis are useful to estimate the extent and duration of hyperglycemia. We recently established that age-adjusted glycated albumin (GA) is a useful indicator of glycemic control, regardless of age. Objective To compare the levels of various glycemic control indicators at diagnosis between NDM and other types of insulin-dependent diabetes mellitus. Patients and Methods We included 8 patients with NDM, 8 with fulminant type 1 diabetes (FT1D), and 24 with acute-onset autoimmune type 1 diabetes (T1AD). Plasma glucose, glycated hemoglobin (HbA1c), GA, and age-adjusted GA (calculated as previously reported) were measured and compared. Results There were no significant differences in the plasma glucose levels of the group of patients with NDM and those with FT1D or T1AD. HbA1c and GA levels in the NDM group were not significantly different from those in the FT1D group, and both indicators were lower than those in the T1AD group. Age-adjusted GA levels in the NDM group did not differ significantly from those in the T1AD group, but were higher than those in the FT1D group. Conclusions These findings suggest that the time-course of plasma glucose elevation in NDM until diagnosis is similar to that in T1AD. In addition, the high age-adjusted GA value at diagnosis of NDM indicates that this test is useful for assessing chronic hyperglycemia in NDM.

The recent clinical application of perfusion technology for the machine preservation of donation after cardiac death (DCD) grafts has some advantages. Oxygenation has been proposed for the preservation of DCD liver grafts. The aim of this study is to clarify whether the use of HbV-containing preservation solution during the subnormothermic machine perfusion (SNMP) of the liver graft improves the graft function of DCD porcine livers in an ex vivo reperfusion model. Pig livers were excised after 60 minutes of warm ischemic time and were preserved under one of three preservation conditions for 4 hours. The preservation conditions were as follows: 4°C cold storage (CS group; N = 5), Hypothermic machine preservation (HMP) with UW gluconate solution (HMP group; N = 5), SNMP (21°C) with UW gluconate solution (SNMP group; N = 5), SNMP (21°C) with HbVs (Hb; 1.8 mg/dl) perfusate (SNMP+HbV group; N = 5). Autologous blood perfusion was performed for 2 hours in an isolated liver reperfusion model (IRM). The oxygen consumption of the SNMP and SNMP+HbV group was higher than the HMP groups (p < 0.05). During the reperfusion, the AST level in the SNMP+HbV group was lower than that in the CS, HMP and SNMP groups. The changes in pH after reperfusion was significantly lower in SNMP+HbV group than CS and HMP groups. The ultrastructural findings indicated that the mitochondria of the SNMP+HbV group was well maintained in comparison to the CS, HMP and SNMP groups. The SNMP+HbVs preservation solution protected against metabolic acidosis and preserved the liver function after reperfusion injury in the DCD liver.

Asplenia syndrome is frequently complicated by a total anomalous pulmonary venous connection. Pulmonary venous obstruction, following total anomalous pulmonary venous connection surgery, is one of the risk factors for morbidity and mortality. In some patients, the pulmonary vasculature is abnormal even in the absence of clinical evidence of pulmonary venous obstruction. We hypothesized that a change in the pulmonary hemodynamics could indicate the abnormality of pulmonary vein in a patient with asplenia, single right ventricle, and total anomalous pulmonary venous connection, following Fontan procedure. Here, we present a case of asplenia, single right ventricle, total anomalous pulmonary venous connection, and right pulmonary venous obstruction in which evidence of a potential left pulmonary venous obstruction was obtained following the administration of inhaled nitric oxide and oxygen.

Protein-losing enteropathy (PLE) is a rare and life-threatening complication that occurs after the Fontan procedure. We herein report the case of an 11-year-old Japanese boy who developed PLE six times after undergoing the Fontan procedure. High-dose spironolactone therapy has been effective for 2 years. His high level of serum aldosterone decreased to a nearly normal range and spironolactone may have a diuretic and anti-inflammatory potential.

Antibody-mediated phagocytosis of platelets using a flow cytometric monocyte-based phagocytosis assay (FMPA) has been shown to predict the outcome of platelet transfusion. The easy adherence between platelets and monocytes even in the absence of an antibody is regarded as one of limitations of the FMPA. To improve the FMPA for prediction of transfusion outcome, we used the pH-sensitive dye pHrodo succinimidyl ester (pHrodo-SE), which has weak fluorescence at neutral pH and has increased fluorescence intensity in low pH conditions such as in lysomes. Platelets stained with pHrodo-SE were sensitized with an HLA class I monoclonal antibody (w6/32 clone) or anti-HLA class I containing antisera. The platelets were incubated with monocyte-enriched mononuclear cells. Phagocytic activity was assessed by the percentage of monocytes that phagocytosed platelets. Sensitization of platelets with w6/32 significantly increased platelet phagocytosis by monocytes in dose- and time-dependent manners. Anti-HLA class I antibody-containing sera caused platelet phagocytosis in a cognate antigen-antibody-dependent manner. There was a significant correlation (r=0.69, p

Context: Myeloid-derived suppressor cells (MDSCs) are a subset of immature myeloid cells that function as immunosuppressive cells in various pathological conditions. Membrane-derived microvesicles are thought to be involved in MDSC induction. Earlier reports have described that injection of considerable amount of liposome into rat can suppress Con A-induced splenic T-cell proliferation. Liposome-internalized cells expressing CD11b/c suppress T-cell proliferation. Nitric oxide (NO) appears to be involved in the suppression. We speculated that, similarly to membrane-derived microvesicles, liposomal microparticles can induce MDSC-like cells in vivo. Objectives: To confirm our speculation we investigated dose-dependency of the suppressive effect, the effect of liposome on the induction of inducible NO synthase (iNOS), and anti-CD3 antibody-stimulated T-cell proliferation and cytokine production. Materials and methods: Liposome particles of 250 nm diameter were prepared and suspended in saline. Then, various amounts of liposomal suspension were injected intravenously into rats. After 24 h, rat spleens were removed and concanavalin A (or anti-CD3 antibody) stimulated-splenic T-cell proliferation and the production of iNOS, NO and cytokines were evaluated. Results: T-cell proliferation was suppressed dose-dependently by liposome injection. The immunosuppressive cell exerts its suppressive activity in a dose-dependent manner. The suppression was eliminated by iNOS inhibitor. iNOS was detected in liposome-loaded splenocytes. Anti-CD3 antibody-stimulated T-cell proliferation was also inhibited. Enhanced production of IL-10 was observed. Conclusions: Liposomal microparticles can induce MDSC-like cells in vivo. The lipids which comprise liposomes might serve an important role in the induction of MDSCs in vivo.

Background Volume-reduced washed platelets (VR-wPLTs), which are prepared by concentrating platelets (PLTs) into a smaller volume of additive solution (AS), may prevent not only circulatory overload, but also adverse reactions caused by plasma components. Although VR-wPLTs may be quickly degraded due to high PLT concentrations, few studies have examined the effects of storage on VR-wPLTs. We examined here the in vitro properties of VR-wPLTs prepared with M-sol AS during their storage for 7 days. Study Design and Methods Platelet concentrates (PCs) were divided into two equal aliquots (control group and test group). After the centrifugation of both aliquots and removal of as much supernatant as possible, the pellet of the control group was resuspended in 160 mL of M-sol while that of the test group was resuspended in 80 or 40 mL of M-sol. The wPLTs of both groups were stored in polyolefin bags with agitation at 20 to 24°C for 7 days. Results The pH values of both groups were maintained at higher than 7.0 during the 7-day storage. Differences in %disk, CD62P, annexin V, percent hypotonic shock response, and aggregation values between the test group and control group were small for at least 2 days after washing. Conclusions The in vitro properties of VR-wPLTs were not markedly degraded for at least 2 days. Therefore, the storage properties of PLTs may be maintained in VR-wPLTs prepared at blood centers until they are administered to patients in hospitals.

Background As the presence of fetal hemoglobin (HbF) affects the accuracy of hemoglobin A1c (HbA1c) analysis methods, HbA1c measurement may not be a good indicator for patients with neonatal diabetes mellitus, whereas glycated albumin (GA) may be a good indicator. Objective To investigate whether total glycated hemoglobin (GHb) or HbF-adjusted HbA1c (adj-HbA1c) can act as a glycemic control marker in infants. Subjects and Methods Plasma glucose (PG), GA, HbF, GHb measured using the affinity method, and HbA1c measured using the latex-immunoturbidimetry (LA) or the high-performance liquid chromatography (HPLC) methods were determined in 26 full-term newborn infants aged 4–234 d. Adj-HbA1c was calculated as HbA1c/(total Hb − HbF). Results GHb, adj-HbA1c measured using the LA and the HPLC methods were 4.8 ± 0.5%, 4.5 ± 0.5%, and 4.7 ± 0.6%, respectively. GA was most positively correlated with PG (r = 0.696, p

Background: Activated platelets form heterogeneous aggregates of platelets and monocytes, which are involved in a variety of inflammatory disorders. Some anti-human leukocyte antigen (HLA) Class I antibodies have been shown to activate platelets. Materials and Methods: Human leukocyte antigen-A2-positive or HLA-A2-negative platelets were incubated with HLA-A2-negative peripheral blood mononuclear cells (PBMNCs) in the presence of anti-HLA-A2 serum at 37°C. The binding of platelets to monocytes was analysed by flow cytometry. The levels of IL-1 β and IL-8 in the culture supernatant were determined by ELISA. Results: Anti-HLA-A2 serum increased the formation of aggregates between monocytes and HLA-A2-positive platelets, but not HLA-A2-negative platelets, in a dose-dependent manner. Antiserum also increased the number of platelets bound to monocytes in a time-dependent manner. The addition of anti-P-selectin glycoprotein ligand (PSGL-1) mAb almost completely inhibited the formation of platelet–monocyte aggregates as well as the number of platelets bound to monocytes. When HLA-A2-positive or HLA-A2-negative platelets were incubated with HLA-A2-negative PBMNCs in the presence of anti-HLA-A2, the level of IL-1β and IL-8 in the supernatant of coculture was significantly higher in HLA-A2-positive platelets than in HLA-A2-negative platelets. The addition of anti-PSGL-1 mAb partially but significantly inhibited the production of both IL-1β and IL-8. Conclusions: The activation of platelets with anti-HLA Class I alloantibody caused the formation of platelet–monocyte aggregates, followed by the production of IL-1β and IL-8, in a cognate antigen–antibody manner. The adhesive interaction of P-selectin and PSGL-1 at least partially contributed to these phenomena.

OBJECTIVE To investigate the interactions between inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in response to ischaemia-reperfusion (I/R) of rabbit bladder. MATERIALS AND METHODS Rabbit bladders were exposed to 2 h of ischaemia by bilaterally clamping the major arteries entering the bladder and then a subsequent 36 h of reperfusion (I/R) with or without intraperitoneal administration of a selective iNOS inhibitor n-(3-(amynomethyl)benzyl)acetamidine (1400W) or a selective COX-2 inhibitor NS-398 given 1 h before killing. The bladder tissues were processed for isometric tension experiments, enzymatic NOS activitiy, tissue contents of nitrite/nitrate (NOX), cyclic guanosine monophosphate (cGMP) and COX activity determined by prostaglandin E2 (PGE2) production. RESULTS iNOS and constitutive NOS (cNOS) activities, NOX and PGE2 contents in the bladder tissues at 36 h after reperfusion were significantly higher than those in the sham group with no significant increase in cGMP. Treatment with 1400W abrogated the increases in iNOS activity and NOX as well as PGE2 without changing cNOS activity. In the tension experiments, a NOS substrate, l-arginine, induced detrusor contraction only in the I/R group, which was inhibited by 1400W or NS-398 but not by a selective soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazole[4,3-a]quinoxalin-1-one (ODQ). 8-Br-cGMP induced detrusor relaxation in the sham and I/R groups. Also, l-arginine increased NOX and PGE2 in the bladder tissues only in the I/R group, which were inhibited by pretreatment with 1400W. While, l-arginine increased cGMP contents in the I/R group and this increase was suppressed by ODQ but not by 1400W. CONCLUSION These results show that NO derived from an up-regulation of iNOS after I/R increases COX-2-derived PG via a cGMP-independent mechanism. NO-mediated activation of COX-2 may be an important mechanism for the modulation of bladder function after I/R injury.

The flowers of Asarum are usually regarded as scentless or sometimes to have a foul odor. Recently, we noticed that Asarum yaeyamense , endemic to Iriomote Island, Japan, has a floral fragrance with a distinct “fruity note.” To determine the chemical characteristics of this fragrance and whether “non-scented” Asarum species emit any volatiles, we collected floral scents of A. yaeyamense and related species ( A. lutchuense , A. hypogynum , A. fudsinoi , A dissitum , A. tokarense , and A. senkakuinsulare ) using headspace methods and analyzed these scents by gas chromatography–mass spectrometry (GC–MS). The results indicated that A. yaeyamense mainly emitted α-cedrene (tentatively identified), an unidentified sesquiterpene, methyl tiglate, and manoyl oxide (tentatively identified). Methyl tiglate may be a source of the “fruity note” in the A. yaeyamense fragrance. We also detected emissions of volatiles, mainly sesquiterpenes, from some “non-scented” Asarum species. This study constitutes a rare case of the detection of the emission of a diterpene (manoyl oxide) as a floral scent volatile.

OBJECTIVE To investigate whether activation of brain and spinal cholinergic pathways affects the micturition reflex in rats. MATERIALS AND METHODS The effects of intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration of neostigmine as a cholinesterase inhibitor and oxotremorine-M (OXO-M) as a muscarinic acetylcholine receptor (mAChRs) agonist, on the micturition reflex were evaluated by infusion cystometrography (CMG) in urethane-anaesthetized untreated rats or rats pretreated with capsaicin. RESULTS Neostigmine injected i.c.v. increased bladder capacity (BC) and pressure threshold (PT) dose-dependently, with an increase in maximum voiding pressure (MVP) and a decrease in voiding efficiency (VE) at higher doses. Also, neostigmine injected i.t. increased the BC and PT dose-dependently without changing MVP or VE, and these effects were not apparent in capsaicin-pretreated rats. In both routes, atropine as an antagonist of mAChRs, but not mecamylamine as a nicotinic-AChR antagonist, almost completely antagonized the effects of neostigmine. The rank order of potencies of the antagonists for increasing effects of BC induced by 1 nmol of neostigmine was: pirenzepine (an M1 mAChR antagonist) = atropine > 4-DAMP (an M3 mAChR antagonist) >> methoctramine (an M2 mAChR antagonist) and tropicamide (an M4 mAChR antagonist) via the i.c.v. route; and atropine > methoctramine > pirenzepine > tropicamide and 4-DAMP via the i.t. route, respectively. OXO-M injected via i.c.v. and i.t. had the same effects on BC, PT, MVP and VE as neostigmine by i.c.v. and i.t., respectively. CONCLUSIONS These results indicate that activation of muscarinic cholinergic mechanisms by the cholinesterase inhibitor in the brain and spinal cord can inhibit the micturition reflex, mainly by affecting afferent pathways. These mAChR-induced inhibitory effects seem to be mediated through M1/M3 receptor subtypes in the brain, while in the spinal cord, the M1/M2 receptor subtypes might be involved in inhibitory effects, which are mediated via inhibition of mechanoceptive C-fibre afferent pathways.

BACKGROUND: Type I allergic reactions such as urticaria-like manifestations constitute a large percentage of transfusion-related adverse events. Along with donor factors, patient factors might be involved in these reactions. Sera from some patients with chronic idiopathic urticaria show histamine-releasing activity (HRA). Sera from patients who develop Type I allergic reaction might possess HRA. STUDY DESIGN AND METHODS: Pretransfusion serum samples were collected. Mast cells were cultured from peripheral blood CD34+ cells and mixed with the serum samples. Cells with elevated intracytoplasmic Ca2+ concentrations were monitored using flow cytometry to evaluate Ca2+ influx–inducing activity (CaIA) in serum. The amount of histamine released into the supernatant was measured using an enzyme immunoassay kit to evaluate HRA. In some assays, cells were incubated with pertussis toxin (Ptx). RESULTS: CaIA values were higher (p

In the stria vascularis (SV), it is known that the Na+-K+-ATPase is expressed abundantly and its activity in the basolateral membrane of marginal cells is high. Ouabain, an inhibitor of the Na+-K+-ATPase, causes not only a decline in the endocochlear DC potential but also acute vacuolar formation in marginal cells. We studied the ionic mechanisms underlying the ouabain-induced vacuolar formation in marginal cells using perilymphatic perfusion in guinea pigs. Perilymphatic perfusion with 1 mM ouabain dissolved in the artificial perilymph for 50 min caused many vacuoles of a wide range of sizes in the apical cytoplasm of marginal cells, the bulging of marginal cells into the scala media and strial volume increase. Removal of K+ from the perilymph reduced the proportion of vacuoles and strial thickening, but the bulging of marginal cells remained. In contrast, the sizes of vacuoles were drastically reduced and extrusion of marginal cells into the scala media could not be observed in the absence of perilymphatic Na+. Furthermore, the total volume of SV was obviously reduced in comparison with the control. These results indicate that perilymphatic Na+ and K+ are responsible for these morphological changes caused by ouabain, and that perilymphatic Na+ plays an important role in the cellular volume regulation in SV in the presence of ouabain. It is supposed that the transport system of perilymphatic Na+ and K+ into marginal cells may contribute to vacuolar formation when ouabain is administered. Regarding Na+, we hypothesize two possibilities for the perilymphatic Na+ transporting pathway as follows. 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. 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. Regarding K+, it is expected that the K+ recycling pathway plays a role in ouabain-induced vacuolar formation in marginal cells.

Dimethylarginine dimethylaminohydrolase (DDAH) is an enzyme that metabolizes asymmetrical N G , N G -dimethyl- l -arginine (ADMA) and N G -monomethyl- l -arginine (MMA), which are competitive endogenous inhibitors of NO synthase. However, it remains unknown whether NO itself influences DDAH activity and/or ADMA/MMA contents to regulate NO generation via a biofeedback mechanism. The present study was designed to examine the effects of NO on intracellular ADMA and MMA contents and DDAH gene expression levels and enzymatic activities in cultured rat aortic endothelial cells. The NO donors SNAP and NOR3 did not influence DDAH-1 expression but increased DDAH-2 mRNA and protein levels in concentration-dependent manners. SNAP upregulated DDAH enzymatic activity and reduced the MMA and ADMA contents but did not affect the symmetrical N G , N ’ G -dimethyl- l -arginine and l -arginine levels, thereby negating a mediatory role for system y + in ADMA/MMA downregulation. The cGMP agonists 8-bromo-cGMP and C-type natriuretic peptide also stimulated DDAH-2 gene and protein expression levels and DDAH activity and increased the amount of nitrite/nitrate released into the culture supernatants. SNAP-induced DDAH-2 gene expression and DDAH activity were significantly inhibited by a protein kinase G inhibitor, KT5823, and a soluble guanylate cyclase inhibitor, ODQ, suggesting a mediatory role for cGMP in NO-induced DDAH-2 expression. Suppression of DDAH-2 mRNA using small interfering RNA technology abrogated NO-induced DDAH-2 expression. These data demonstrate that NO acts on endothelial cells to induce DDAH-2 expression via a cGMP-mediated process to reduce ADMA/MMA. Thus, the DDAH-2-ADMA/MMA-endothelial NO synthase regulatory pathway and NO-induced cGMP constitute a positive feedback loop that ultimately serves to maintain NO levels in the endothelial environment.

In the present experiments, we tried to elucidate whether changes in arginase activity, protein expression of arginase-I and -II, and NO production are involved in accelerating the intimal hyperplasia following administration of cigarette smoke extract (CSE). The intimal hyperplasia was caused by removing endothelial cells with the aid of balloon embolectomy catheter in the right carotid artery of the male rabbit. The left carotid artery underwent sham operation and served as control. CSE was prepared by bubbling a stream of cigarette smoke into phosphate buffered saline. Rabbits were given subcutaneously with CSE once a day for 5 weeks from 1 week before to 4 weeks after the surgery. The specimens were assessed histologically and the intima/media ratio (%) was evaluated as an index of the intimal hyperplasia. The accelerated intimal hyperplasia with CSE was accompanied by the augmentation of the impaired cyclic GMP production, enhanced overall arginase activity and up-regulation of arginase-I. Pearson's correlation coefficient analyses revealed the close relationships among the arginase activities in endothelial cells and smooth muscle layer, the intimal/media ratio and cyclic GMP production. These results suggest that the enhanced arginase activity together with facilitated up-regulation of arginase-I with CSE, which was associated with the augmented impairment of NO production, shed a new light on the processes associated with accelerating the intimal hyperplasia in rabbit carotid arteries following CSE.

In the present experiments, we tried to elucidate whether changes in arginase activity and protein expression of arginase I and II are involved in the occurrence of intimal hyperplasia in premenopausal human uterine arteries. They were obtained from thirty-four patients undergoing total abdominal hysterectomy with informed consent for the present study. All specimens were assessed histologically and the intima/media ratio (%) was evaluated as an index of the intimal hyperplasia. Thirteen patients out of 34 had histologically normal arteries (intima/media ratio = 18.1 ± 0.7%), whereas the remaining 21 patients had various degrees of intimal hyperplasia (intima/media ratio = 32.7 ± 2.3%), and these specimens were categorized as hyperplasic. Intimal hyperplasia was accompanied by impaired cyclic GMP production, enhanced overall arginase activity, and up-regulations of arginase I and II in endothelial cells and of arginase II in the smooth muscle layer. Pearson’s correlation coefficient analyses revealed the close relationships among the arginase activities in endothelial cells and smooth muscle layer, the intimal /media ratio, and cyclic GMP production. These results suggest that the enhanced arginase activity and expressions of two arginase subtypes shed new light on the processes associated with the occurrence of intimal hyperplasia in premenopausal human uterine arteries. Keywords:: intimal hyperplasia, arginase I and II, cyclic GMP, nitric oxide, human uterine artery

We designed the present experiments to investigate the involvement of endogenous nitric oxide synthase (NOS) inhibitors, dimethylarginine dimethylaminohydrolase (DDAH) as a hydrolyzing enzyme of the NOS inhibitors, NOS, arginase which shares l-arginine as a common substrate with NOS, and endothelin-1 (ET-1) in the pulmonary dysfunction after induction of experimental subarachnoid hemorrhage (SAH) in the rabbit. SAH was induced by injecting autologous blood into the cisterna magna, and controls were injected with saline. On day 2, pulmonary arteries were isolated for determinations. A significant impairment of the endothelium-dependent relaxation (EDR) caused by acetylcholine was found in 20 cases (43.5%) out of 46 SAH animals, and the same animals exhibited accompanying the significantly impaired cyclic GMP production, accumulated endogenous NOS inhibitors, attenuated DDAH activity, enhanced arginase activity and accumulated ET-1 within the vessel wall. Meanwhile, there were no differences in endothelial NOS activity per se and sodium nitroprusside-induced relaxation between the animals with an impaired EDR and those without such a change. ET-1 content within aortic wall was increased with concomitant decrease in cyclic GMP production after the intraperitoneal application of authentic monomethylarginine as a NOS inhibitor in the rat. The current results suggest that accumulated endogenous NOS inhibitors and enhanced arginase activity possibly bring about the impaired NO production, thereby attenuating the EDR and contributing to the accumulation of ET-1 within the vessel wall. The accumulated endogenous NOS inhibitors at least partly result from the decreased DDAH activity. These alterations may be relevant to the pulmonary dysfunction after induction of SAH.

Cigarette smoking is an independent risk factor for vasculogenic erectile dysfunction (ED). Nitric oxide (NO) has been demonstrated to be the principal mediator of cavernous smooth muscle relaxation and penile erection. Therefore, we examined whether or not enzyme activities and factors involved in the NO generation pathway are affected in rabbit corpus cavernosum after administration of nicotine- and tar-free cigarette smoke extract (CSE). CSE was prepared by bubbling a stream of cigarette smoke into phosphate-buffered saline. CSE was injected subcutaneously into adult male rabbits once a day for 5 wk. In the CSE group, significantly decreased cyclic GMP production as a marker of NO generation was associated with attenuated overall nitric oxide synthase (NOS) activity, enhanced arginase activity, accumulation of endogenous NOS inhibitors such as monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), and decreased dimethylarginine dimethylaminohydrolase (DDAH) activity as an metabolizing enzyme of endogenous NOS inhibitors. Neuronal NOS (nNOS) and DDAH I protein expression were decreased without altering endothelial NOS expression, while arginase I expression was upregulated. These results suggest that impaired NO production would result from blunted NOS activity, which is possibly brought about by the downregulation of nNOS protein, accumulation of endogenous NOS inhibitors, and enhanced arginase activity together with upregulation of arginase I protein in cavernous tissue. The impaired DDAH activity due to decreased expression of DDAH I protein would result in an accumulation of endogenous NOS inhibitors with CSE. These alterations may be relevant to induction of the erectile dysfunction following CSE.

The present study was designed to investigate the roles of enhanced arginase activity due to up-regulated arginases and the decreased hydroxyarginine for accelerating intimal hyperplasia with hyperglycemia. Thirteen weeks after injection of alloxan or physiological saline, endothelial denudation of the carotid artery was performed to induce intimal hyperplasia. The intimal hyperplasia occurred on 4 weeks following denudation was significantly accelerated by hyperglycemia. The method to measure L-arginine, endogenous NOS inhibitors such as monomethylarginine and asymmetric dimethylarginine, and hydroxyarginine as an intermediate of NO production simultaneously was established with the aid of high-performance liquid chromatography. In hyperglycemia group, the impaired cyclic GMP production as an indicator of NO production in endothelial cells was accompanied by the enhanced arginase activity together with increased expression of arginase I and II proteins, accumulated endogenous NOS inhibitors, reduced concentration of hydroxyarginine, and decreased DDAH activity in endothelial cells. However, NOS activity per se remained unchanged in the hyperglycemia group. Authentic hydroxyarginine inhibited arginase activity in a concentration-dependent manner. The inhibition of arginase with hydroxyarginine at a reduced concentration with hyperglycemia became significantly lower than that for the control. These results suggest that the accelerated intimal hyperplasia with hyperglycemia is closely related to the impaired NO production in endothelial cells, which results from accumulation of endogenous NOS inhibitors and accelerated arginase activity together with up-regulation of arginase I and II proteins. Decreased DDAH activity would bring about the accumulation of endogenous NOS inhibitors. Furthermore, reduced concentration of hydroxyarginine with hyperglycemia possibly results in an enhanced arginase activity in vivo, implicating partly in the impairment of NO production.

The present study was designed to investigate whether endothelial intracellular calcium concentration ([Ca(2+)](i)), endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) generation altered in association with impaired endothelium-dependent relaxation (EDR) in pulmonary artery (PA) specimens from experimental subarachnoid hemorrhage (SAH) rabbits. Injecting non-heparinized autologous arterial blood into cisterna magna induced the SAH. Simultaneous measurements of endothelial [Ca(2+)](i) and isometric tension of PA specimens were performed using fura 2. The subjects included normal control rabbits (group N), SAH rabbits with normal EDR (group A) and with impaired EDR (group B). When treated with 10(-7) M acetylcholine (ACh), endothelial [Ca(2+)](i) was significantly lower in group B (74.1+/-8.5 nM) than that in groups A (153.0+/-28.0 nM, p0.05) and N (184.8+/-27.8 nM, p0.01). Basal and ACh-stimulated cyclic GMP productions as a marker of NO generation were also significantly (p0.005) decreased in group B as compared to those in the other two groups. Meanwhile, there were no differences in eNOS activity per se among the three groups. These results suggest that the attenuated endothelial [Ca(2+)](i) elevation leads to the impaired NO generation in PA endothelium, which in turn impairs the EDR and possibly increases the vascular resistance of PA following SAH.

BACKGROUND: To reduce adverse reactions due to platelet (PLT) transfusion, medical solutions on the market, such as saline and ACD-A, are used to replace the plasma of PLT concentrates in Japan; however, they are not strongly preservative. Here, an attempt was made to develop a novel additive solution (M-sol) having the ability to preserve PLTs stably, with only approved solutions for clinical use. STUDY DESIGN AND METHODS: M-sol is a mixture of solutions for medical use, which consists of 77 mmol per L NaCl, 3 mmol per L KCl, 1 mmol per L CaCl2, 21 mmol per L Na acetate, 15 mmol per L glucose, 9.4 mmol per L Na3 citrate, 4.8 mmol per L citric acid, 44 mmol per L NaHCO3, and 1.6 mmol per L MgSO4. The in vitro variables of PLTs stored in M-sol, Seto-sol, PASIIIM, or 100 percent plasma were compared during 14 days of storage. RESULTS: The in vitro parameters (pH, P-selectin, %hypotonic shock response, %disk, mean PLT volume, aggregability) of PLTs were better maintained in M-sol containing 3 percent plasma than in 100 percent plasma, PASIIIM with 31 percent plasma, and Seto-sol with 3 percent plasma during 14 days of storage. CONCLUSION: The 2-week storage of PLTs in M-sol is feasible in terms of the in vitro PLT function. Our results here show that the additive solution, with a high ability to preserve PLTs, can be prepared by mixing solutions approved for clinical use that are not specifically for PLT storage.

The mechanism by which UV-C irradiation inactivates M13 bacteriophage was studied by analyzing the M13 genome using agarose gel electrophoresis and South-Western blotting for pyrimidine dimers. The involvement of singlet oxygen (1O2) was also investigated using azide and deuterium oxide and under deoxygenated conditions. With a decrease in M13 infectivity on irradiation, single-stranded circular genomic DNA (sc-DNA) was converted to Form I and Form II, which had an electrophoretic mobility between that of sc-DNA and linear-form DNA. However, the amount of sc-DNA remaining was not correlated with the survival of M13. The formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)PP) increased as a function of irradiation dose. The decrease in M13 infectivity was highly correlated with the increase in CPD and (6-4)PP, whereas no change was seen in M13 coat protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 8-Oxo-7,8-dihydro-2'-deoxyguanosine did not form in the M13 genome after UV-C irradiation. Inactivation of M13 was neither enhanced by deuterium oxide nor inhibited by azide. Deoxygenation of the M13 suspension did not affect the inactivation, indicating that 1O2 did not participate in the inactivation of M13 by UV-C irradiation under these conditions. These results indicated that UV-C irradiation induced not only CPD and (6-4)PP formation but also additional tertiary structural change in DNA inside the M13 virions, resulting in primary damage and a loss of infectivity. The indirect effect of UV-C irradiation such as 1O2 production followed by oxidative damage to nucleic acids and proteins might have contributed less, if at all, to the inactivation of M13 than the direct effect of UV-C.

Hemoglobin vesicle (HbV), a liposomal oxygen carrier containing human hemoglobin, was intravenously infused into rats. After the infusion of saline, the HbV or empty vesicle (EV), numbers of red cells, leukocytes and platelets in peripheral blood were unchanged during the observation period of one week in addition to each time point among three groups. However, the lymphocyte ratio transiently decreased and the granulocyte ratio increased in the HbV and EV groups at 6 h after the infusion. Those changes returned to the initial value one day after the infusion and those were maintained for the subsequent observation period. No dramatic change was seen in the ratio of CD4(+)/CD8(+) T cells. A transient decrease of the complement titer was observed three days after the infusion of HbV and EV, although the consumption of complement titer was not detected in rat serum by mixing HbV or EV in vitro, indicating that the transient decrease of complement titer in vivo was not due to the consumption of complement due to the interaction with HbV or EV. Multiple infusions of HbV caused the decrease of complement titer only after the first infusion and no allergic reaction was observed. No anaphylactic shock was observed in rats administered with EV several times, while ovalbumin (OVA) sensitized rats died with symptoms of respiratory distress after the second OVA administration. These results indicate that HbV could be administered without serious clinical symptoms or adverse reactions.

The genus Cycas (Cycadaceae; gymnosperm) have circumstantially been considered to be wind-pollinated. The cones of Cycas revoluta Thunb., however, emit a strong unpleasant odor. The chemical profiles of floral scents often correlate with various pollination modes (pollinators). We collected and analyzed the volatiles emitted from male and female cones of C. revoluta native to Iriomote and Yonaguni Islands, Japan. The analyses indicated that estragole (4-allylanisole) dominated in the volatiles (67.0-92.7%), with small amounts of other benzenoids, e.g., anethole, methyl salicylate, methyl eugenol, and ethyl benzoate. Several fatty acid esters were also detected in the samples from Iriomote Island. The function of estragole in the reproductive biology of C. revoluta is discussed.

Hemoglobin vesicles (HbVs), cellular-type artificial oxygen carriers containing human hemoglobin, were assessed for their biocompatibility by mixing with human plasma in vitro. Among three kinds of HbVs (PEG-DPEA-HbV, PEG-DPPG-HbV and DPPG-HbV), PEG-DPEA-HbV did not affect the extrinsic or intrinsic coagulation activities of the plasma, while PEG-DPPG-HbV and DPPG-HbV tended to shorten the intrinsic coagulation time. The kallikrein-kinin cascade of the plasma was slightly activated by PEG-DPPG-HbV and DPPG-HbV, but not by PEG-DPEA-HbV. The complement consumption of the plasma was observed by incubation with DPPG-HbV, but not with PEG-DPEA-HbV or PEG-DPPG-HbV. These results indicate that PEG-DPEA-HbV has a higher biocompatibility with human plasma.

There is evidence to suggest that water homeostasis in the inner ear is regulated via the vasopressin (VP)-aquaporin 2 (AQP2) system in the same fashion as in the kidney. The VP-AQP2 system in the kidney is well known to be inhibited by lithium, resulting in polyuria due to a decrease in reabsorption of water in the collecting duct of the kidney. Therefore, lithium is also likely to inhibit the VP-AQP2 system in the inner ear, and consequently exert some influence on inner ear fluid homeostasis. In this study, we investigated the effects of lithium on AQP2 expression in the rat inner ear, and on the cochlear fluid volume in hydropic ears of guinea pigs. A quantitative PCR study revealed that lithium reduced AQP2 mRNA expression in the cochlea and endolymphatic sac. Lithium application also decreased the immunoreactivity of AQP2 in the cochlea and endolymphatic sac. In a morphological study, lithium intake significantly reduced endolymphatic hydrops dose-dependently. These results indicate that lithium acts on the VP-AQP2 system in the inner ear, consequently producing a dehydratic effect on the endolymphatic compartment.

Arginase shares L-arginine as a common substrate with nitric oxide (NO) synthase (NOS). We examined whether increased arginase activity is involved in impaired cavernous relaxation with aging in the rabbit.Young adult (3 to 5 months old) and aged (36 to 48 months old) rabbits were used for the current experiments. Cavernous tissues obtained from the 2 groups were processed for isometric tension experiments, cyclic guanosine monophosphate determination, measurements of NOS and arginase activities, endogenous methylarginines and L-arginine.Carbachol (CCh) produced an endothelium dependent and NO mediated relaxation that was significantly impaired in aged cavernous specimens without change in sodium nitroprusside induced relaxation. Stimulated cyclic guanosine monophosphate production with CCh was significantly decreased in aged cavernous specimens. Ca dependent NOS was predominant in rabbit cavernous specimens. Ca dependent and independent NOS activities remained unchanged in the 2 groups. The tissue contents of N-monomethyl-L-arginine and asymmetric N,N-dimethyl-L-arginine as endogenous NOS inhibitors, symmetrical N,N'-dimethyl-L-arginine and L-arginine as a substrate of NOS were decreased in aged cavernous specimens. Arginase activity was significantly higher in aged cavernous specimens. Impaired CCh induced relaxation in aged cavernous specimens was normalized in the presence of N-hydroxy-L-arginine as an arginase inhibitor or by the supplementation of excess L-arginine.These results strongly suggest that impaired endothelium dependent and NO mediated cavernous relaxation with aging is due to decreased NO production, which would result from increased arginase activity and probably from decreased L-arginine content.

Epstein-Barr virus (EBV) -specific cytotoxic T lymphocytes (CTLs) were induced from PBMCs of a sero-positive healthy donor by stimulation with an autologous EBV-transformed B-lymphoblastoid cell line (EBV-LCL). CD4+ lymphocytes with high cytotoxic activities were found in the cultured fraction. The purified CD4+ bulk CTLs were expanded with immobilized anti-CD3 and anti-CD28 antibodies in the presence of IL-2. The CD4+ CTLs showed vigorous proliferation, up to 6, 000-fold cumulative expansion. They maintained high killing activity against autologous target cells while exhibiting no NK and LAK activities, even after expansion. These data indicate that ex vivo expansion with immobilized antibodies is useful to obtain a large number of CTLs without losing their specific activities. The cytotoxity mediated by the CD4+ bulk CTLs seemed to depend mainly on the perforin/granzyme pathway because a H+-ATPase inhibitor, concanamycin A, strongly inhibited the killing. The CD 4+ CTLs proliferated in response to autologous monocyte-derived dendritic cells pulsed with one of the EB viral proteins, EBNA 1. This finding suggests the existence of EBNA 1-recognizing clone (s) in the cell fractions. When stimulated with autologous EBV-LCL or activated with PMA and ionomycin, the CD4+ CTLs predominantly produced IFN-γ, not IL-4, indicating that they belong to the T helper 1 (Th1) type.

Arginase shares the common substrate L-arginine with nitric oxide synthase (NOS). We investigated the roles of NOS and arginase for modulating intrinsic and vasoconstrictor tone in rabbit corpus cavernosum (RCC).Isolated RCC tissues were used for isometric tension experiments, and NOS and arginase activities. The endothelium lining RCC lacunar spaces was disrupted and/or removed by saponin treatment.Following stretch of approximately 1gm NG-nitro-L-arginine methyl ester (L-NAME) as a NOS inhibitor caused endothelium dependent contraction, while the potent and specific arginase inhibitor N omega-hydroxy-nor-L-arginine (nor-NOHA) caused endothelium dependent relaxation. Relaxation with nor-NOHA was reversed by L-NAME. In the presence of 10 mM L-arginine 0.1 mM nor-NOHA was ineffective. Pretreatment with 0.1 mM L-NAME and 0.1 mM nor-NOHA did not significantly affect the vasoconstrictor response to phenylephrine. The magnitude of contraction with 0.1 mM L-NAME and relaxation with 0.1 mM nor-NOHA during contraction induced by phenylephrine were not significantly different from changes with L-NAME and nor-NOHA under intrinsic basal tone. In the enzymatic study NOS and arginase were detectable in cavernous homogenates. Nor-NOHA inhibited arginase but not NOS activity.Results indicate that basal nitric oxide production from the endothelium regulates intrinsic cavernous tone and endogenous arginase activity in the endothelium modulates tone by inhibiting nitric oxide production, presumably through competition with constitutive NOS for the common substrate L-arginine.

To investigate the properties of endocytosis of the epithelial cells in the intermediate portion of the endolymphatic sac (ES), we infused cationized ferritin (CF), microperoxidase (MPO), and horseradish peroxidase (HRP), as endocytosis tracers, into the endolymphatic space of the ES. Thirty minutes after infusion, the tissues were fixed and the distributions of HRP, MOP and CF were observed by transmission electron microscopy. The results at 30 min after tracer infusion were as follows. Concerning CF, endocytosis showed different activity depending on the area of the ES. In one area, CF was hardly observed within the epithelial cells, while in another area, it was observed within the epithelial cells. The CF-loaded vesicles were mainly coated vesicles. These results suggested that infusion of the artificial endolymphatic sac endolymph with CF may not stimulate the whole signal system of the ES. Concerning HRP, it bound to the apical membrane and was observed in the vesicles and the sorting endosomes. The diameters of these vesicles were of two types: one had a smaller diameter (less than 200 nm), while the other had a larger diameter (over 200 nm) and originated from macropinocytosis. Concerning MPO, it was much less observed in the vesicles than HRP. We discuss the different activities of endocytosis among these tracers, and suggest that a large molecular weight substance, i.e. HRP, might stimulate the fluid-phase endocytosis and/or adsorptive pinocytosis.

We examined whether endogenous nitric oxide (NO) synthase (NOS) inhibitors are involved in the impaired trigonal relaxation with ischemia in rabbits.Rabbits were divided into control and ischemia groups. Two weeks after partial vessel occlusion strips of trigone and detrusor were processed to determine endogenous methylarginines and L-arginine by automated high performance liquid chromatography. We also compared NOS activity and NO mediated functional responses to electrical field stimulation between 2 groups.Neurogenic and NO but not sodium nitroprusside induced mediated relaxation in the trigone were significantly impaired following ischemia. Ca2+ dependent NOS activity, and baseline and stimulated cyclic guanosine monophosphate production with electrical field stimulation were significantly decreased following ischemia. The contents of L-NMMA (NG-monomethyl-L-arginine) and asymmetrical ADMA (NG, NG-dimethyl-L-arginine) but not L-arginine or symmetrical SDMA (NG, N'G-dimethyl-L-arginine) were increased in the trigone following ischemia. Authentic L-NMMA and ADMA but not SDMA inhibited neurogenic relaxations in a concentration dependent manner without affecting the relaxation produced by sodium nitroprusside in control tissue. Excess L-arginine abolished L-NMMA and ADMA inhibition.These results suggest that impaired NO mediated trigonal relaxation following ischemia is closely related to decreased NOS activity and the increased accumulation of L-NMMA and ADMA.

BACKGROUND: 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. STUDY DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Present experiments were designed to investigate whether the facilitated polyol pathway is involved in the augmentation of intimal hyperplasia with hyperglycemia. Twelve weeks after a single bolus intravenous injection of alloxan (100 mg/kg) or saline, rabbits underwent a unilateral endothelial denudation of the carotid artery. Intimal hyperplasia was evident 4 weeks after denudation and significantly augmented in hyperglycemic animals treated with alloxan. This effect was accompanied by the enhanced accumulation of endogenous NOS inhibitors (N(G)-monomethyl-l-arginine [l-NMMA] and asymmetric, N(G),N(G)-dimethyl-l-arginine [ADMA]) in regenerated endothelial cells, impairment of NO production and release, and enhanced accumulation of endothelin-1 (ET-1) within the vessel wall. Sorbitol levels in aortic endothelial cells and within the smooth muscle layer were significantly increased with hyperglycemia. All these changes associated with hyperglycemia were significantly reduced in animals treated with the selective aldose reductase inhibitor fidarestat (3 mg/kg/d). These findings suggest that the facilitated polyol pathway possibly plays an important role for the augmentation of intimal hyperplasia caused by the hyperglycemic state.

Although only 5% of steady-state peripheral blood (PB) CD34+ cells were found to express chemokine receptor CXCR4, 45% of the cells became CXCR4+ after incubation at 37 degrees C for 4 hours. In contrast, there were no remarkable differences between PB CD34+ cells before and after the 37 degrees C incubation in their expression of selectin ligand, VLA-4, and VLA-5 or in their affinity for VCAM-1 or fibronectin. This increase in CXCR4 expression level was inhibited by the addition of brefeldin A, actinomycin D, or cycloheximide. When PB CD34+ cells with CXCR4 expression levels enhanced by a 4-hour preincubation at 37 degrees C or bone marrow (BM) CD34+ cells were exposed overnight to stromal cell-derived factor 1 (SDF-1), the expression levels of CXCR4 were greatly reduced, and when SDF-1 was removed, CXCR4 levels were thereafter up-regulated. The reexpressed CXCR4 was able to elicit integrin-dependent migration of hematopoietic progenitor cells. There was no difference in the severe combined immunodeficient mouse repopulating cell activity between PB CD34+ cells with and cells without a 37 degrees C preincubation.

Hemoglobin vesicles (HbV), a type of liposome-encapsulated hemoglobin (LEH), have been recently developed as an artificial oxygen carrier. The efficacy of HbV has been demonstrated in the transfusion of HbV into rodent models of hemorrhagic shock. It is important to evaluate the compatibility of HbV with human blood cells. We examined the effects of HbV on human platelet activation in vitro by estimating the platelet release reaction and expression of platelet surface activation markers in the presence or absence of agonists. HbV concentration in the reaction volume was prepared at 20% or 40%. Preincubation of platelet-rich plasma (PRP) with HbV had no adverse effects on RANTES or serotonin release from platelets. Preincubation of whole blood with HbV also had no effects on exposure of P-selectin on platelets. However, binding of PAC-1, a monoclonal antibody that detects the activation-dependent conformational change of αIIbβ3 to platelets, was amplified by preincubation of whole blood with HbV in the presence of relatively low concentration of ADP. These results suggest that HbV enhances the binding of PAC-1 to platelets. The clinical meaning of this increased binding of PAC-1 needs to be addressed.

A simple and highly sensitive reverse transcriptase (RT) assay was developed by combining a previously reported non-radioisotopic RT assay with the use of a template-primer-immobilized microplate, an enzyme capture protocol, product digestion and a chemiluminescent substrate. The assay was able to detect directly the RT activity in serum samples, plasma and cell culture medium without the need for concentration and extraction of the enzyme. The assay was able to detect RT activity equivalent to 100 virions/ml of HIV-1. These results suggest that this highly sensitive chemiluminescent RT assay can be used not only for virological investigation but also for routine screening of biopharmaceuticals.

We assessed whether the responsiveness of the detrusor is changed in rabbits with alloxan induced hyperglycemia.Hyperglycemia was induced by a bolus intravenous injection of alloxan (60 mg./kg.) in Japanese White male rabbits. At 16 weeks after alloxan detrusor muscle strips prepared from age matched normoglycemic and hyperglycemic rabbits were mounted in organ chambers. Contractile responses to KCl, carbachol, adenosine triphosphate, 5-hydroxytryptamine and electrical field stimulation were compared in the 2 groups. The effect of sarpogrelate as a selective antagonist of 5-hydroxytryptamine 2A receptor on the contractile response to 5-hydroxytryptamine was also compared.The current experiments demonstrated that hyperglycemia caused significant decreases in neurogenic and carbachol induced contractions accompanied by unchanged adenosine triphosphate and KCl induced contractions. Neurogenic bladder contraction in the hyperglycemic rabbit was significantly potentiated by exogenously applied 5-hydroxytryptamine. Potentiation was detectable even after the desensitization of purinoceptors but undetectable in the presence of atropine. Hyperglycemia resulted in enhancement of the 5-hydroxytryptamine induced bladder contraction. Sarpogrelate tended to normalize the enhanced contraction.The decrease in neurogenic bladder contraction possibly accompanied by the decreased density of muscarinic receptors, the potentiation of neurogenic bladder contraction with 5-hydroxytryptamine probably due to facilitated cholinergic transmission and the enhanced contractility to 5-hydroxytryptamine would be at least in part involved in bladder dysfunction associated with hyperglycemia.

We investigated the possible role of endogenous methylarginine derivatives, such as NG-monomethyl-L-arginine (L-NMMA), asymmetrical NG, NG-dimethyl-L-arginine (ADMA) and symmetrical NG, N'G-dimethyl-L-arginine (SDMA), for regulating nitric oxide synthase in the rabbit lower urinary tract.Strips of detrusor, trigone and proximal urethra were processed for the determination of endogenous methylarginines and L-arginine by automated high performance liquid chromatography. We also compared nitric oxide synthase activity and nitric oxide mediated functional responses to electrical field stimulation in the 3 regions. Nitric oxide synthase activity was measured by determining the conversion of [3H] L-arginine to [3H] L-citrulline.L-NMMA, ADMA and SDMA were detectable by high performance liquid chromatography in the detrusor, trigone and proximal urethra. Electrical field stimulation induced nitric oxide mediated prominent relaxation in the trigone and proximal urethra, while no relaxation response was observed in the detrusor. Exogenously applied 1 to 100 microM. L-NMMA and 1 to 100 microM. ADMA but not 100 microM. SDMA dependently inhibited Ca2+ dependent nitric oxide synthase activity in all regions, and electrical field stimulation induced relaxation in the trigone and proximal urethra. Inhibition with L-NMMA and ADMA was blocked in the presence of 3 mM. L-arginine but not by 3 mM. D-arginine.Three methylarginines and nitric oxide synthase are localized throughout the rabbit lower urinary tract. Endogenous L-NMMA and ADMA may be involved in regulating nitric oxide biosynthesis of the micturition reflex in the normal or disease state.

In an earlier publication (Takeuchi et al., Biophys. J. 79 (2000) 2572–2582), we proposed that K + channels in intermediate cells within the stria vascularis may play an essential role in the generation of the endocochlear potential (EP), and we presented an extended version of the five-compartment model of the stria vascularis. In search of further evidence supporting the five-compartment model, we studied the effects of Cs + added to the perilymph on guinea pig EP. Cs + is known as a competitive K + channel blocker. Both the scala tympani and the scala vestibuli of four cochlear turns were perfused at a flow rate of 10 μl/min, and the EP was recorded from the second cochlear turn. Cs + at 30 mM caused a biphasic change in the EP; the EP increased transiently from a control level of 89.6 mV to 94.8 mV within 10 min, and then decreased to a steady level of 24.5 mV within the next 40 min. We propose that the initial transient increase in the EP results from Cs + -mediated blockade of K + conductance in the basolateral membrane of hair cells, and that the subsequent EP decrease is due to effects of Cs + on the stria vascularis. We believe that Cs + in the perilymph is able to access the stria vascularis by being taken up by fibrocytes in the spiral ligament and then being transported to intermediate cells because it is known that Cs + is taken up via Na + ,K + -ATPase and that gap junctions connect fibrocytes in the spiral ligament to basal cells and basal cells to intermediate cells. To clarify the effect of intracellular Cs + on the electrophysiological properties of intermediate cells, these cells were dissociated from guinea pigs and studied by the whole-cell patch-clamp method. Intracellular Cs + depolarized intermediate cells in a dose-dependent manner. In addition, efflux of Cs + from the intermediate cell was much less than the efflux of K + . Thus, Cs + may accumulate in the intermediate cell, which depolarizes the cell, which in turn decreases the EP. We conclude that the five-compartment model of the stria vascularis can explain the EP decrease caused by Cs + in the perilymph.

The present experiments were designed to investigate the ability to produce nitric oxide, concentrations of N(G)-monomethyl-L-arginine (L-NMMA), and asymmetric N(G), N(G)-dimethyl-L-arginine (ADMA) in endothelial cells, endothelin-1 within the vessel wall, and the degree of intimal hyperplasia (intima/media ratio) in perimenopausal human uterine arteries. According to the tentative classification based on basal cyclic GMP levels, 16 arteries could be grouped into groups I and II consisting of eight each. Net production of the nucleotide was significantly higher in group I than that in group II. Concentration of L-NMMA plus ADMA and endothelin-1 content were significantly higher in group II. All specimens from group I were histologically normal, whereas mild to severe intimal hyperplasia was observed in group II specimens. Although considerable individual variations were detectable in the intima/media ratio, L-NMMA plus ADMA and endothelin-1 (n = 35 each), there were significant and positive correlations between three parameters, indicating that intimal hyperplasia became greater as L-NMMA plus ADMA and endothelin-1 were increased. These results suggest that endogenous nitric oxide synthase inhibitors in endothelial cells and endothelin-1 within the vessel wall are important markers of intimal hyperplasia.

New coordination polymers of copper(II) propionate, [Cu 4 (pro) 4 -(CH 3 O) 4 ] (Hpro = propionic acid) ( 1 ), [Cu 4 (pro) 4 (CH 3 O) 4 (pyz)] n (pyz=pyrazine) ( 2 ), and [Cu 2 (pro) 2 (CH 3 O) 2 (CH 3 OH) 2 (bpy)] n ·2 n CH 3 OH (bpy = 4,4'-bipyridine) ( 3 ), have been synthesized and characterized structurally. The crystal structures of 1 and 2 consist of methoxo- and propionato-bridged tetranuclear copper(II) units which are connected by propionato-oxygen atoms of adjacent units or pyrazine molecules to form a two-dimensional network. The crystal structure of 3 shows a one-dimensional chain structure. The magnetic properties of 1 and 2 are antiferromagnetic. coordination polymers copper(II) complexes crystal structures magnetic property