Beraprost sodium regulates cell cycle in vascular smooth muscle cells through cAMP signaling by preventing down-regulation of p27(Kip1).

OBJECTIVE: Beraprost sodium (BPS), a prostacyclin (PGI(2)) analogue, has been reported to exhibit beneficial effects on atherosclerosis in both human and animal models. To clarify the underlying mechanism, we investigated the effects of BPS on neointimal formation after balloon injury in the canine coronary artery. Furthermore, we determined its anti-atherosclerotic effects in cultured smooth muscle cells (SMCs). METHODS: Adult beagle dogs (10-12 kg) were fed on a high-cholesterol diet (10 g/day) and underwent balloon-denudation of the coronary artery. The dogs were divided into two groups: a BPS-treated group (20 microg/kg per day) and a control group. Twenty-eight days after injury, the dogs were killed and the coronary arteries were examined morphometrically. Three days after injury, the proliferative activity in the medial layer of the coronary artery was evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation, and p27(Kip1), a cyclin-dependent kinase (cdk) inhibitor, expression was examined by immunohistochemistry. We also examined the effects of BPS on SMC proliferation based on BrdU incorporation and cell cycle analysis. In addition, p27(Kip1) regulation was evaluated in primary-cultured SMCs. RESULTS: BPS administration decreased the intima/media ratio (I/M) by 88% in the control group. Three days after injury, BPS attenuated the proliferation rate of the cells in the media of the coronary artery by 35%, and maintained p27(Kip1) expression, which declined in the control cells. In the cultured proliferating SMC, BPS prevented the down-regulation of p27(Kip1). The 8-bromo-cyclic adenosine monophosphate (8-br-cAMP), a cAMP analogue, had similar actions as BPS in the regulation of p27(Kip1). The proliferation of cultured SMC was inhibited in a dose-dependent manner, and cell cycle arrest in the G1 phase was induced by BPS. CONCLUSIONS: Our data suggest that BPS inhibits neointimal formation after balloon denudation in the coronary artery through its inhibitory effect on SMC proliferation by preventing p27(Kip1) down-regulation.

Inhibition of smooth muscle cell migration by the p21 cyclin-dependent kinase inhibitor (Cip1).

In vascular smooth muscle cells (SMCs), proliferation and migration contribute to lesion formation after arterial injury. In the cell cycle, several cyclin-dependent kinases (cdks) inhibitors are implicated in the regulating of cyclin-cdk activity such as p21Cip1, p16Ink4 and p27Kip1. Although Cip1 inhibits SMC proliferation, its effects on SMC migration are unknown. To test the hypothesis that Cip1 inhibits SMCs migration and proliferation, we transfected the Cip1 gene into a strain of rabbit aortic SMCs (SM3 cells). Both the spreading and the attachment of Cip1-transfected SM3 cells to extracellular matrices (ECMs) were inhibited compared to that of vector-transfected cells. In the modified Boyden's chamber assay the effect of fibronectin on the migratory activity of Cip1-transfected SM3 cells was significantly less than that of vector transfected cells in response to PDGF-BB. These data suggested that Cip1 inhibited both the migration and proliferation of SMC.

Iodine-123 iodoamphetamine brain scan in a patient with auditory hallucination.

The case of an alcoholic patient with auditory hallucination is reported in which [123I] iodoamphetamine (IMP) brain imaging demonstrated increased accumulation in the left superior temporal lobe which corresponded to the left primary and secondary auditory areas. Thus, IMP brain scans seem to have the potential to objectively localize the brain abnormalities in auditory hallucination.

Isolation and characterization of lectins specific for mannose/fucose/N-acetylglucosamine from rat peritoneal macrophages.

Rat peritoneal macrophages were shown to have two distinct mannose/fucose/N-acetylglucosamine-specific lectins. The major lectin of 180 kDa, which is similar in size to the mannose receptor first isolated from alveolar macrophages (Wileman, T.E., Lennartz, M.R., & Stahl, P.D. (1986) Proc. Natl. Acad. Sci. U.S. 83, 2501-2505), was shown to occur as a dimer under nondenaturing conditions. The 29 and 32 kDa lectins were identified as members of the liver mannan-binding protein family on the basis of their immunochemical crossreactivity, collagenase sensitivity, and molecular sizes (Oka, S., Ikeda, K., Kawasaki, T., & Yamashina, I. (1988) Arch. Biochem. Biophys. 260, 257-266). Despite the similarity in the sugar binding specificity, these two types of lectin were clearly differentiated with regard to the binding to IgM molecules. The 29 and 32 kDa lectins bound to IgM most likely through high-mannose type oligosaccharides on IgM, whereas the 180 kDa lectin did not.

Self-retaining abdominal retractor for minilaparotomy.

BACKGROUND: We describe our experience using a new self-retaining atraumatic abdominal retractor for minilaparotomy performed either alone or in conjunction with laparoscopy. TECHNIQUE: Minilaparotomy was performed by a conventional technique and the self-retaining abdominal wall retractor (Dexterity Protractor) was placed for the duration of the open surgery. EXPERIENCE: The device was employed in 72 consecutive cases in which minilaparotomy was performed either alone or in conjunction with laparoscopy. Indications for minilaparotomy included extensive myomectomies and tubal ligation reversals in fertility patients, hysterectomies, adnexal surgery, appendicitis, and either planned or incidental concomitant bowel surgery. No allergic reactions to the plastic device were noted. Postoperative evaluation demonstrated no evidence of wound infections, nerve injuries, seromas or dehiscence in the short term, nor hernia formation or other complications in the long term. CONCLUSION: The Dexterity Protractor permitted effective retraction of the abdominal wall. The device provided gentle retraction around the entire incision, excellent surgical exposure, and a barrier to specimens containing bacterial or potentially malignant cells. The method of application was uncomplicated and well-suited to abdominal walls of varying thickness. The utilization of this device was not associated with wound-related complications in this study of patients.

beta-Amyloid protein-dependent nitric oxide production from microglial cells and neurotoxicity.

beta-Amyloid protein (A beta) is the major component of the senile plaques in Alzheimer's disease (AD), and microglial cells have been shown to be closely associated with these plaques. However, the roles of A beta and microglial cells in pathogenesis of AD remain unclear. Incubation of rat microglial cells with A beta(1-40) caused a significant increase in nitrite, a stable metabolite of nitric oxide (NO), in culture media, while there was no detectable increase in nitrite in astrocyte-rich glial cells or cortical neurons after incubation with A beta(1-40). Nitrite production by microglial cells was also induced by A beta(1-42), but not A beta(25-35). An inhibitor of NO synthase, NG-monomethyl-L-arginine (NMMA), as well as dexamethasone and actinomycin D, dose-dependently inhibited this nitrite production. Among the various cytokines investigated such as interleukin-1, interleukin-6, tumor necrosis factor-alpha and interferon-gamma (IFN-gamma), only IFN-gamma markedly enhanced A beta-dependent nitrite production. Cultured cortical neurons were injured by microglial cells stimulated with A beta in a dose-dependent manner in the presence of IFN-gamma. Neurotoxicity caused by the A beta plus IFN-gamma-stimulated microglial cells was significantly attenuated by NMMA. Thus, although further investigations into the effect of A beta on human microglial cells are needed, it is likely that A beta-induced NO production by microglial cells is one mechanism of the neuronal death in AD.

Isolation and characterization of a receptor lectin specific for galactose/N-acetylgalactosamine from macrophages.

Rat-peritoneal macrophages are shown to be able to take up glycoproteins terminated by galactose as well as those by mannose and/or N-acetylglucosamine. A lectin responsible for the uptake of galactose-terminated glycoproteins was isolated by affinity chromatography on a column of Sepharose 4B-asialoorosomucoid. The macrophage lectin isolated shared many properties in common with the well-established hepatic lectin specific for galactose/N-acetylgalactosamine. Thus, the lectin bound to asialoglycoproteins specifically, and the binding was inhibited by galactose and N-acetylgalactosamine. The lectin had a single major component of mol. wt. 42,000 as well as two minor components of 60,000 and 65,000, and required calcium for binding. In addition, the macrophage lectin was immunologically crossreactive with the hepatic lectin. Despite these similarities, however, the macrophage lectin was differentiated from the hepatic lectin in molecular size, relative preponderance of minor components, and titration profile with the antibodies raised against the hepatic galactose/N-acetylgalactosamine-specific lectin.

[An empirical research for demand of influenza vaccination].

PURPOSE: This article examines the demand for influenza vaccination in Japan. METHODS: Original date were obtained from a survey conducted by the authors. Two approaches, usual demand analysis and conjoint analysis, were employed. The second approach, conjoint analysis, uses people's statements on how they would respond to different hypothetical situations. In this research, we ask people whether they wish to be vaccinated given different circumstances such as costs of vaccination, degree of convenience, and outbreak news. RESULTS: In the demand analysis, the vaccination rate during the 1999-2000 season was found to have increased by 0.8 percentage points compared to that of the previous season. The rate increased by 1.0 to 3.5 percentage points among the group of people who experienced influenza in the previous season. The vaccination rate also increased by 31-47 percentage points for those who were vaccinated in the previous season. A 10 percentage increase in household income decreased the demand for vaccination by 2 percentage points. Although household income was significant in only with the largest sample, this result may indicate that the time or opportunity cost for vaccination decreases the vaccination demand. In the conjoint analysis, the financial cost was significantly negative. When the cost was reduced from the current level of 6,000 yen to free of charge, the vaccination rate would increase by 43.5 percentage points. Were vaccination available at night or during holidays,! or at school or work, the rate would increase by 11 percentage points, or 16 percentage points, respectively. Most of all, news of influenza prevalence was very influential in increasing the desire for vaccination by 33 percentage points. Vaccination experience and last year's influenza experience were both significantly positive, increasing the rate by 22 and 8 percentage points, respectively. CONCLUSIONS: In the demand analysis, influenza experience and history of vaccination during the 1999-2000 season were found to be influential regarding the decision for vaccination. From the conjoint analysis, providing vaccination of night or during holidays, as well as at work or at schools would increase the demand. News of influenza outbreaks were also found to increase the vaccination demand. Higher income, however, was found to have a negative influence, suggesting that opportunity costs may be an important factor for some individuals. Habit formation effects through a history of vaccination plays quite an important role in vaccination demand.

Analysis of binding site for the novel small-molecule TLR4 signal transduction inhibitor TAK-242 and its therapeutic effect on mouse sepsis model.

BACKGROUND AND PURPOSE: TAK-242, a novel synthetic small-molecule, suppresses production of multiple cytokines by inhibiting Toll-like receptor (TLR) 4 signalling. In this study, we investigated the target molecule of TAK-242 and examined its therapeutic effect in a mouse sepsis model. EXPERIMENTAL APPROACH: Binding assay with [(3)H]-TAK-242 and nuclear factor-kappaB reporter assay were used to identify the target molecule and binding site of TAK-242. Bacillus calmette guerin (BCG)-primed mouse sepsis model using live Escherichia coli was used to estimate the efficacy of TAK-242 in sepsis. KEY RESULTS: TAK-242 strongly bound to TLR4, but binding to TLR2, 3, 5, 9, TLR-related adaptor molecules and MD-2 was either not observed or marginal. Mutational analysis using TLR4 mutants indicated that TAK-242 inhibits TLR4 signalling by binding to Cys747 in the intracellular domain of TLR4. TAK-242 inhibited MyD88-independent pathway as well as MyD88-dependent pathway and its inhibitory effect was largely unaffected by lipopolysaccharide (LPS) concentration and types of TLR4 ligands. TAK-242 had no effect on the LPS-induced conformational change of TLR4-MD-2 and TLR4 homodimerization. In mouse sepsis model, although TAK-242 alone did not affect bacterial counts in blood, if co-administered with ceftazidime it inhibited the increases in serum cytokine levels and improved survival of mice. CONCLUSIONS AND IMPLICATIONS: TAK-242 suppressed TLR4 signalling by binding directly to a specific amino acid Cys747 in the intracellular domain of TLR4. When co-administered with antibiotics, TAK-242 showed potent therapeutic effects in an E. coli-induced sepsis model using BCG-primed mice. Thus, TAK-242 may be a promising therapeutic agent for sepsis.

Insertion of mitochondrial DNA-encoded F1F0-ATPase subunit 8 across the mitochondrial inner membrane in vitro.

Cytochrome oxidase subunits I, II, and III, the mitochondrial DNA-encoded proteins, are inserted across the inner membrane by the Oxa1p-containing translocator in a membrane potential-dependent manner. Oxa1p is also involved in the insertion of the cytoplasmically synthesized precursor of Oxa1p itself into the inner membrane from the matrix via the conservative sorting pathway. The mechanism of insertion of the other mitochondrially synthesized proteins, however, is unexplored. The insertion of the mitochondrial DNA-encoded subunit 8 of F(1)F(0)-ATPase (Su8) across the inner membrane was analyzed in vitro using the inverted inner membrane vesicles and the Escherichia coli lysate-synthesized substrate. This assay revealed that the N-terminal segment of Su8 inserted across the membrane to the intermembrane space and assumed the correct trans-cis topology depending on the mitochondrial matrix fraction. This translocation reaction was similar to those of Sec-independent, direct insertion pathways of E. coli and chloroplast thylakoid membranes. (i) It required neither nucleotide triphosphates nor membrane potential, and hydrophobic forces drove the process. (ii) It did not require protease-sensitive membrane components facing the matrix space. (iii) It could be inserted across liposomes in the correct topology in a matrix fraction-dependent manner. Thus, a novel mechanism conserved in bacteria and chloroplasts also functions in the insertion of Su8 across the mitochondrial inner membrane.

Structural similarity between the macrophage lectin specific for galactose/N-acetylgalactosamine and the hepatic asialoglycoprotein binding protein.

The rat peritoneal macrophage lectin specific for galactose/N-acetylgalactosamine was shown to be a homologue of the hepatic asialoglycoprotein binding protein (rat hepatic lectin, RHL). The macrophage lectin was immunochemically crossreactive with the major form of RHL (RHL-1) but not with the minor forms (RHL-2 and -3). The overall homology between the macrophage lectin and RHL-1 was confirmed by peptide maps of their lysyl endopeptidase digests on reverse-phase HPLC. Despite these similarities, however, the macrophage lectin was distinct from HRL-1 as revealed by the differences in the NH2-terminal 20 amino acid sequences of these two lectins.

A case of cold-dependent exercise-induced anaphylaxis.

Exercise-induced anaphylaxis (EIA) is a form of physical urticaria that is induced by exercise. A 16-year-old Japanese boy had a 4-year history of recurrent wealing and dyspnoea after physical exercise such as jogging, playing handball or riding a bicycle in winter. The episodes were not associated with ingestion of foods including wheat or soya bean. A provocation test, with 15 min of exercise and 2 min of cold stimulation immediately before or immediately after the exercise, elicited a weal that was localized to the test area. A challenge test with ingestion of boiled soya beans and exercise did not elicit a weal. Therefore, in this case, cold exposure, but not food ingestion, was essential for inducing EIA. Cold-dependent EIA is different from cold urticaria, food-dependent EIA, cholinergic urticaria and cold-induced cholinergic urticaria, and may be a distinct entity.

Expression of a functional asialoglycoprotein receptor through transfection of a cloned cDNA that encodes a macrophage lectin.

The Gal/GalNAc-specific lectin on rat peritoneal macrophages (macrophage asialoglycoprotein binding protein, M-ASGP-BP) is structurally similar to rat hepatic asialoglycoprotein-binding protein (ASGP-BP) or rat hepatic lectin (RHL) and is highly homologous with the major component of RHL, RHL-1 (Ii, M, Kurata, H., Itoh, N., Yamashina, I., and Kawasaki, T. (1990) J. Biol. Chem. 265, 11295-11298). We found in this study that transfection with a cDNA clone that encodes a single polypeptide, M-ASGP-BP, was sufficient for the expression of an endocytic receptor for asialoorosomucoid (ASOR) on the COS-1 cell surface. The Kuptake value for ASOR for the transfected cells was 12.5 nM, which is similar to that for peritoneal macrophages (23 nM), and the number of ASOR bound on the cell surface was 1-8 x 10(5)/cell, this value being hundreds of times larger than that for peritoneal macrophages. 125I-ASOR bound on the surfaces of the transfected cells was rapidly internalized on incubation at 37 degrees C, and after 90 min of incubation, most of the radioactivity was recovered in acid-soluble degraded products from the medium. These results confirmed that the cDNA cloned in our previous study does in fact encode M-ASGP-BP and also that the single polypeptide chain can form a homooligomeric receptor (probably a hexamer or octamer) exhibiting high affinity for ASOR. The latter property was distinct from that of the hepatic ASGP-BP in that simultaneous transfection of two cloned cDNAs that encode RHL-1 and RHL-2/3 was required to produce an active ASOR receptor (McPhaul, M., and Berg, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8863-8867). This M-ASGP-BP expression system may serve as a simple model with which to investigate the molecular mechanisms underlying carbohydrate-mediated endocytosis.

Technique for detecting changes in fluorescence lifetime by means of optoelectronic circuit auto-oscillation.

We present a new, simple, inexpensive, and highly precise approach to excited-state fluorescence-lifetime-based measurements. The detection system consists of a closed-loop optoelectronic arrangement containing a radio frequency resonance amplifier, a fluorescence excitation light source, a fiber-optic delay line, and a photodetector. The system exhibits auto-oscillations in the form of intensity modulation. The oscillation frequency varies with the modulation phase shift of the fluorescent light. This frequency is used as the detection parameter, which is advantageous because frequency may be measured easily, inexpensively, and with high precision. This technique is well suited for chemical or biosensor applications.