AAV serotype 2/1-mediated gene delivery of anti-inflammatory interleukin-10 enhances neurogenesis and cognitive function in APP+PS1 mice.

Brain inflammation is a double-edged sword. It is required for brain repair in acute damage, whereas chronic inflammation and autoimmune disorders are neuropathogenic. Certain proinflammatory cytokines and chemokines are closely related to cognitive dysfunction and neurodegeneration. Representative anti-inflammatory cytokines, such as interleukin (IL)-10, can suppress neuroinflammation and have significant therapeutic potentials in ameliorating neurodegenerative disorders such as Alzheimer's disease (AD). Here, we show that adeno-associated virus (AAV) serotype 2/1 hybrid-mediated neuronal expression of the mouse IL-10 gene ameliorates cognitive dysfunction in amyloid precursor protein+ presenilin-1 bigenic mice. AAV2/1 infection of hippocampal neurons resulted in sustained expression of IL-10 without its leakage into the blood, reduced astro/microgliosis, enhanced plasma amyloid-ß peptide (Aß) levels and enhanced neurogenesis. Moreover, increased levels of IL-10 improved spatial learning, as determined by the radial arm water maze. Finally, IL-10-stimulated microglia enhanced proliferation but not differentiation of primary neural stem cells in the co-culture system, whereas IL-10 itself had no effect. Our data suggest that IL-10 gene delivery has a therapeutic potential for a non-Aß-targeted treatment of AD.

Saccades and prefrontal hemodynamics in basketball players.

We investigated saccade performance and prefrontal hemodynamics in basketball players with different skill levels. Subjects were 27 undergraduate basketball players and 13 non-athlete undergraduates (control group: CON). The players were divided into two groups: those who had played in the National Athletic Meet during high school or played regularly (n=13, elite group: ELI) and those who were bench warmers (n=14, skilled group: SKI). Horizontal eye movement and oxy-, deoxy-, and total-hemoglobin (Hb) concentration in the prefrontal cortex during pro- and anti-saccade were measured using electro-oculography and near-infrared spectroscopy, respectively. Only error rate in anti-saccade was less in ELI (4.8+/-4.0%) than SKI (13.7+/-12.6%) and CON (13.9+/-8.3%) (p<0.05). In ELI alone, oxy- (-0.15+/-0.18 mmol*mm) and total-Hb (-0.12+/-0.15 mmol*mm) during anti-saccade decreased significantly compared with that during rest (p<0.05), while those in CON significantly increased (oxy-Hb: 0.17+/-0.15 mmol*mm, total-Hb: 0.14+/-0.14 mmol*mm) (p<0.05). These results suggest that inhibition of eye movement to a visual target changes from voluntary to automatic through the motor learning of basketball.

Drug evolution concept in drug design: 2. Chimera method.

The drug evolution method represents a novel approach towards efficient rational drug design by implementing the drug evolution concept to the creation and development of general chemical libraries with the purpose of allowing the identification of drug candidates with improved odds and lesser costs than the traditional drug design strategies. As another example of successful translation of the biological evolution into chemical evolution, the chimera method comprises the grafting of selected building blocks, identified through a basic search within a drug library, onto the same substitution sites on a rationally chosen scaffold. The method allows the creation of a library containing both drugs and prospective drug candidates without any priorly required knowledge on the pursued disease or molecular target. Two libraries having scaffolds derived from para-aminobenzoic acid and salicylic acid have exemplified the application of the chimera method. The validation of the method has been achieved through the high number of recognized drugs within the library, which exhibit in the same time a wide variety of therapeutic activities and interact with a broad spectrum of molecular targets. The drug-enriched chimera libraries are expected to provide a highly efficient access to novel drug candidates whose unspecified therapeutic effects should be further revealed through high-throughput screening.

Recombinant human soluble thrombomodulin delivers bounded thrombin to antithrombin III: thrombomodulin associates with free thrombin and is recycled to activate protein c.

Recombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (Kd = 2.6 nM) or PC activation (Kd = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (Kd < 0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

Evidence that the protein C activation pathway amplifies the inhibition of thrombin generation by recombinant human thrombomodulin in plasma.

Thrombomodulin (TM) is a cofactor for the thrombin-catalyzed activation of anticoagulant protein C. However, we have no evidence that thrombomodulin actually activates protein C during blood coagulation processing, nor do we know whether this activated protein C acts as an anticoagulant. We studied the inhibitory action of recombinant human soluble TM (rhs-TM) on thrombin generation in whole plasma. Human plasma was activated with small amounts of tissue factor using phospholipid vesicles in place of activated platelets. Thrombin generation was observed. The addition of only 2 nM of rhs-TM prevented rapid generation of thrombin and reduced the total amount of thrombin generated. In order to study the influence of the protein C activation pathway on this inhibitory action of rhs-TM, protein C-depleted plasma was used. rhs-TM had little inhibitory effect on protein C-depleted plasma. However, the addition of protein C caused a delay in thrombin generation and a reduction of the maximum thrombin concentration. We concluded that the anticoagulant activity of rhs-TM was amplified by the protein C activation pathway.

Effects of recombinant human soluble thrombomodulin (rhs-TM) on a rat model of disseminated intravascular coagulation with decreased levels of plasma antithrombin III.

We reported that recombinant human soluble thrombomodulin (rhs-TM) is effective for disseminated intravascular coagulation (DIC) in vivo, in mice and rats. In the present work, we investigated the effects of decreased plasma antithrombin III (ATIII) levels on anticoagulant effects of rhs-TM, as compared to findings with heparin, of which effect is lowered by the decreased plasma ATIII levels in patients with DIC. Rat plasma ATIII levels decreased when we mixed plasma with anti-rat ATIII antibody and the potential of heparin to prolong APTT or PT was markedly diminished. The potential of rhs-TM to prolong APTT and PT was not affected. In rats injected with anti-rat ATIII antibody, plasma ATIII levels decreased immediately. When the rats were infused with tissue factor (TF), DIC was induced. At doses of rhs-TM and heparin which were equally effective at inhibiting the decrease in platelet count and fibrinogen level in control rats treated with TF, only rhs-TM remained effective in preventing DIC in rats with reduced ATIII levels. Heparin was not effective when administered to these rats with reduced ATIII levels. Therefore, rhs-TM effectively inhibits coagulation independent of ATIII levels, in contrast to heparin, which depends on the ATIII level.

Antithrombotic effect of recombinant human soluble thrombomodulin on endotoxin-induced disseminated intravascular coagulation in rats.

Thrombomodulin (TM) is an endothelial cell membrane glycoprotein which neutralizes thrombin procoagulant activity and accelerates the thrombin-catalyzed activation of protein C. We expressed recombinant human soluble TM (rhs-TM) in Chinese hamster ovary cells and compared the effects of rhs-TM and heparin on endotoxin-induced experimental disseminated intravascular coagulation (DIC) in rats. Experimental DIC was induced by a continuous intravenous infusion of endotoxin for four hours. rhs-TM or heparin was infused simultaneously with endotoxin. Treatment with rhs-TM significantly reversed the endotoxin-induced changes in significantly reversed the endotoxin-induced changes in following parameters: platelet count, fibrinogen level and fibrinogen and fibrin degradation products. Furthermore, glomerular fibrin deposits elevated by endotoxin treatment were reduced by the rhs-TM administration. Heparin showed the similar effects to rhs-TM. Activated partial thromboplastin time (APTT) in rats receiving rhs-TM were slightly longer than APTT in endotoxin-treated rats, but rats receiving heparin had much more prolonged APTT. From these results, we concluded that rhs-TM may be useful for the clinical treatment of DIC while having only minor adverse effects on APTT.

Taxines from the needles of Taxus wallichiana.

A taxine, 5 alpha O-(3'-dimethylamino-3'-phenylpropionyl) taxinine M (1) together with two known compounds 7-O-acetyltaxine A (2) and 2 alpha-acetoxy-2' beta-deacetylaustrospicatine (3) were isolated from the needles of the Himalayan yew, Taxus wallichiana Zucc. Their structures were elucidated on the basis of the NMR spectral data, ESI-MS/MS analysis and chemical methods. Compounds 1 and 3 showed moderate cytotoxic activity against the lung cancer cell line A549 in vitro.

The antithrombotic effects of recombinant human soluble thrombomodulin (rhsTM) on tissue factor-induced disseminated intravascular coagulation in crab-eating monkeys (Macaca fascicularis).

We evaluated the antithrombotic effects of recombinant human soluble thrombomodulin (rhsTM) in plasma and in a monkey model. rhsTM dose-dependently prolonged activated partial thromboplastin time (APTT) in the following order: humans > monkeys > rats >> rabbits. The prolongation of APTT by rhsTM was also observed in protein C-deficient plasma. rhsTM activated protein C and inactivated factor Va in human and monkey plasma, but not in rat plasma. These findings suggest that the antithrombotic activities of rhsTM are fully expressed in human and monkey. Therefore, to evaluate the whole activity of rhsTM in a clinical model, tissue factor (TF) was intravenously infused into crab-eating monkeys to induce disseminated intravascular coagulation (DIC). Pretreatment with rhsTM reduced fall in fibrinogen with a biphasic and moderate dose-dependency curve, and reduced thrombin-antithrombin III (TAT) levels with a flat linear dose-dependency, while heparin prevented fall in fibrinogen with a steep linear dose-dependency curve without reducing TAT levels. Further evidence suggesting that rhsTM activates protein C in vivo was also obtained. Taken together, the data indicate that rhsTM fully expresses its antithrombotic activities in human and monkey but not in rat and rabbit, and rhsTM prevents TF-induced DIC in monkeys by suppressing thrombin generation.

Effects of autonomic agents and chemical mediators on ion transport by canine tracheal epithelium.

Active ion transport by the airway epithelium plays an important role in maintaining the effective defense mechanisms of the airway by regulating the volume and composition of the airway fluid. We investigated the abilities of adrenergic agents, cholinergic agents, and chemical mediators to modulate ion transport in canine tracheal epithelium, using Ussing-type chambers. Transepithelial electric potential difference (PD), resistance (R), and short circuit current (SCC) of the tracheal epithelium were measured before and during exposure to a drug or after a change in the perfusate composition. The mean values and S.E. (N = 41) of PD, R, and SCC during the control period were -18 +/- 4 mV (luminal negative to submucosa), 240 +/- 42 omega.cm2, and 50 +/- 5 microA/cm2, respectively. Ouabain (10(-4) M), an inhibitor of Na+-K+-ATPase, in the mucosal bath abolished PD and SCC. Replacement of luminal Na by choline reversibly reduced PD and SCC. These findings suggest that PD and SCC of the tracheal epithelium are maintained by the transcellular transport of luminal Na toward the mucosa. Isoproterenol (10(-5) M), epinephrine (10(-4) M), and norepinephrine (10(-4) M) markedly increased both PD and SCC. Acetylcholine (10(-4) M) and histamine (10(-4) M) did not alter SCC significantly. Prostaglandin E1 (10(-6) M) and F2 alpha (10(-5) M) slightly increased PD and SCC. These results indicate that adrenergic and cholinergic agents induce different patterns of effect on ion transport (adrenergic-dominant) in the tracheal epithelium. Thus, the effects of autonomic agents and chemical mediators on ion transport may explain, in part, the pathogenesis of airway disorders observed in many respiratory diseases.

Unsatisfying functions and multiobjective fuzzy satisfaction design using genetic algorithms.

This paper describes a new fuzzy satisfaction method using genetic algorithms (GA) for multiobjective problems. First, an unsatisfying function, which has a one-to-one correspondence with the membership function, is introduced for expressing "fuzziness". Next, the multiobjective design problem is transformed into a satisfaction problem of constraints by introducing an aspiration level for each objective. Here, in order to handle the fuzziness involved in aspiration levels and constraints, the unsatisfying function is used, and the problem is formulated as a multiobjective minimization problem of unsatisfaction ratings. Then, a GA is employed to solve the problem, and a new strategy is proposed to obtain a group of Pareto-optimal solutions in which the decision maker (DM) is interested. The DM can then seek a satisfaction solution by modifying parameters interactively according to preferences.

[Detection of methicillin-resistant Staphylococcus aureus by the Showa disk].

The Showa disk susceptibility test using two penicillinase-resistant penicillins (PRPs: methicillin and oxacillin) and four cephalosporins (cefazolin, cefmetazole, ceftizoxime, and latamoxef) was evaluated to discriminate between the strains of Staphylococcus aureus resistant to methicillin (MRSA) and those susceptible (MSSA). Among 129 MRSA and 112 MSSA strains, significant numbers of MRSA found to be false-susceptible to PRPs, especially when incubated at 37 degrees C. Using agar plates supplemented with 5% NaCl, oxacillin disk correctly categorized all the MRSA and MSSA strains, but two MSSA strains were mischaracterized with methicillin disk. All the MSSA strains found to be susceptible against cephalosporins tested, but significant numbers of MRSA were also interpreted to be susceptible. Only the result of ceftizoxime disk susceptibility test highly correlated with methicillin resistance, when incubated at 35 degrees C. All the MRSA did not produce any significant bacterial growth inhibitory zone around the disk, whereas all the MSSA found to be susceptible (19 mm or more), e.g., 100% correlation. With these data, we recommend that ceftizoxime disk susceptibility test, incubated at 35 degrees C, provides a more reliable method to characterize the strains of MRSA and MSSA. The interpretive breakpoint would be used as; less than or equal to 16 mm (greater than or equal to 25 micrograms/ml) for MRSA.

Multicenter evaluation of a colorimetric microplate antimycobacterial susceptibility test: comparative study with the NCCLS M24-P.

A colorimetric test method using the microplate culture technique for the determination of susceptibility of Mycobacterium tuberculosis against antimycobacterial agents was developed and evaluated by the multicenter study. The test method utilizes an oxidation-reduction dye, 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC), as an indicator of mycobacterial growth. When compared to the presently available test method, some modifications were also included; lower inoculum density (10-fold dilution), inclusion of an inoculum diluted 1:100 as a growth control, and the preparation of inoculum preincubated in Middlebrook 7H9 broth and spectrophotometrically adjusted to McFarland #1 turbidity. The test method evaluated was highly precise and reliable to detect antimycobacterial resistances when the ATCC reference strains were tested. Also, the interpretations of the test result were highly comparable to those determined by the method of NCCLS M24-P, the % agreements ranging from 76.1% (ethambutol) to 91.3% (streptomycin). The test results were also comparable to those determined by Ogawa media; > 90% agreed with susceptible, intermediate, or resistant. The appearance of mycobacterial colonies on the test media was easily read, and the test results were more comparable to those of NCCLS M24-P. With these results, it can be concluded that the colorimetric microplate susceptibility test method described will be more suitable for clinical mycobacteriology laboratories.

[Antitumor effects of Behenoyl-ara-C (BH-AC) in combination with Idarubicin (IDA) in P 388 leukemic cell bearing mice].

The antitumor effects of Behenoyl-ara-C (BH-AC) in combination with Idarubicin (IDA) on leukemia were studied. First, a combination of IDA with Ara-C, which is the main metabolite of BH-AC, was evaluated with regard to its in vitro cytotoxic activity on mouse P 388 leukemic cells. The effect of this combination proved to be additive according to isobologram analysis. Secondly, the antitumor activity of an intravenous bolus-administration of a combination of BH-AC and IDA was evaluated by the life span of P 388 bearing mice, and compared with the activity of the Ara-C and IDA combination. The antitumor activity of Ara-C administered alone was clearly dependent on the administration schedule and was most intense when Ara-C was administered with the most frequent injections (3 bolus injections/day x 3 days), whereas antitumor activity of BH-AC was less dependent on the schedule. IDA administered alone showed dose-dependency in its antitumor activity up to 3 mg/kg. The maximum effects of IDA were observed with amounts of 3 - 4 mg/kg. In the same leukemia model, the combination of frequent injections of BH-AC and a single injection of IDA (increased life span: ILS>300%; cure ratio: CR = 3/5) conferred a more potent effect compared to the results of BH-AC (ILS = 133%, CR = 0/5) or IDA (ILS = 67%, CR = 2/5) alone. The effect of BH-AC and IDA combination was comparable or superior to that of the Ara-C and IDA (ILS = 233%, CR = 2/5) combination. These results indicated the possibility of clinical usefulness with a combination therapy of BH-AC and IDA against leukemia.

[Anti-emetic treatment with metoclopramide and other drugs during CDDP therapy].

The present trial was designed to compare the effect of metoclopramide(MCP) + dexamethasone(DM) (Method A) with that of MCP + DM + lorazepam(Lor) (Method B) in the treatment of CDDP (cis-diamminedichloroplatinum)-induced nausea and vomiting in a randomized fashion. The results were collected by questionnaire given to 50 patients. The dosage of CDDP was 80-100 mg/m2. In addition, MMC and VDS, or VP-16 were used concurrently. Within 24 hours after the administration of CDDP, vomiting was not observed in 72% and 88% of the patients treated with Method A and Method B, respectively, and nausea was not noted in 48% and 68%, respectively. Marked malaise was observed in 36% of patients in the Method A group and in 12% in the Method B group. With respect to the extent of comfort, 16% of patients in the Method A group and 56% in the Method B group felt good. Sixteen percent in the Method A group and 56% in the Method B group were satisfied with the anti-emetic treatment. Thus, Method B was significantly superior to Method A with regard to the degree of comfort and treatment satisfaction.