Joint instability causes catabolic enzyme production in chondrocytes prior to synovial cells in novel non-invasive ACL ruptured mouse model.

OBJECTIVE: The Anterior Cruciate Ligament (ACL)-deficient model helps to clarify the mechanism of knee osteoarthritis (OA); however, the conventional ACL injury model could have included concurrent onset factors such as direct compression stress to cartilage and subchondral bone. In this study, we established a novel Non-invasive ACL-Ruptured mouse model without concurrent injuries and elucidated the relationship between OA progression and joint instability. DESIGN: We induced the ACL-Rupture non-invasively in twelve-week-old C57BL/6 male mice and evaluated histological, macroscopical, and morphological analysis at 0 days. Next, we created the ACL-R, controlled abnormal tibial translation (CATT), and Sham groups. Then, the joint stability and OA pathophysiology were analyzed at 2, 4, and 8 weeks. RESULTS: No intra-articular injuries, except for ACL rupture, were observed in the ACL-R model. ACL-R mice increased anterior tibial displacement compared to the Sham group (P < 0.001, 95% CI [-1.509 to -0.966]) and CATT group (P < 0.001, 95% CI [-0.841 to -0.298]) at 8 weeks. All mice in the ACL-R group caused cartilage degeneration. The degree of cartilage degeneration in the ACL-R group was higher than in the CATT group (P = 0.006) at 8 weeks. The MMP-3-positive cell rate of chondrocytes increased in the ACL-R group than CATT group from 4 weeks (P = 0.043; 95% CI [-28.32 to -0.364]) while that of synovial cells increased at 8 weeks (P = 0.031; 95% CI [-23.398 to -1.021]). CONCLUSION: We successfully established a Non-invasive ACL-R model without intra-articular damage. Our model revealed that chondrocytes might react to abnormal mechanical stress prior to synovial cells while the knee OA onset.

The difference in joint instability affects the onset of cartilage degeneration or subchondral bone changes.

OBJECTIVE: It has been debated whether the onset of knee osteoarthritis is initiated in cartilage or subchondral bone. The purpose of this study was to clarify the effects of increasing or decreasing joint instability on cartilage degeneration and subchondral bone changes in knee OA by comparing different models of joint instability. DESIGN: We used the anterior cruciate ligament transection (ACL-T) model and the destabilization of the medial meniscus (DMM) model. In addition, we created a controlled abnormal tibial translation (CATT) model and a controlled abnormal tibial rotation (CATR) model. We performed joint instability analysis, micro-computed tomography analysis, histological and immunohistological analysis in 4 and 6 weeks. RESULTS: The CATT group suppressed joint instability in the ACL-T group (6 weeks; P = 0.032), and the CATR group suppressed joint instability in the DMM group (6 weeks; P = 0.032). Chondrocyte hypertrophy in the ACL-T and DMM groups was increased compared to the Sham group (6 weeks; [ACL-T vs Sham], P = 0.002, 95%CI [5.983-33.025]; [DMM vs Sham], P = 0.022, 95%CI [1.691-28.733]). In the subchondral bone, the BV/TV in the DMM and CATR groups was increased compared to the ACL-T and CATT groups (6 weeks; [DMM vs ACL-T], P = 0.002, 95%CI [7.404-37.582]; [DMM vs CATT], P = 0.014, 95%CI [2.881-33.059]; [CATR vs ACL-T], P = 0.006, 95%CI [4.615-34.793]; [CATR vs CATT], P = 0.048, 95%CI [0.092-30.270]). CONCLUSIONS: This study showed that joint instability promotes chondrocyte hypertrophy, but subchondral bone changes were influenced by differences in ACL and meniscus function.

Very-low-voltage operation of Mach-Zehnder interferometer-type electroabsorption modulator using asymmetric couplers.

We have proposed and developed a new type of electroabsorption modulator (EAM) that employs both optical absorption and interferometric extinction. The EAM operates at a record low voltage of 0.2 V at 25.8-Gbit/s modulation, which can reduce optical transmitter power consumption and allows the adoption of cost-effective CMOS drivers.

Measurement of psychological state changes at low dopamine transporter occupancy following a clinical dose of mazindol.

RATIONALE: The beneficial effects of psychostimulant drugs in the treatment of psychiatric disorders occur because they increase the extracellular dopamine concentration by inhibiting re-uptake of extracellular dopamine at dopamine transporters. However, the psychological effects at low dopamine transporter occupancy have not been well demonstrated. OBJECTIVES: The purpose of the study was to evaluate the psychological effects, dopamine transporter occupancy, and dopamine release induced by a single oral administration of a clinical dose of mazindol. METHODS: Ten healthy male volunteers were orally administered a placebo and a clinical dose of mazindol (1.5 mg) on separate days. The psychological effects of mazindol were assessed using a visual analogue scale to detect alterations in the state of consciousness. The amount of blockade of dopamine transporters was assessed using positron emission tomography with [18F]FE-PE2I and extracellular dopamine release was measured as the amount of change in [11C]raclopride binding. RESULTS: Following administration of a clinical dose of mazindol, the dopamine transporters were blocked by 24-25 %, and the binding potential of [11C]raclopride was reduced by 2.8-4.6 %. The differences of a score measuring derealisation and depersonalization associated with a positive basic mood were significantly correlated with the change in the [11C]raclopride binding in the limbic striatum. CONCLUSIONS: A subtle alteration in the state of consciousness was detected with a correlation to the changes in the [11C]raclopride binding, which implies that a subtle alteration in extracellular dopamine concentration in the limbic striatum by a small amount of dopamine transporter occupancy can affect the state of consciousness. TRIAL REGISTRATION HTTPS://UPLOAD.UMIN.AC.JP/CGI-OPEN-BIN/CTR_E/CTR_VIEW.CGI?RECPTNO=R000009703 : UMIN000008232.

Cross-linking of a synthetic partial-length (1-28) peptide of the Alzheimer beta/A4 amyloid protein by transglutaminase.

Cerebral deposits of beta/A4 amyloid protein is a pathologic sign of Alzheimer's disease. A synthetic partial-length (1-28) peptide of this protein contains one glutamine and two lysine residues. Here we show that this peptide can be a substrate of transglutaminase, which catalyzes cross-linking between glutamine and lysine residues in peptides, by demonstrating the formation of multimeric peptides due to the action of this enzyme. A modified (Lys28 to L-norleucine) version of the synthetic peptide was also cross-linked, but another modified version (Lys16 to L-norleucine) was very poorly cross-linked, indicating that Lys16 is involved exclusively in the cross-linking of the partial-length peptide catalyzed by transglutaminase.

The inhibitory effects of okadaic acid on platelet function.

Okadaic acid (OA), a potent inhibitor of protein phosphatases type 1 and type 2A, inhibited thrombin-induced platelet aggregation (IC50 = 0.8 microM), [14C]serotonin release and increase in intracellular Ca2+ ([Ca2+]i) in the same dose dependence. In the absence of thrombin OA increased the phosphorylation of 50-kDa protein and 20-kDa myosin light chain (MLC20). The 50-kDa protein phosphorylation was accomplished within a shorter time period and at a lower concentration than was the MLC20. OA decreased the thrombin-induced phosphorylation of 47-kDa protein and MLC20, although phosphorylation of MLC20 reincreased at higher concentrations of OA (5-10 microM). Since type 2A phosphatase is more sensitive to OA than type 1, these results suggest that type 2A phosphatases are involved in the regulation of Ca2+ signaling in thrombin-induced platelet activation.

Purification and partial characterization of CD9 antigen of human platelets.

CD9 antigen (p24) was purified from human platelets and partially characterized. The yield was 75 micrograms from 10 units of platelet concentrates. p24 (38,000 copies/platelet) has intramolecular disulfide bond(s) and, in SDS-PAGE, consists of major 24-kDa molecule and minor 26- to 31-kDa molecules. The N-terminal sequence of p24, PVKGGTKXIKYLLFGFNFIF, indicates that the protein has not previously been characterized and amino terminus (position 12-20) is hydrophobic.

The platelet activation induced by wheat germ agglutinin.

In human platelets, wheat germ agglutinin (WGA) induced serotonin release without cell agglutination. WGA induced the phosphorylation of both 40-kDa and 20-kDa proteins in a parallel manner, and at least, the phosphorylation of 40-kDa protein was preceded by transient formation of endogenous diacylglycerol (DG) accompanied by a decrease in phosphatidylinositol (PI). Both phosphorylation of these two proteins and serotonin release were inhibited by prior treatment of platelets with dibutyryl cyclic AMP, W-7, or TMB-8. These results suggest that both phosphatidylinositol turnover and Ca2+ mobilization play an essential role in WGA-induced platelet activation.

(-)-1-(Benzofuran-2-yl)-2-propylaminopentane enhances locomotor activity in rats due to its ability to induce dopamine release.

"Catecholaminergic and serotoninergic activity enhancer" effects are newly found mechanisms of action of a class of compound that enhance impulse propagation-mediated release of catecholamines and serotonin in the brain. In the present study, (-)-1-(benzofuran-2-yl)-2-propylaminopentane hydrochloride [(-)-BPAP HCl], a compound with selective and potent "catecholaminergic and serotoninergic activity enhancer" effects, was tested for its efficacy to potentiate locomotor activity in normal rats and to attenuate hypolocomotion in reserpine-treated rats. (-)-BPAP HCl potentiated locomotor activity in non-habituated rats during a 2-h observation period dose-dependently (0.3-10 mg/kg). (-)-BPAP HCl (1-3 mg/kg) was also effective to reverse reserpine-induced hypolocomotion. The effects of (-)-BPAP HCl in normal and reserpine-treated rats were attenuated by the dopamine D1 receptor antagonist, R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390), suggesting that the effects of (-)-BPAP HCl were mediated by activation of the dopaminergic system. In addition, the administration of (-)-BPAP HCl increased ipsilateral turning in unilaterally 6-hydroxydopamine-lesioned rats, implying presynaptic activation of nigrostriatal dopaminergic terminals by (-)-BPAP HCl. Furthermore, although antiparkinsonian agents, such as apomorphine and amantadine, failed to improve reserpine-induced ptosis, (-)-BPAP HCl significantly improved ptosis. These findings suggested that a "catecholaminergic and serotoninergic activity enhancer" compound, (-)-BPAP, stimulates motor function in rats and improves motor deficits in animal models of Parkinson's disease due to its ability to induce dopamine release.

PGI3 production from eicosapentaenoic acid in 3T3 fibroblast cells and cultured bovine pulmonary artery endothelial cells.

Production of PGI3 from eicosapentaenoic acid (EPA) in 3T3 fibroblast cells and in cultured bovine pulmonary artery endothelial cells (CPAE) which had an ability to produce PGI2 from arachidonic acid (AA), was investigated by bioassay, gas chromatography-mass spectrometry (GC-MS) and thin layer chromatography (TLC). Inhibition of platelet aggregation was observed in the supernatant obtained from a culture medium of 3T3 fibroblast cells after incubation with EPA. This active substance in the supernatant could be identified as PGI3, since the inhibitory effect of the supernatant was blocked by tranylcypromine, a potent inhibitor of prostacyclin synthetase. The inhibitory effect on platelet aggregation by the supernatant from culture medium with EPA was low compared with that with AA. More delta 17-6-keto PGF1 alpha from EPA was produced than 6-keto PGF1 alpha from AA at the same concentration of substrates, when the formation of prostacyclins from EPA and AA was measured as their stable metabolites, delta 17-6-keto PGF1 alpha and 6-keto PGF1 alpha by GC-MS respectively. It is suggested that the anti-aggregatory ability of PGI3 is lower than that of PGI2. On the contrary, the amount of PGI3 produced from EPA was significantly less than that of PGI2 produced from AA in CPAE. The production of 6-keto PGF1 alpha from exogenous AA was decreased by increasing the ratio of EPA to AA in the culture medium and the production of delta 17-6-keto PGF1 alpha from EPA also decreased by an addition of AA to the culture medium. This result suggests that AA and EPA compete each other at the site of action of cyclooxygenase.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of forskolin on platelet deaggregation and cyclic AMP generation.

The diterpene, forskolin, induced a partial deaggregation of ADP- or collagen-aggregated human platelets in vitro. An increase in platelet cyclic AMP by forskolin was assumed to mediate the platelet deaggregation. PGE1 also deaggregated these platelets, and a combination of forskolin and PGE1 produced deaggregation greater than the maximum which could be obtained with each agent alone. A greater than additive effect was observed on the platelet cyclic AMP level in the presence of both forskolin and PGE1. No additive effect was observed in the phosphorylation of molecular weight (Mr) 21K polypeptide using forskolin (0.1 mmol/l) and PGE1 (5 mumol/l) suggesting that although cyclic AMP is responsible for the deaggregation process a mechanism other than phosphorylation through cyclic AMP-dependent protein kinase may be responsible for the effect of forskolin on platelet deaggregation.

Spectrophotometric determination of trace amounts of hemoglobin using the oxidative decomposition reaction of a copper(II)-phthalocyanine complex.

A catalytic effect of hemoglobin was found in the oxidative decomposition of a copper(II)-phthalocyanine complex (Cu-pts) with peroxomonosulfate. Based on this finding, a simple, rapid, highly sensitive and high-precision spectrophotometric method for hemoglobin was developed. In this indicator system, Cu-pts as the indicator and hemoglobin as the catalyst were simultaneously decomposed with peroxomonosulfate. Therefore, the slopes of the absorbance-time curve of the sample solution become equal to that of the blank solution after 5 min. The calibration curve was linear over the range of 12-258 microg/l. Detection limits (3sigma) were 8.13 microg/l, and the relative standard deviation was 2.05% (10 determinations) for 130 microg/l of hemoglobin. The metal ions did not significantly produce an interference in this method. The albumin had some influence but was permissible up to 6 times the weight of hemoglobin. The proposed method was applied to human blood samples with satisfactory results.

Growth inhibition of capsaicin on HeLa cells is not mediated by intracellular calcium mobilization.

The effects of capsaicin on cellular growth and intracellular calcium mobilization were examined in human cervical carcinoma derivation, HeLa cells. Capsaicin inhibited cellular growth and increased intracellular calcium level in HeLa cells. This capsaicin-induced intracellular calcium concentration rise was blocked by capsazepin, vanilloid (capsaicin) receptor antagonist. But, an intracellular calcium chelator BAPTA/AM did not block the inhibitory effect of capsaicin on cellular growth. These observations suggest that intracellular calcium mobilization is not required for the capsaicin-induced inhibition of cellular growth.

Bioactive peptides derived from food proteins preventing lifestyle-related diseases.

Many kinds of bioactive peptides which might prevent lifestyle-related diseases are released from food proteins after enzymatic digestion. Inhibitory peptides for angiotensin I-converting enzyme (ACE) having anti-hypertensive effect have been isolated from enzymatic digests of various food proteins. LKPNM, which was isolated from the thermolysin digest of dried bonito was activated 8-fold by ACE itself and showed a prolonged effect after oral administration. Two vasorelaxing peptides, ovokinin and ovokinin(2-7), showing antihypertensive effect after oral administration were obtained from ovalbumin digests. We found that low molecular weight peptides derived from food proteins lowered serum cholesterol without increasing excretion of cholesterol and bile acids. An immunostimulating peptide isolated from an enzymatic digest of soybean protein prevented alopecia induced by cancer chemotherapy.

[Method-performance studies of notified analytical method for fenbutatin oxide and cyhexatin].

Method-performance studies were conducted for the notified revised analytical method of fenbutatin oxide (FBTO) and cyhexatin (CHT). FBTO and CHT spiked into rice, soybeans, spinach, orange, tea powder and tea extract at the level of 0.5 microgram/g for FBTO and 0.1 microgram/g for CHT were analyzed in replicate in 6 laboratories. Means recoveries of FBTO were 85.2-96.5% and those of CHT were 83.5-89.2% except from soybeans (46.5%). Repeatability relative standard deviation values of FBTO and CHT in each crop were in the ranges of 2.3-9.4% and 3.2-6.3%, respectively. Reproducibility relative standard deviations were 3.9-12.6% for FBTO and 8.3-12.9% for CHT. Detection limits were 0.015-0.05 microgram/g for FBTO and 0.005-0.02 microgram/g for CHT.

[Method-performance studies of notified analytical method for clofentezine].

Method-performance studies were conducted for the notified revised analytical method of clofentezine. Clofentezine spiked in azuki beans, apple, orange, banana, grape, tea powder and tea extract at the level of 0.2 microgram/g (2 micrograms/g for tea) was analyzed in replicate in 6 laboratories. Mean values of recovery from 7 crops ranged from 78.4 to 85.2%. Repeatability relative standard deviation values ranged from 2.2 to 4.6% and reproducibility standard deviation values ranged from 4.8 to 10.3%. The detection limits were 0.005-0.01 microgram/g. These results show the notified analytical method has good performance.

[Effects of selegiline hydrochloride on intracellular Ca2+ contents in cultured neuronal cells].

The effects of Selegiline hydrochloride (Selegiline HCl) on the intracellular Ca2+ contents of primarily cultured rat striatal, mesencephalic neuronal cells and PC-12 cells were examined by the use of a Ca2+ imaging analyzer. In the former two cell types, Selegiline HCl (10(-5)-10(-6) M) induced a transient inflow of extracellular Ca2+ through the voltage-dependent N-type Ca2+ channel. In addition, all cells indicating an increase in the intracellular Ca2+ content were found to be catecholaminergic neurons which showed a positive reaction with anti-tyrosine hydroxylase antibodies. Furthermore, a transient intracellular influx of Ca2+ was observed in the NGF-pretreated PC-12 cells. From these results, it is suggested that Selegiline HCl elicits various functions, including antioxidation, activation of neurotrophic factor biosynthesis and neuronal protection probably via an unidentified specific proteins of tyrosine hydroxylase-positive neurons.

[Effect of PSK on prostaglandin metabolism (I)].

The effect of PSK (Krestin) on the metabolism of prostaglandins was investigated. The effect of PSK on the thromboxane A2 (TXA2) level, which stimulates tumor proliferation and platelet aggregation, was examined using platelets. PSK suppressed platelet aggregation and the production of malondialdehyde (MDA) and TXB2 which is a stable metabolite of TXA2. The effect of PSK on the production of prostacyclin (PGI2), which is an anti-tumor PG, was then examined using rat arterial rings. It was found that PGI2 production was stimulated by PSK. The in vivo inhibition of platelet activation by PSK was then examined using two thrombosis models in which platelet aggregation was mainly involved. PSK exerted its anti-platelet effect by regulation of PG production. It was concluded that not only an immune regulating effect but also PG regulation are involved in the pharmacological action of PSK.