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.

Controlling joint instability after anterior cruciate ligament transection inhibits transforming growth factor-beta-mediated osteophyte formation.

OBJECTIVE: Abnormal joint instability contributes to cartilage damage and osteophyte formation. We investigated whether controlling joint instability inhibited chronic synovial membrane inflammation and delayed osteophyte formation and examined the role of transforming growth factor-beta (TGF-ß) signaling in the associated mechanism. DESIGN: Rats (n = 94) underwent anterior cruciate ligament (ACL) transection. Anterior tibial instability was either controlled (CAM group) or allowed to continue (SHAM group). At 2, 4, and 8 weeks after surgery, radiologic, histopathologic, immunohistochemical, immunofluorescent, and enzyme-linked immunosorbent assay examinations were performed to evaluate osteophyte formation and TGF-ß signaling. RESULTS: Joint instability increased cartilage degeneration score and osteophyte formation, and cell hyperplasia and proliferation and synovial thickening were observed in the synovial membrane. Major findings were increased TGF-ß expression and Smad2/3 following TGF-ß phosphorylation in synovial membarene, articular cartilage, and the posterior tibial growth plate (TGF-ß expression using ELISA: 4 weeks; P = 0.009, 95% CI [260.1-1340.0]) (p-Smad2/3 expression density: 4 weeks; P = 0.024, 95% CI [1.67-18.27], 8 weeks; P = 0.034, 95% CI [1.25-25.34]). However, bone morphogenetic protein (BMP)-2 and Smad1/5/8 levels were not difference between the SHAM model and the CAM model. CONCLUSIONS: This study showed that the difference between anterior tibial instability caused a change in the expression level of TGF in the posterior tibia and synovial membrane, and the reaction might be consequently involved in osteophyte formation.

Controlling joint instability delays the degeneration of articular cartilage in a rat model.

OBJECTIVE: Joint instability induced by anterior cruciate ligament (ACL) transection is commonly considered as a predisposing factor for osteoarthritis (OA) of the knee; however, the influence of re-stabilization on the protection of articular cartilage is unclear. The aim of this study was to evaluate the effect of joint re-stabilization on articular cartilage using an instability and re-stabilization ACL transection model. DESIGN: To induce different models of joint instability, our laboratory created a controlled abnormal joint movement (CAJM) group and an anterior cruciate ligament transection group (ACL-T). Seventy-five Wistar male rats were randomly assigned to the CAJM (n = 30), ACL-T (n = 30), or no treatment (INTACT) group (n = 15). Cartilage changes were assessed with soft X-ray analysis, histological and immunohistochemistry analysis, and real-time polymerase chain reaction (PCR) analysis at 2, 4, and 12 weeks. RESULTS: Joint instability, as indicated by the difference in anterior displacement between the CAJM and ACL-T groups (P < 0.001), and cartilage degeneration, as evaluated according to the Osteoarthritis Research Society International (OARSI) score, were significantly higher in the ACL-T group than the CAJM group at 12 weeks (P < 0.001). Moreover, joint re-stabilization maintained cartilage structure (thickness [P < 0.001], surface roughness [P < 0.001], and glycosaminoglycan stainability [P < 0.001]) and suppressed tumor necrosis factor-alpha (TNF-α) and caspase-3 at 4 weeks after surgery. CONCLUSION: Re-stabilization of joint instability may suppress inflammatory cytokines, thereby delaying the progression of OA. Joint instability is a substantial contributor to cartilage degeneration.

Usefulness of Muscle Synergy Analysis in Individuals With Knee Osteoarthritis During Gait.

OBJECTIVE: To clarify whether there are any muscle synergy changes in individuals with knee osteoarthritis, and to determine whether muscle synergy analysis could be applied to other musculoskeletal diseases. METHODS: Subjects in this study included 11 young controls (YC), 10 elderly controls (EC), and 10 knee osteoarthritis patients (KOA). Gait was assessed on a split-belt treadmill at 3 km/h. A non-negative matrix factorization (NNMF) was applied to the electromyogram data matrix to extract muscle synergies. To assess the similarity of each module, we performed the NNMF analysis assuming four modules for all of the participants. Further, we calculated joint angles to compare the kinematic data between the module groups. RESULTS: The number of muscle modules was significantly lower in the EC (2-3) and KOA (2-3) groups than in the YC group (3-4), which reflects the merging of late swing and early stance modules. The EC and KOA groups also showed greater knee flexion angles in the early stance phase. Contrarily, by focusing on the module structure, we found that the merging of early and late stance modules is characteristic in KOA. CONCLUSION: The lower number of modules in the EC and KOA groups was due to the muscle co-contraction with increased knee flexion angle. Contrarily, the merging of early and late stance modules are modular structures specific to KOA and may be biomarkers for detecting KOA. SIGNIFICANCE: Describing the changes in multiple muscle control associated with musculoskeletal degeneration can serve as a fundamental biomarker in joint disease.

Analysis of expressed sequence tags of flower buds in Lotus japonicus.

In order to study gene expression in a reproductive organ, we constructed a cDNA library of mature flower buds in Lotus japonicus, and characterized expressed sequence tags (ESTs) of 842 clones randomly selected. The EST sequences were clustered into 718 non-redundant groups. From BLAST and FASTA search analyses of both protein and DNA databases, 58.5% of the EST groups showed significant sequence similarities to known genes. Several genes encoding these EST clones were identified as pollen-specific genes, such as pectin methylesterase, ascorbate oxidase, and polygalacturonase, and as homologous genes involved in pollen-pistil interaction. Comparison of these EST sequences with those derived from the whole plant of L. japonicus, revealed that 64.8% of EST sequences from the flower buds were not found in EST sequences of the whole plant. Taken together, the EST data from flower buds generated in this study is useful in dissecting gene expression in floral organ of L. japonicus.

Characterization of potentially chemopreventive phenols in extracts of brown rice that inhibit the growth of human breast and colon cancer cells.

Rice is a staple diet in Asia, where the incidence of breast and colon cancer is markedly below that in the Western world. We investigated potential colon and breast tumor-suppressive properties of rice, testing the hypothesis that rice contains phenols that interfere with the proliferation or colony-forming ability of breast or colon cells. Brown rice, its white milled counterpart, and bran from brown rice were boiled and extracted with ethyl acetate. The extracts were analyzed by high pressure liquid chromatography-mass spectrometry. Eight phenols, protocatechuic acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid, vanillic acid, methoxycinnamic acid, and tricin, were identified in the extracts of bran and intact brown rice. These extracts were separated into nine fractions by column chromatography. The effect of bran extract and its fractions at 100 microg/ml on cell viability and colony-forming ability of human-derived breast and colon cell lines was assessed. Bran extract decreased numbers of viable MDA MB 468 and HBL 100 breast cells and colon-derived SW 480 and human colonic epithelial cells as judged by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 -sulfophenyl)-2H-tetrazolium assay. It also reduced colony formation of SW 480 colon and MDA MB 468 breast cells. Of the eight phenols identified in the brown rice bran, when applied at 50 microM, caffeic acid decreased numbers of all cell types except HBL 100. Tricin, ferulic acid, and methoxycinnamic acid interfered with cell viability in one or more cell lines. Tricin (50 microM) and the other phenols (200 microM) inhibited colony formation of SW 480 cells. Clonogenicity of MDA MB 468 cells was inhibited by caffeic acid, ferulic acid, and tricin (50 microM). Tricin was the most potent anticlonogenic of the compounds with IC50s of 16 microM in the SW 480 colon cells and 0.6 microM in the MDA MB 468 breast cells. The results suggest that: (a) brown rice and bran contain compounds with putative cancer chemopreventive properties; (b) certain phenols contained in brown rice bran, e.g., tricin, may be associated with this activity; and (c) these phenols are present at much lower levels in white than in brown rice. Thus, the consumption of rice bran or brown rice instead of milled white rice may be advantageous with respect to cancer prevention.

Architecture of Purkinje cells of the reeler mutant mouse observed by immunohistochemistry for the inositol 1,4,5-trisphosphate receptor protein P400.

P400 protein, which is identical to the inositol 1,4,5-trisphosphate receptor protein, is a glycoprotein closely associated with the membranes of Purkinje cells. Three types of monoclonal antibodies against P400 protein were employed for the immunohistochemical detection of Purkinje cells in the cerebellum and brainstem of the normal and reeler mouse. Purkinje cells in both types of mice were immunoreactive against anti-P400 antibodies, and the soma, dendrites, axon and even terminal boutons in the cerebellar and vestibular nuclei could be clearly visualized. In the cerebellum of the reeler mutant, the heterotopic Purkinje cells both within and below the granule cell layer were also immunopositive and could be clearly differentiated from the deep cerebellar nuclei, in which neurons were immunonegative. The molecular layer of the reeler cerebellum varied in thickness and certain parts were completely defective. The dendrites within the molecular layer extended from Purkinje cells whose cell bodies were located in the normal position, abnormally in the granule cell layer, or at the surface of the central mass. Outside the cortex of the cerebellum, ectopic Purkinje cells were demonstrated in 3 cerebellar nuclei, the cerebellar medulla and peduncle, and brainstem of the normal and reeler mouse.

Plant--fungal interactions: the search for phytoalexins and other antifungal compounds from higher plants.

A brief review is given of some biological, chemical and chemotaxonomic aspects of phytoalexin research. Emphasis is placed on the search for antifungal compounds in the plant families Leguminosae and Rosaceae, and in rice, Oryza sativa. The possible role of phytoalexins in the resistance of rice plants against the fungus Pyricularia oryzae (= Magnaporthe grisea) is discussed, and the future prospects of phytoalexin research are outlined.

Distribution of 8-oxygenated leaf-surface flavones in the genus Ocimum.

Nine species of Ocimum (Lamiaceae) were surveyed for leaf-surface flavonoids by means of HPLC with diode array detection and atmospheric pressure chemical ionisation (APCI) mass spectrometry. The analysis revealed the presence of 23 different flavones, most of which were identified by comparing their UV and mass spectra with those of standards. Almost all taxa investigated contained flavones methoxylated in the 6- and 8-positions, such as nevadensin, xanthomicrol and gardenin B. The same taxa also produced flavones methoxylated in the 6-position but hydroxylated in the 8-position, including isothymusin (5,8,4'-trihydroxy-6,7-dimethoxyflavone), pedunculin (5,8-dihydroxy-6,7,4'-trimethoxyflavone) and a new flavone, 5,7,8-trihydroxy-6,4'-dimethoxyflavone, which was given the trivial name pilosin. This compound was isolated from O. americanum var. pilosum and also detected as a minor constituent in O. x citriodorum leaf extracts. Its molecular structure was elucidated by means of NMR spectroscopy. 8-Oxygenated flavones were absent only from O. lamiifolium. APCI mass spectrometry of the flavonoids revealed that the product ions formed by collision induced dissociation of the protonated molecule provided structural information about the substitution pattern of the A-ring. The chemotaxonomic and biogenetic implications of the results are discussed.

Simple in-office closure of small intractable tympanic membrane perforations after myringoplasty.

Perforations produced following myringoplasty are very difficult to close by ordinary cautery or patching even if they are small. Therefore we decided to close these intractable perforations with a skin graft from the cartilaginous ear canal. Our method is an in-office operation under local anesthesia. After the removal of epithelium from around the perforation a skin graft was placed by the over-lay method. A total of 11 patients were treated, of which 81.8% (9/11) demonstrated successful closure in the initial surgery and two re-perforations were successfully closed by a second and third operation, respectively. The technique used in this surgery is relatively easy and appropriate for in-office operations. The method's advantages include decreased invasiveness with minimal anesthesia and no requirement for ear canal packing or fixatives, and cosmetic benefit. This method also can be applied to perforations caused by chronic otitis media, traumatic perforations, and perforations after inserting tubes.

[A morphological study on the brain in wriggle mouse Sagami, a new neurological mutant with dystonic disorders, with special reference to the abnormality of the cerebellum].

The 'Wriggle Mouse Sagami (WMS)' is a new neurological mutation that was discovered in an inbred strain of BALB/c mice at the Ohmura Institute for Laboratory Animals (Address: Zama 228, Kanagawa, Japan) in 1984. The affected characteristics are transmitted by an autosomal recessive gene (wri). The clinical symptoms are characterized by dystonic involuntary movements, such as fine tremors of the forelimbs, hypertonic musculature of the extremities, difficulty in maintaining an upright posture, writhing of the trunk, wriggling of the neck up-and-down and from side to side without any coordination between the movements of the limbs and trunk, and inability to walk a staigho that begin at 10 days to 2 weeks after birth and are progressive until 12 weeks of age. Here a morphological study on the brain of the WMS with special reference to the cerebellum was intended. The results were as follows: (1) In spite of these severe clinical symptoms, no marked abnormalities were observed in the cyto- and myeloarchitecture of the central nervous system, although the size of the whole brain was somewhat reduced and the molecular layer of the cerebellum was relatively hypotrophic compared with the granule cell layer. (2) The motor tracts investigated were normally detected by the retrograde HRP-labeling method. (3) Slight abnormality of the dendritic trees and spines, and remarkable axonal swellings of the Purkinje cells were demonstrated by a sensitive immunohistochemical method for inositol 1,4,5-trisphophate receptor protein (P400 protein). These changes were also evaluated by the Golgi method. (4) In electron microscopy of the molecular layer of the WMS cerebellum, parallel fibers seemed to be reduced and shrunken, and their synaptic contacts on the dendritic spines of the Purkinje cells were clearly reduced even at 17 days after birth. (5) Consequently, the Purkinje cells were possibly affected by failure of accurate connection with input fibers or by synaptic dysfunction, which might occur over the entire central nervous system.

Antimicrobial activity of some Ganoderma species from Nigeria.

The crude n-hexane:diethyl ether, chloroform:acetone and methanol extracts of four species of Ganoderma (Ganoderma colossum (Fr.) C. F. Baker, G. resinaceum Boud., G. lucidum (cf.) (Curtis) P. Karst. and G. boninense (cf.) Pat.), from Nigeria, were tested for antimicrobial activity. The three solvent extracts of all the species of Ganoderma were active against Pseudomonas syringae and Bacillus subtilis, whereas none of the extracts were active against Cladosporium herbarum. Preliminary thin layer chromatography chemical tests on these extracts of Ganoderma showed that they contained compounds that stained blue-violet and blue or green when sprayed with anisaldehyde-sulphuric acid or Dragendorff, respectively. The profile of compounds in the extracts showed some variation among the four species.

Change in membrane permeability induced by amyloid beta-protein fragment 25-35 in brain neurons dissociated from rats.

Effects of amyloid beta-protein fragment 25-35, A beta P(25-35), on the membrane permeability of organic molecules were examined in the brain neurons dissociated from rats by using an argon laser (equipped in flow cytometer and laser microscope) and a combination of two fluorescent dyes, fluo-3-AM and ethidium bromide. A beta P(25-35) at concentrations of 1 microM or greater induced both leakage of fluo-3 from the neurons and permeation of ethidium across the membrane in a dose-dependent manner, although both dyes are highly impermeant to the intact plasma membrane. Thus, A beta P(25-35) seems to increase not only membrane permeability of inorganic ions such as Ca2+, Na+ and K+, as previously suggested, but also that of organic molecules. Therefore, the brain neuron membrane is suggested to lose its integrity in the presence of A beta P(25-35) that leads to neuronal death.

Surgical results of tympanoplasty with hokudai-shaped partial and total ossicular replacement prostheses.

The surgical results of tympanoplasty using a ceramic partial ossicular replacement prosthesis (PORP) or a ceramic total ossicular replacement prosthesis (TORP) were analyzed. The present study included 25 ears in 22 patients who underwent tympanoplasty between December 1986 and September 1994 and were followed up for more than 6 months postoperatively. Because the ceramic prosthesis was extruded from 2 of the 25 ears, the surgical results in the other 23 ears were analyzed, Surgical results were assessed according to a criterion that permits comparison with data from around the world. The criterion considers a postoperative airbone gap within 20 dB as a success. According to this criterion, the success rate was 90.9% for PORP, 50.0% for TORP, and 69.6% in total. These results are comparable with those found at other centers.

Thyroplasty type I with ceramic shim.

To prevent side effects from a silicone shim in Isshiki thyroplasty type I, we used a ceramic shim in 10 patients with unilateral recurrent laryngeal nerve paralysis. No published reports have described the use of ceramic in this type of surgery. According to the degree of glottic insufficiency, ceramic shims of various heights were inserted into the fenestration made in the thyroid ala. All patients experienced subjective improvement of voice postoperatively. Laryngoscopies in most cases showed that glottic insufficiency improved postoperatively. In the postoperative examination, the maximum phonation time improved an average of 3.7 s, and the mean flow rate improved an average of 331 ml/s. We have analyzed the relationship of these improvements to the degree of glottic insufficiency and have compared our results with those of other investigators.

Detection of abnormal high-frequency components in the QRS complex by the wavelet transform in patients with idiopathic dilated cardiomyopathy.

In order to investigate whether increased fine, fractionated signals within the QRS complex can detect arrhythmogenic substrates and how these fine signals link with ventricular mechanical dysfunction, wavelet analysis was performed on averaged QRS complexes obtained from the left precordial lead in 26 patients with idiopatic dilated cardiomyopathy (IDCM) and in 12 normal subjects. The number of local maxima and the duration of the wavelet transform were significantly greater in patients with IDCM than in normal subjects; the number at 100 Hz was 8.8+/-3.1 vs 6.0+/-1.1 (p<0.01), and the duration at 100Hz was 93+/-15 vs 75+/-7ms (p<0.01). Both of these indices were greater in the patients with than in those without late potentials, repetitive ventricular premature beats or cardiac death. In addition, significant inverse curvilinear relationships were observed between the left ventricular ejection fraction and both the number of local maxima and the duration of the wavelet transform. In conclusion, fine fragmented signals in the QRS complex detected by wavelet analysis would be an important marker for potentially arrhythmogenic substrates and seemed to progress in parallel with left ventricular mechanical dysfunction in IDCM.