High MMP-1, MMP-2, and MMP-9 protein levels in osteoarthritis.

Our study examined the relationship between the expression of matrix metalloproteinases (MMP)-1, MMP-2, and MMP-9 proteins and the pathogenesis of osteoarthritis (OA). We employed rigorous inclusion and exclusion criteria in computer-based bibliographic databases to extract published studies relevant to this investigation. The STATA 12.0 software was used for the statistical analyses. A total of 1408 studies were initially searched, and 10 studies with 458 OA patients and 295 healthy controls were included in this meta-analysis. The meta-analysis results suggested that the protein levels of MMP-1, MMP-2, and MMP-9 were higher in patients with OA than those in the control group. A subgroup analysis according to ethnicity showed that the protein levels of MMP-1 and MMP-2 were higher in Asian patients with OA than in controls. Caucasians showed no statistically significant differences in protein expression of MMP-1 and MMP-2 between the OA patient group and the control group. Interestingly, the protein levels of MMP-9 in patients with OA were higher than those in the control group in both Asians and Caucasians. A sample-source analysis suggested that the serum levels of MMP-2 and MMP-9 proteins were higher in patients with OA than in controls, while MMP-1 and MMP-9 protein expressions were higher in the synovial joint fluid of patients with OA than in controls. In conclusion, our meta-analysis results suggested that the increased expression of MMP-1, MMP-2, and MMP-9 proteins might be associated with the pathogenesis of OA.

Toroidal soft x-ray array on the EXL-50 spherical tokamak.

A toroidal soft x-ray array system for spectrum and intensity measurements on the EXL-50 spherical tokamak is described. Silicon drift detectors and digital multichannel analyzers are adopted for all 21 channels of the array, and an average energy resolution of 147 eV at 5.89 keV has been achieved at count rates over 500 kcps. In total, 20 channels of the array are symmetrically observed in both co- and counter-current directions on the EXL-50 mid-plane with a spatial resolution of around 10 cm, and the remaining one serves as a background reference channel. Tungsten emissions from tungsten coating of the limiters on the central post are observed. The influence of hard x rays on measured soft x-ray spectra and system operation is discussed.

Analysis of ovariectomy and estrogen effects on body composition in rats by X-ray and magnetic resonance imaging techniques.

Resistance of bone to fracture--bone strength--has been shown to depend on both the amount of bone and its architectural spatial organization. In vivo magnetic resonance (MR) techniques have the capability of imaging bone tissue, including the trabecular microarchitecture and the marrow composition. We have applied in vivo and ex vivo MR methods to the tibia in an ovariectomized rat model of osteoporosis. Specifically, in vivo high-resolution three-dimensional MR imaging and localized MRS were facilitated by specialized coils and high field magnets, resulting in enhanced sensitivity of detection. As a result, in vivo and ex vivo differences in marrow composition were found between sham-ovariectomized, ovariectomized, and ovariectomized animals treated with 17-beta-estradiol. Estrogen effects were detected in vivo 7 days after surgery (3 days into treatment) as a decrease in the tibial fat signal level. The in vivo effects of ovariectomy were observed 56 days after surgery as an increase in MR image fat signal level and spectral fat/water ratio in the proximal tibia. Ex vivo measurements of tibial marrow water signal discriminated clearly between the sham and ovariectomized groups and showed increased individual variations in the treatment group. Imaging further showed that the highest fat content is observed in the epiphysis. Computed tomography confirmed ovariectomy-induced loss of bone in the proximal tibial metaphysis compared with the sham group. This loss of cancellous bone with ovariectomy is consistent with the MR observations of increases in both fat and water in the metaphysis. These data showed that MR techniques complement X-ray techniques in the bone, water, and fat compositional analysis of the appendicular skeleton in response to ovariectomy and pharmacological treatment.

Biosynthetic human parathyroid hormone (1-34) effects on bone quality in aged ovariectomized rats.

For the first time, PTH (1-34) was found to significantly affect bone quality, femora length, and body weight of aged, ovariectomized rats. Specifically, we examined the effects of biosynthetic human PTH (1-34) in 9 month-old rats that were ovariectomized and dosed for the ensuing 6 months with 8 or 40 microg/kg PTH. Bone content, architecture, and quality of axial and appendicular skeletal sites were analyzed by computed tomography (QCT), histomorphometry, and biomechanical testing. The large sample size (n = 26-35) of this study was useful in confirming the anabolic, dose-dependent effects of PTH at trabecular and cortical bone sites. Longitudinal analysis of tibias by QCT confirmed a small age-dependent reduction in bone density, with further reductions observed for ovariectomized controls (OVX). Subtle but deleterious effects of ovariectomy on bone quality are described. Additionally, the strength of the femoral neck was shown not to differ between baseline, sham-operated controls, or OVX in this model, suggesting limited utility of this measurement in aged rats. Both doses of PTH induced substantial gains above OVX, in bone mass and connectivity for the proximal tibia, distal femur, proximal femur, and spine. Ovariectomy significantly decreased the toughness of vertebral bone. However, PTH at 8 microg/kg reversed this deleterious effect on bone quality, while 40 microg/kg PTH significantly improved both toughness and brittleness beyond baseline controls. Cortical bone analyses at the femoral or tibial diaphysis confirmed the PTH stimulation of endosteal and periosteal bone formation with resulting increase in cortical thickness, moment of inertia, strength, and stiffness of the femur. PTH treatment significantly improved the intrinsic properties, Young's modulus and toughness, of the femur compared with OVX. At 40 microg/kg PTH, bone mass and strength were typically greater than sham or baseline controls, confirming that PTH is functionally anabolic at trabecular and cortical bone sites. Interestingly, PTH dose-dependently increased the femora length, in the absence of differences between baseline, sham, and OVX controls. PTH slightly increased body weight above OVX. In addition, PTH did not interfere with the beneficial effects of ovariectomy on rat longevity.

LY353381 x HCl: an improved benzothiophene analog with bone efficacy complementary to parathyroid hormone-(1-34).

LY353381 x HCl is a benzothiophene analog that is structurally related to raloxifene with potent selective estrogen receptor modulator activity in the ovariectomized rat model of postmenopausal osteoporosis. The effects of LY353381 x HCl on bones, body weight, and uterine weight were evaluated in 7-month-old rats with osteopenia that was induced by ovariectomizing animals for 1 month before initiation of treatment with several agents individually, in combination, or in sequence. LY353381 x HCl was administered daily by itself for 90 days, in combination with the amino-terminal fragment of PTH-(1-34) (PTH) for 90 days, or sequentially after PTH when PTH was discontinued after 45 days of treatment. Additionally, comparisons were made of animals treated with PTH alone, 17alpha-ethynyl estradiol alone, equine estrogens (Premarin) alone, raloxifene alone, or combinations of PTH and equine estrogens or raloxifene. Ovariectomy induced increases in the rate of bone turnover and body weight while decreasing bone mineral density, bone mineral content, bone strength, trabecular bone volume, trabecular thickness, trabecular number, and uterine weight. LY353381 x HCl at 0.01-1 mg/kg had marginal effects on body weight and no effect on uterine weight compared with those in ovariectomized controls, in contrast to 17alpha-ethynyl estradiol or equine estrogens. LY353381 x HCl prevented further bone loss due to ovariectomy in tibia, femora, and lumbar vertebra, like 17alpha-ethynyl estradiol but unlike equine estrogens. LY353381 x HCl prevented the resorption of trabecular bone spicules, like 17alpha-ethynyl estradiol, but inhibited bone formation activity to a lesser extent than 17alpha-ethynyl estradiol. In this model, 17alpha-ethynyl estradiol appeared to be more efficacious after 3 months of treatment than equine estrogens in the proximal tibia metaphysis, suggesting efficacy differences between metabolites of 17beta-estradiol in bone. PTH at 10 microg/kg had no effect on body weight or uterine weight, but significantly increased bone mass to beyond those in sham-operated controls, baseline controls, and groups receiving other individual treatments at both axial and appendicular sites. The combination of LY353381 x HCl and PTH increased bone mass at a faster rate and to a greater extent than PTH alone or the combinations of equine estrogens/PTH and raloxifene/PTH at trabecular bone sites. The LY353381 x HCl/PTH combination improved bone mass and quality beyond any agent alone in regions enriched for cancellous bone, but was not significantly better than PTH alone on cortical bone. Additionally, when PTH was discontinued at 45 days, LY353381 x HCl prevented the rapid loss of bone observed in controls. Therefore, LY353381 x HCl appears to be useful by itself, in combination, or in sequence with PTH to replace lost bone in postmenopausal women.

Cyclic AMP synthesis in Escherichia coli strains bearing known deletions in the pts phosphotransferase operon.

A series of isogenic strains harboring known deletions in the pts operon of Escherichia coli have been constructed by reverse genetics. Strains bearing deletions for the whole pts operon failed to grow on maltose or on carbon sources of the same class. In these strains the total cAMP synthesis was significantly lower than in a strain deleted only for the crr gene. This indicated that enzyme I or phosphorylated histidine-containing phosphotransferase protein in addition to its role in phosphorylating enzyme IIIGlc, is involved in adenylate cyclase (AC) activation or cAMP excretion. It was further shown that deletions in the pts operon do not affect synthesis of AC.

Mutational analysis of the enzyme IIIGlc of the phosphoenolpyruvate phosphotransferase system in Escherichia coli.

The phosphoenolpyruvate phosphotransferase system (PTS) component EIIIGlc is responsible for transport and phosphorylation of glucose via EIIGlc. It also regulates the catabolism of other carbon sources, such as lactose and maltose, by modulating both the intracellular concentrations of the corresponding inducers and of cAMP. Mutational analysis of EIIIGlc was performed in order to identify crucial residues mediating the interactions between EIIIGlc and its target proteins. Such mutations were isolated by in vitro hydroxylamine mutagenesis of the cloned EIIIGlc gene, crr. Five mutated EIIIGlc impaired in the function of inducer exclusion were obtained. However, these mutations did not abolish the function of EIIIGlc in the transport and phosphorylation of glucose, nor in activation of adenylate cyclase. A single amino acid change was found for each mutation, which is located in a restricted area of the polypeptide chain: Gly47-->Ser47 for the HA2 and HA5 mutations, Ala76-->Thr76 for HA4 mutation and Ser78-->Phe78 for HA3 mutation, indicative of quaternary interactions between the corresponding region of EIIIGlc and its target protein(s).

[Drug antagonism of TNF induced proliferation of bovine cerebromicrovascular smooth muscle cells].

Effects of tumor necrosis factor (TNF) on the proliferation of bovine cerebromicrovascular smooth muscle cells (BCSMC) were investigated. At concentrations from 50 to 5000 U.ml-1, TNF was shown to induce proliferation of cultured BCSMC in a dose-dependent manner. After 24 h incubation of the cells, TNF significantly stimulated the proliferation of BCSMC and reached maximal effects after 48 h incubation, then the effects slightly decreased. At concentrations from 10(-6) to 10(-4) mol.L-1, both imperatorin (Imp) and iso-imperatorin (Isi) were found to antagonize the TNF induced proliferation of BCSMC. Their maximal inhibitory effects were 42.2 and 36.1% at 10(-4) mol.L-1 respectively. 6-(alpha,alpha-diphenylacetylpiperazinly) phenyl-5-methyl-4,5-dihydro-3 (2H)-pyridazinone (DMDP) and 6-(alpha-phenylacetylpiperazinyl) phenyl-5-methyl-4,5-dihydro-3(2H)-pyridazinone (PMDP) were also found to possess similar effect at lower concentration (10(-6) mol.L-1), but no significant effect was observed when the drug concentration was higher.

Effects of dauricine and anisodamine on LTs and PAF induced DNA synthesis in bovine anterior cerebral arterial smooth muscle cells.

The effects of leukotrienes (LTs) and platelet activating factor (PAF) on DNA synthesis in bovine anterior cerebral arterial smooth muscle cells were studied. The results showed that LTB4, LTD4 and PAF were all stimulatory to the cells. The maximal rates of stimulation were 32%, 29% and 77%, respectively, for LTB4, LTD4 and PAF at 10(-7) mol/L. Both dauricine and anisodamine at concentrations of 10(-7) to 10(-4) mol/L exhibited strong inhibitory effects on LTs and PAF induced DNA synthesis.

[Effects of calcimycin on platelet adhesion to cultured bovine cerebral microvascular endothelial cells and its antagonism by drugs].

Calcimycin induced rabbit platelet adhesion to cultured bovine cerebral microvascular endothelial cells (CMEC) and its inhibition by triazelodiazepine (WEB 2086), 1,5-bis-(3,4-dimethoxyphenyl)-tetrahydro-(4H)-pyran (DMPP) and tetrandrine (Tet) were investigated. The results showed that calcimycin significantly increased platelet adhesion to CMEC. The platelet adhesion to CMEC was increased by 17.1% vs control after stimulation with calcimycin 0.01 mumol.L-1 for 25 min. WEB 2086 0.1, 1.0 and 10.0 mumol.L-1 or DMPP 0.1, 1.0 and 10.0 mumol.L-1 or Tet 0.1, 1.0 and 10.0 mumol.L-1 inhibited the calcimycin induced platelet adhesion to CMEC by 9.0, 22.9 and 23.1% or 9.7, 15.6 and 22.1% or 7.8, 15.6 and 24.6%, respectively. This indicates that DMPP and Tet may have perspectives in the prevention and treatment of cerebral vascular diseases.

Effects of dauricine and tetrandrine on [3H] WEB 2086 specific binding to bovine anterior cerebral arterial smooth muscle cells in vitro.

By using [3H] WEB 2086, a PAF antagonist, specific binding sites of PAF on bovine anterior cerebral arterial smooth muscle cells was identified. Two populations of binding sites with different dissociation constants on the cells were found. The Kd-1 = 22.8 +/- 5.0 nmol.L-1, Kd-2 = 186 +/- 20.5 nmol.L-1 at 25 C. The total number of binding sites were Bmax-1 = 2.1 +/- 0.3 pmol/10(6) cells and Bmax-2 = 12.1 +/- 1.5 pmol/10(6) cells. Dauricine and tetrandrine, two active compounds with similar chemical structure extracted from traditional Chinese herbs, were found to inhibit [3H] WEB 2086 specific binding significantly in culture cells.

[Effects of 6-(alpha alpha-diphenylacetylpiperazinyl) phenyl-5-methyl-4,5-dihydro-3 (2H)-pyridazinone on rabbit platelet aggregation and TXB2, cAMP production].

6-(alpha alpha-diphenylacetylpiperazinyl) phenyl-5-methyl-4,5-dihydro-3 (2H)-pyridazinone (DMDP) is a new synthetic pyridazinone derivative. This compound was shown to inhibit AA, ADP and PAF-induced rabbit platelet aggregation, and its IC50s were found to be 1.12 +/- 0.1, 4.19 +/- 0.5 and 2.97 +/- 0.1 mumol/L, respectively. At the concentration range of 1-500 mumol/L, the compound was found to depress TXB2 content and to increase cAMP levels in washed rabbit platelets in a dose-dependent manner. These might be the mechanisms of the compound on the inhibition of rabbit platelets.

[Effects of platelet activating factor on the cerebrovascular permeability in rats and the protection by drug].

The action of platelet activating factor in the rat brain was detected by Evans blue staining after injection of PAF into the rat brain. The results show that PAF increased the Evans blue staining of the brain, but no staining was observed without prior injection of PAF. Meanwhile, PAF was shown to stimulate the release of 14C-arachidonic acid (14C-AA) in the cerebral microvascular smooth muscle cells (CMSMC). SZ-1, a new synthetic drug, dose-dependently inhibited the Evans blue staining of the rat brain and the PAF induced 14C-AA release in CMSMC. These results indicate that the action of PAF in the rat brain might be related to the stimulation of AA release. SZ-1 may antagonize the PAF receptor and protect the brain from PAF induced damage.

Effects of anisodamine and dauricine on proliferation, DNA synthesis, and calcium influx in bovine anterior cerebral arterial smooth muscle cells in culture.

The in vitro culture of bovine anterior cerebral arterial smooth muscle cells (BACASMC) was first established in our laboratory. Anisodamine and dauricine inhibited the proliferation, DNA synthesis and calcium influx in the cells in dose-dependent manners. At 0.01 mmol.L-1, both drugs inhibited the proliferation by 17.6% and 8.3%, the DNA synthesis by 11.9% and 56.8%, the calcium influx by 26.6% and 31.4%, respectively. The results indicated that anisodamine and dauricine might have prospect in the prevention and treatment of cerebrovascular diseases.

[Inhibitory effect of dauricine on platelet activating factor released from calcimycin-induced mouse peritoneal macrophages].

The effects of dauricine (Dau) on the release of platelet activating factor (PAF) from mouse peritoneal macrophages stimulated by calcimycin (A-23187) was studied. The method of sodium [3H]acetate incorporating into macrophages to synthesize PAF was set up for the first time. Calcimycin (0.2 mumol/L) significantly induced mouse peritoneal macrophages to utilize sodium [3H]acetate to synthesize PAF. PAF released from macrophages medium fluid increased as the concentration of sodium [3H]acetate increased. The maximal amount of PAF released from macrophages was attained by incubating macrophages with sodium [3H]acetate (250 mumol/L) and calcimycin (2 mumol/L) over 30 min. Extracted by CHCl3:CH3OH:H2O (2:2:1.8), separated by thin layer chromatography (TLC) and determined by liquid scintillation counting, PAF released was inhibited significantly by Dau both in time (10-30 min) and dose (1-1000 mumol/L) dependent manners. The IC50 of Dau for the formation of PAF was 2.5 mumol/L. On the same condition PAF release was also significantly inhibited by quinacrine at 500 mumol/L. The results indicate that Dau is a potent inhibitor of PAF synthesis in mouse peritoneal macrophages.

[Inhibitory effects of dauricine and anisodamine on production of prostaglandins on bovine cerebral arterial smooth muscle cells].

The effects of dauricine (Dau), aniso amine (Ani), platelet activating factor (PAF), leukotriene C4 (LTC4) and leukotriene D4 (LTD4) on the production of TXB2 and 6-keto-PGF1 alpha (the stable metabolites of TXA2 and PGI2, respectively) in bovine anterior cerebral arterial smooth muscle cells were studied. The normal quantities of TXB2 and 6-keto-PGF1 alpha produced by bovine anterior cerebral arterial smooth muscle cells were 16 +/- 5 and 464 +/- 24 pg/10(5) cells, respectively, when measured by radioimmunoassay (RIA). The levels of TXB2 and 6-keto-PGF1 alpha in bovine anterior cerebral arterial smooth muscle cells decreased significantly when the cells were treated with Dau or Ani over 20 min. Both drugs inhibited the production of TXB2 and 6-keto-PGF1 alpha in dose (1-100 mumol/L) dependent manner. The bovine anterior cerebral arterial smooth muscle cells were stimulated markedly by LTC4 and LTD4 to produce TXB2 and 6-keto-PGF1 alpha on the same condition even at 0.01 mumol/L. When the cells were treated with PAF over 20 min, TXB2 increased significantly, but 6-keto-PGF1 alpha remained unchanged. If the cells were preincubated with Dau or Ani 20 min before PAF, LTC4 or LTD4 stimulation, the production of TXB2 and 6-keto-PGF1 alpha especially TXB2 were inhibited significantly compared with that of PAF, LTC4 or LTD4 group, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

[Studies of murine ectoplacental cone cells interaction with laminin].

Implantation of the mouse embryo is dependent on the interactions between the trophoblast cells and the surrounding uterine environment. The initial invasion by primary trophoblast stimulates the uterine stromal fibroblasts differentiate into decidual cells, which deposit a pericellular matrix consisting of LN, FN and Col IV. The secondary trophoblast giant cells (TGCs) from ectoplacental cone (EPC) invade the decidua to form the fetal portion of the placenta. We used synthetic peptides cyclic YIGSR (cYIGSR) and RGDS to study the mechanisms of EPC cells interaction with LN. The results indicated that cYIGSR and RGDS promoted EPC attachment and had synergistic effect, and cYIGSR also promoted EPC outgrowth and secondary TGCs migration. LN supported EPC attachment and outgrowth, as well as secondary TGCs migration from EPC. Biologically active domains RGD of LN A chain and YIGSR of LN B1 chain participated synergistically in EPC attachment, outgrowth, as well as secondary TGCs migration. Since synthetic peptides cYIGSR and RGDS can't competitively inhibit EPC attachment with LN completely, there must be other binding sites involved in the interaction.

Transcription study of the genes encoded in the region of the junction between the large single copy and the inverted repeat A of spinach chloroplast DNA.

The expression of the psbA, trnH-GUG and rps19' genes from spinach chloroplasts, coding respectively for the 32 kDa protein, the tRNA(His) (GUG), and the putative ribosomal protein CS19', has been studied by cloning, Northern hybridization and 3' and 5' S1 mapping experiments.It is demonstrated that the putative transcription termination signal of the psbA gene does not function as a rho-independent terminator of transcription in E. coli, whatever its orientation.Evidence is presented suggesting that, in spinach, the psbA and trnH-GUG genes are probably cotranscribed. The 3'-OH extremities of transcripts observed downstream from the putative psbA terminator are interpreted as resulting from processing of the psbA precursor.Using different approaches, it is shown that the rps19' gene, located on the other strand and overlapping the trnH-GUG gene, is not expressed.

Cloning and mutagenesis of the rabbit ApoB mRNA editing protein. A zinc motif is essential for catalytic activity, and noncatalytic auxiliary factor(s) of the editing complex are widely distributed.

Apolipoprotein (apo) B mRNA editing is the specific deamination of cytidine (nucleotide 6666) to uridine in apoB mRNA. We isolated a full-length cDNA clone encoding the rabbit apoB mRNA editing protein (REPR), a subunit of the editing complex. Rabbit REPR is analogous to a rat enterocyte 27-kDa protein that has been shown to have cytidine deaminase activity. Like rat REPR, rabbit REPR edited synthetic apoB RNA when mixed with chicken enterocyte extract. Surprisingly, the REPR also acquired editing activity when mixed with extracts from various organs of the rabbit (liver, gallbladder, stomach, intestine, adrenals, thyroid, testes, spleen, kidney, and lung) or the chicken (kidney and liver). In contrast, the rabbit REPR mRNA was found only in the small and large intestine. Thus, the auxiliary protein(s) of the apoB mRNA editing complex, which are essential for editing activity, exist in organs devoid of significant apoB mRNA editing or apoB synthesis. REPR requires zinc for its catalytic activity. We mutated putative zinc-coordinating residues (His61, Cys93, Cys96) and 2 additional residues (Glu63, Pro92) of the rabbit REPR that are conserved in other cytidine or deoxycytidylate deaminases and in rat REPR. The wild-type and mutant REPR cDNAs each produced 28-kDa proteins when transcribed and translated in vitro. Compared with the wild-type editing activity, the mutations of His61-->Ala, Glu63-->Ala, Cys93-->Ala, and Cys96-->Ala abolished or greatly reduced editing activity, whereas the mutations of His61-->Cys (which also can coordinate zinc) and Pro92-->Ala had a lesser effect. These results indicate that His61, Cys93, and Cys96 are essential for editing activity, probably because they coordinate zinc, whereas Glu63 also is essential, because it may be involved in the deaminase reaction. In addition, the widespread distribution of the auxiliary factor(s) portends their involvement in other RNA editing reactions.