Influences of miR-320a on proliferation and apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis through targeting MAPK-ERK1/2.

OBJECTIVE: To study the expression of micro ribonucleic acid (miR)-320a in synovial tissues of patients with rheumatoid arthritis (RA) and explore the influences of miR-320a on the proliferation and apoptosis of fibroblast-like synoviocytes (FLSs) in RA and its mechanism. PATIENTS AND METHODS: The expression level of miR-320a in synovial tissues of 40 healthy people and 32 RA patients was detected via reverse transcription-polymerase chain reaction (RT-PCR). The FLSs were isolated from RA patients, cultured in vitro and divided into Control group and miR-320a mimic group. The proliferation and apoptosis of FLSs in each group were observed. Finally, the expression level of mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) 1/2 in each group was detected via Western blotting. RESULTS: The expression level of miR-320a in synovial tissues of RA patients was significantly lower than that in healthy people (p < 0.05). After miR-320a mimic was transfected into FLSs cultured in vitro, EdU staining and flow cytometry analysis were performed. The results revealed that the proportion of EdU-positive cells significantly declined in miR-320a mimic group, the proportion of cells in G0/G1 phase was increased, while the cells in G2/M and S phases were significantly decreased (p < 0.05). Above data indicated that the cell proliferation ability was significantly inhibited. In addition, the results of flow cytometry also showed that the apoptosis rate of FLSs in miR-320a mimic group was significantly higher than that in Control group (p < 0.05). The results of Western blotting manifested that the Bcl-2 associated X protein (Bax)/Bcl-2 ratio in miR-320a mimic group was also obviously increased (p < 0.05). According to further studies, the phosphorylation level of ERK1/2 in miR-320a mimic group was remarkably inhibited (p < 0.05). CONCLUSIONS: The expression level of miR-320a significantly declined in synovial tissues of RA patients. MiR-320a attenuated proliferation and promoted apoptosis of FLSs through inhibiting the activation of the MAPK-ERK1/2 signaling pathway.

The effect of medicaments used in endodontic regeneration on root fracture and microhardness of radicular dentine.

AIM: To investigate the effect of medicaments used in endodontic regeneration on root fracture resistance and microhardness of radicular dentine. METHODOLOGY: The root canals of mandibular premolars (n = 180) were instrumented and randomized into three treatment groups and an untreated control group. Each treatment group received either triple antibiotic paste (TAP), double antibiotic paste (DAP) or calcium hydroxide paste [Ca(OH)2] intracanal medicament. Teeth were kept in saline for 1 week, 1 month or 3 months. After each time-point, 15 teeth were randomly selected from each group and two root cylinders were obtained from each tooth. One cylinder was subjected to a fracture resistance test, and the other cylinder was used for a microhardness test. Two-way ANOVA and Tukey's pairwise comparisons were used for statistical analysis. RESULTS: For the microhardness test, the two-way interaction between group and time was significant (P < 0.001). The intracanal application of TAP and DAP caused significant and continuous decrease in root dentine microhardness after one (P < 0.05) and 3 months (P < 0.001), respectively. The three-month intracanal application of Ca(OH)2 significantly increased the microhardness of root dentine (P < 0.05). The time factor had a significant effect on fracture resistance (P < 0.001). The three intracanal medicaments caused significant decreases in fracture resistance ranging between 19% and 30% after 3-month application compared to 1-week application. CONCLUSION: In this laboratory study, the 3-month application of triple antibiotic paste, double antibiotic paste or calcium hydroxide paste medicaments significantly reduced the root fracture resistance of extracted teeth compared to a 1-week application.

Dopamine use is an indicator for the development of threshold retinopathy of prematurity.

AIM: To assess whether treatment of premature infants with dopamine is a risk factor for development of retinopathy of prematurity (ROP). METHODS: A retrospective case series analysis of two groups was utilised with a minimum follow up of 6 months. Clinical profiles and patient risk factors were identified along with an evaluation of ROP progression and an analysis of clinical outcome. All infants were seen in a single community neonatal intensive care unit (NICU). 41 consecutive high risk infants were identified during a 36 month period whose birth weight was less than 1000 grams and who remained in the NICU without transfer until at least 28 days of age. Dilated indirect ophthalmoscopy fundus examinations were performed on all infants to identify the degree of and progression to threshold ROP. RESULTS: 18 of 41 infants were treated with dopamine for hypotension. The group of infants requiring dopamine differed statistically from the non-dopamine treated group by having a slightly higher birth weight, a greater incidence of hypotension and colloid treatment, and in manifesting more advanced respiratory disease. Within the dopamine treated group, 12 of 18 infants (67%) reached prethreshold ROP and seven infants (39%) reached threshold ROP requiring laser treatment. In contrast, only three of the infants (13%) who did not require dopamine for hypotension progressed to prethreshold (p = 0.001) and only one of these infants (4%) progressed to threshold ROP (p = 0.02). Logistic regression analysis among other variables demonstrated that dopamine use and gestational age are important factors in this low birthweight population for predicting the development of threshold ROP (dopamine use: adjusted odds ratio = 119.88, p = 0.0061; gestational age: adjusted odds ratio = 0.061, p = 0.0043). CONCLUSIONS: Dopamine use in low birthweight infants may therefore be a risk factor for the development of threshold ROP. More vigilant screening of high risk infants requiring dopamine therapy for systemic hypotension may be warranted.

Suprachoroidal hemorrhage.

Suprachoroidal hemorrhage is a feared complication of all types of intraocular surgery. Although rare, it is typically associated with severe visual disability, and this has prompted efforts to better understand the pathogenesis of this condition, to identify the patients at risk for this event, and to improve treatment of patients who develop this condition either intraoperatively or postoperatively. Controversy still exists regarding the best course of treatment for these patients. Although the introduction of perfluorocarbon liquids as a surgical adjunct during vitrectomy surgery may assist in the removal of suprachoroidal hemorrhage, the visual outcomes still remain disappointing.

Posterior vitreoschisis. An echographic finding in proliferative diabetic retinopathy.

PURPOSE: To describe the echographic characteristics of splitting the outer posterior cortical vitreous in patients with proliferative diabetic retinopathy and vitreous hemorrhage. METHODS: The authors retrospectively reviewed the echographic findings in 270 patients who were evaluated at the Doheny Eye Institute between January 1983 to December 1989 for proliferative diabetic retinopathy and vitreous hemorrhage. None of the eyes had undergone pars plana vitrectomy before echographic examination. RESULTS: Forty-five patients (17%) had echographic evidence of splitting of the outer posterior vitreous cortex, a finding the authors have termed posterior vitreoschisis. In all patients, differentiation of the posterior vitreoschisis from a true posterior hyaloid detachment was possible, either on the initial or on serial echographic examination, by the separate detachment of the inner wall of the vitreoschisis cavity and the true posterior hyaloid from the retinal surface. The vitreoschisis cavities often were found to contain unclotted blood. In some eyes, the inner wall of the vitreoschisis cavity was adherent to the apex of the most highly elevated area of traction retinal detachment, suggesting that posterior vitreoschisis may itself result in clinically significant vitreoretinal traction, independent of the presence or extent of true posterior hyaloid separation. CONCLUSIONS: The authors' finding suggest that spontaneous splitting of the outer posterior vitreous cortex may occur in patients with proliferative diabetic retinopathy and vitreous hemorrhage, which may mimic a true posterior cortical vitreous detachment on echographic examination. Preoperative recognition of posterior vitreoschisis may be important in the surgical management of these patients.

Scleromalacia after retinal detachment surgery.

PURPOSE/METHODS: A 34-year-old man developed severe scleral thinning with imminent prolapse after conventional retinal detachment repair, including pars plana vitrectomy, endolaser photocoagulation, and perfluoropropane injection. Cadaveric dura mater was used to repair the scleral defect. RESULTS/CONCLUSION: The cause of scleromalacia in this case remains to be determined. Commercially prepared cadaveric dura mater can be successfully used as a tectonic graft for the treatment of scleral thinning.

Immediate pars plana vitrectomy in the management of inadvertent intracameral injection of gentamicin. A rabbit experimental model.

PURPOSE: Acute retinal toxicity secondary to inadvertent intracameral injection of massive doses of gentamicin is a devastating complication of cataract surgery, for which no treatment, to date, either surgical or medical, has been shown to be effective. A rabbit experimental model was used to examine the effect of immediate pars plana vitrectomy in the management of inadvertent intracameral injection of gentamicin. METHODS: Twenty-seven rabbit eyes were subjected to lensectomy with or without preservation of the lens capsule. Aphakic rabbit eyes and rabbit eyes with an intact lens capsule were then subjected to injections of massive doses (4 mg and 20 mg) of gentamicin into the anterior chamber. Experimental eyes underwent an immediate pars plana vitrectomy and posterior segment lavage. The rabbits were killed after 7 days. Light microscopic examination was then performed to ascertain the degree of retinal damage caused by the injection. RESULTS: Rabbit eyes receiving immediate pars plana vitrectomy exhibited far less structural damage to the retina, as seen by light microscopy, than did corresponding control eyes. Furthermore, there appeared to be a protective effect of an intact lens capsule with regard to retinal damage. CONCLUSIONS: Immediate pars plana vitrectomy and posterior segment lavage may alleviate the effects of intracameral injection of gentamicin. This preliminary study demonstrates the need to perform further studies with a vascularized primate retina to evaluate immediate pars plana vitrectomy in the management of inadvertent intracameral injection of gentamicin.

Deep venous thrombosis following immobilization after retinal detachment surgery.

Although deep venous thrombosis (DVT) and pulmonary embolism are potentially fatal complications of numerous surgical procedures, DVT associated with ophthalmic surgery has rarely been reported in the current literature. We report the development of a DVT in a patient following complicated vitreoretinal surgery.

Massive suprachoroidal hemorrhage with central retinal apposition. A clinical and echographic study.

We reviewed the charts of 18 patients diagnosed with a massive suprachoroidal hemorrhage (MSCH) with central retinal apposition (kissing configuration). Four cases occurred intraoperatively (expulsive), eight after a surgical procedure (delayed), and six associated with blunt or perforating injury (traumatic). In this study, echography was used to monitor the course of MSCH; the mean time for clot lysis was 14 days, and the mean duration of central retinal apposition was 15 days. The expulsive MSCHs were all allowed to resolve spontaneously, with good initial visual outcome in three of the four eyes in which they occurred. In the delayed MSCH group, the majority of patients attained their prehemorrhage visual acuity, with or without early surgical intervention. In the traumatic MSCH group, retinal detachment was a constant complication in all patients. All six patients in the traumatic MSCH group had a poor visual outcome, despite early surgical intervention in five patients. The results of this study suggest that not all MSCHs need to be drained surgically and that, when surgical drainage is indicated, echography may be a useful adjunct in determining the optimal time of drainage.

Soluble metalloendopeptidase from rat brain: action on enkephalin-containing peptides and other bioactive peptides.

A soluble metalloendopeptidase identified in rat brain, preferentially cleaves bonds in peptides having hydrophobic amino acid residues in the P1, P2, and P3' positions. (The nomenclature of T. Schechter and A. Berger is used to describe the interaction between enzyme and substrate. The amino acid residues in the substrate are designated as P1, P2, P3 etc. in the N-terminal direction and P1', P2', P3' etc. in the C-terminal direction from the bond undergoing cleavage. The corresponding subsites in the enzyme are identified by the letter S.) The degradation of a series of biologically active peptides and their affinity toward the enzyme was studied. Dynorphin-(1-8), alpha-neo-endorphin, and beta-neo-endorphin are rapidly hydrolyzed to form leu-enkephalin, whereas bovine adrenal medulla dodecapeptide is hydrolyzed to form met-enkephalin. The enzyme, however, does not cleave a larger precursor molecule of metenkephalin, such as bovine adrenal medulla docosapeptide. Several other bioactive peptides are also cleaved at sites consistent with our previously reported specificity studies. Met- and leu-enkephalin are resistant to hydrolysis. The binding affinity [as expressed by inhibitory constant (Ki) or Michaelis-Menten constant (Km) values] of several bioactive peptides such as dynorphin-(1-8), beta-neo-endorphin, neurotensin, angiotensin I, and bradykinin was found to be in the micromolar range. These peptides were also rapidly hydrolyzed by the enzyme, showing, as a result, high specificity constants (kcat/Km values). The highest enzyme activity was found in brain, testis, and in the anterior and posterior lobes of the pituitary, while the activity in such tissues as spleen, liver, kidney, lung, adrenals, and thyroid amounted to only 10-20% of that found in brain. This distribution of enzyme activity, together with its preference for oligopeptides as substrates, its ability to generate leu- and met-enkephalin from several larger peptide precursors, and its affinity toward several other bioactive peptides, suggests that the enzyme functions in the metabolism of neuropeptides.

Active site directed N-carboxymethyl peptide inhibitors of a soluble metalloendopeptidase from rat brain.

A soluble metalloendopeptidase isolated from rat brain preferentially cleaves bonds in peptides having aromatic residues in the P1 and P2 position. An additional aromatic residue in the P3' position greatly increases the binding affinity of the substrate, suggesting the presence of an extended substrate recognition site in the enzyme, capable of binding a minimum of five amino acid residues [Orlowski, M., Michaud, C., & Chu, T.G. (1983) Eur. J. Biochem. 135, 81-88]. A series of N-carboxymethyl peptide derivatives structurally related to model substrates and containing a carboxylate group capable of coordinating with the active site zinc atom were synthesized and tested as potential inhibitors. One of these inhibitors, N-[1(RS)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, was found to be a potent competitive inhibitor of the enzyme with a Ki of 1.94 microM. The two diastereomers of this inhibitor were separated by high-pressure liquid chromatography. The more potent diastereomer had a Ki of 0.81 microM. The inhibitory potency of the less active diastereomer was lower by 1 order of magnitude. Decreasing the hydrophobicity of the residue binding the S1 subsite of the enzyme by, for example, replacement of the phenylethyl group with a methyl residue decreased the inhibitory potency by almost 2 orders of magnitude. Deletion of the carboxylate group decreased the inhibitory potency by more than 3 orders of magnitude. Shortening the inhibitor chain by a single alanine residue had a similar effect. Binding of the inhibitor to the enzyme increased its thermal stability.(ABSTRACT TRUNCATED AT 250 WORDS)

A soluble metalloendopeptidase from rat brain. Purification of the enzyme and determination of specificity with synthetic and natural peptides.

A metalloendopeptidase, optimally active at a neutral pH, was purified from the soluble fraction of brain homogenates. The enzyme (molecular weight about 67000) is strongly inhibited by metal chelators such as EDTA and o-phenanthroline. An EDTA-treated enzyme can be reactivated by several divalent metal ions including Zn2+, Co2+ and Mn2+. The specificity and kinetic parameters of the enzyme were studied with a series of model synthetic substrates. The enzyme preferentially cleaves peptide bonds in which the carbonyl group is contributed by an aromatic amino acid residue in the P1 position. The lowest Km values and the highest Kcat/Km ratios were obtained with substrates having aromatic residues in the P'3 and P1 position or in the P'3 and both the P1 and P2 positions. Lower kcat/Km ratios were obtained with substrates having arginine residues in position P1, and even lower values with those substrates having a glycine or aspartyl residue in this position. Introduction of a D-amino acid residue in either position P1 or P'1 renders the substrate totally resistant to hydrolysis. The specificity studies suggest that the active site of the metalloendopeptidase can accommodate at least five amino acid residues, with two of those residues binding on the N-terminal side and three binding on the C-terminal side of the hydrolyzed bond. Several biologically active peptides are cleaved by the enzyme at sites consistent with the specificity deduced from studies with model synthetic substrates.