Global microRNA expression profiles in insulin target tissues in a spontaneous rat model of type 2 diabetes.

AIMS/HYPOTHESIS: MicroRNAs regulate a broad range of biological mechanisms. To investigate the relationship between microRNA expression and type 2 diabetes, we compared global microRNA expression in insulin target tissues from three inbred rat strains that differ in diabetes susceptibility. METHODS: Using microarrays, we measured the expression of 283 microRNAs in adipose, liver and muscle tissue from hyperglycaemic (Goto-Kakizaki), intermediate glycaemic (Wistar Kyoto) and normoglycaemic (Brown Norway) rats (n = 5 for each strain). Expression was compared across strains and validated using quantitative RT-PCR. Furthermore, microRNA expression variation in adipose tissue was investigated in 3T3-L1 adipocytes exposed to hyperglycaemic conditions. RESULTS: We found 29 significantly differentiated microRNAs (p(adjusted) < 0.05): nine in adipose tissue, 18 in liver and two in muscle. Of these, five microRNAs had expression patterns that correlated with the strain-specific glycaemic phenotype. MiR-222 (p(adjusted) = 0.0005) and miR-27a (p(adjusted) = 0.006) were upregulated in adipose tissue; miR-195 (p(adjusted) = 0.006) and miR-103 (p(adjusted) = 0.04) were upregulated in liver; and miR-10b (p(adjusted) = 0.004) was downregulated in muscle. Exposure of 3T3-L1 adipocytes to increased glucose concentration upregulated the expression of miR-222 (p = 0.008), miR-27a (p = 0.02) and the previously reported miR-29a (p = 0.02). Predicted target genes of these differentially expressed microRNAs are involved in pathways relevant to type 2 diabetes. CONCLUSION: The expression patterns of miR-222, miR-27a, miR-195, miR-103 and miR-10b varied with hyperglycaemia, suggesting a role for these microRNAs in the pathophysiology of type 2 diabetes, as modelled by the Gyoto-Kakizaki rat. We observed similar patterns of expression of miR-222, miR-27a and miR-29a in adipocytes as a response to increased glucose levels, which supports our hypothesis that altered expression of microRNAs accompanies primary events related to the pathogenesis of type 2 diabetes.

Potentiation of organophosphorus compound-induced delayed neurotoxicity (OPIDN) in the central and peripheral nervous system of the adult hen: distribution of axonal lesions.

Clinical manifestations of mild organophosphorus compound-induced delayed neurotoxicity (OPIDN) produced by diisopropylphosphorofluoridate (DFP) in adult hens are potentiated by posttreatment with phenylmethanesulfonyl fluoride (PMSF). The purpose of this study was to assess whether potentiation of mild OPIDN produces a pattern of axonal lesions in the central and peripheral nervous system similar to that seen in severe OPIDN. Groups of 6 hens each were given the following priming/challenge doses sc at 0 and 4 h, respectively: 0.20 ml/kg corn oil/0.50 ml/kg glycerol formal (GF) (control); 0.50 mg/kg DFP/GF (low-dose DFP); 0.50 mg/kg DFP/60 mg/kg PMSF (potentiated DFP); 60 mg/kg PMSF/GF (PMSF alone); 60 mg/kg PMSF/1.5 mg/kg DFP (protected DFP); and 1.5 mg/kg DFP/GF (high-dose DFP). Two hens from each group were used to assay brain neurotoxic esterase (NTE) 24 h after the challenge dose, and the remaining hens were scored for deficits in walking, standing, and perching ability on d 18. Three hens from each group were perfusion-fixed on d 22 and neural tissues were prepared for histologic evaluation. DFP and/or PMSF caused > 88% brain NTE inhibition in all treated groups, compared to control. Protected DFP yielded no clinical deficits and a distribution and frequency of axonal lesions similar to control. PMSF alone produced a small increase in the frequency of lesions in the cervical spinal cord and peripheral nerves compared to control. Low-dose DFP caused minimal ataxia and increased frequency of axonal lesions in dorsal and lateral cervical spinal cord, ventral lumbar spinal cord, and inferior cerebellar peduncles (ICP) compared to control. Potentiated DFP and high-dose DFP produced maximal ataxia and essentially identical increases in the frequency of lesions in dorsal and ventral thoracic spinal cord, lateral lumbar spinal cord, and peripheral nerves compared to low-dose DFP. The results indicate that PMSF potentiation of mild OPIDN induced in adult hens by low-dose DFP results in an overall pattern of axonal degeneration like that produced by a threefold higher dose of DFP alone, and support the hypothesis that potentiation causes an increase in the frequency of axonal lesions in central and peripheral loci normally affected by OPIDN.

Inhibition of neurotoxic esterase in vitro by novel carbamates.

Carbamyl sulfonate (CS) compounds are a novel class of carbamates derived from amino acid methyl esters. They have the general structure RCH(COOCH3)NH(CO)SO-3K+, where R is the sidechain of the parent amino acid. These compounds were developed as active site-directed inhibitors of human leukocyte elastase (HLE). The purpose of this study was to characterize the inhibition of hen brain neurotoxic esterase (neuropathy target esterase, NTE), horse serum butyrylcholinesterase (BuChE), and bovine erythrocyte acetylcholinesterase (AChE) by CS analogs derived from the methyl esters of L-ala, D-norval, L-norval, L-phe, L-val, L-norleu, D-met, and L-met. Bimolecular rate constants of inhibition (ki) for NTE ranged from 0.571 for L-ala-CS to 17.7 mM-1 min-1 for L-norleu-CS (10-min I50 values of 123 and 3.92 microM, respectively). Potency against NTE increased with chain length for straight-chain R-groups of L-CS compounds. Unlike HLE, NTE was only weakly stereoselective for CS compound enantiomers. The L-isomers were weaker inhibitors of BuChE than NTE (10-min I50 range of 742 to 35.6 microM). In contrast to the L-enantiomers, the I50 plots of D-met-CS and D-norval-CS were not linear for BuChE, suggesting a possible stereospecific mechanistic shift for inhibition of this enzyme, AChE was not effectively inhibited by any of the CS compounds (I50 values > 750 microM). The specificity and charged nature of CS compounds give these unusual NTE inhibitors potential advantages for mechanistic studies of organophosphorus compound-induced delayed neurotoxicity (OPIDN) and its protection or potentiation.

Long-term outcome of 42 knees with chronic infection after total knee arthroplasty.

The outcome of treatment in 40 patients (42 knees) with chronic infections after total knee arthroplasty was reviewed. Eighteen knees were treated with a 2-stage reimplantation. Sixteen of these 18 knees were treated with antibiotic-containing beads between debridement and reimplantation, and 7 of these were also treated with antibiotics in the cement at reimplantation. Infection did not recur in any of these 18 knees. Clinically, the 2-stage reimplantation group averaged a score of 90 points on the Knee Society Clinical Rating System. Average function score was 86.5 points, with average range of motion from 2 degrees to 109 degrees. Sixteen knees were treated with an arthrodesis: 9 with a 1-stage technique with a uniplanar external fixator and 7 with a 2-stage technique with intramedullary nail internal fixation. Infection did not recur in 6 of 9 knees treated with the 1-stage technique, but only 2 had a solid arthrodesis. All 7 treated with the 2-stage intramedullary nail technique had no recurrence of infection and achieved a solid fusion. Reimplantation or arthrodesis was not attempted in 8 other knees because of recalcitrant infection, vascular complications, or medical infirmity. Of the 42 knees, 11 (26%) had a severely morbid outcome. The infection could not be eradicated in 7 knees: 6 required amputation and 1 had a solid fusion but chronic drainage. In 3 knees, the infection was cured but resection arthroplasties were required, and in 1 patient an amputation was needed as a result of an intraoperative vascular complication.

Chlorpyrifos: assessment of potential for delayed neurotoxicity by repeated dosing in adult hens with monitoring of brain acetylcholinesterase, brain and lymphocyte neurotoxic esterase, and plasma butyrylcholinesterase activities.

Previous work has shown that acute exposures to chlorpyrifos (CPS; diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate) cannot produce > 70% inhibition of brain neurotoxic esterase (NTE) and cause organophosphorus compound-induced delayed neurotoxicity (OPIDN) unless the dose is well in excess of the LD50, necessitating aggressive therapy for cholinergic toxicity. The present study was carried out to determine if repeated doses of CPS at the maximum tolerated daily dose without prophylaxis against cholinergic toxicity could cause cumulative inhibition of NTE and OPIDN. Adult hens were dosed daily for 20 days with CPS (10 mg/kg/day po in 2 ml/kg corn oil) or corn oil (vehicle control) (2 ml/kg/day po) and observed for an additional 4 weeks. Brain acetylcholinesterase (AChE), brain and lymphocyte NTE, and plasma butyrylcholinesterase (BuChE) activities were assayed on Days 0 (control only), 4, 10, 15, 20, and 48. During Days 4-20, brain AChE and plasma BuChE activities from CPS-treated hens were inhibited 58-70% and 49-80% of contemporaneous controls, respectively. At 4 weeks after the end of dosing, brain AChE activity in treated birds had recovered to 86% of control and plasma BuChE activity was 134% of control. Brain and lymphocyte NTE activities of treated animals throughout the study were 82-99% and 85-128% of control, respectively. Neither brain nor lymphocyte NTE activities in treated hens exhibited cumulative inhibition. The 18% inhibition of brain NTE seen on days 10 and 20 was significant, but substantially below the putative threshold for OPIDN.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of hen brain acetylcholinesterase and neurotoxic esterase by chlorpyrifos in vivo and kinetics of inhibition by chlorpyrifos oxon in vitro: application to assessment of neuropathic risk.

Chlorpyrifos (CPS; O,O-diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate; Dursban) is a widely used broad-spectrum organophosphorus (OP) insecticide. Because some OP compounds can cause a sensory-motor distal axonopathy called OP compound-induced delayed neurotoxicity (OPIDN), CPS has been evaluated for this paralytic effect. Early studies of the neurotoxicity of CPS in young and adult hens reported reversible leg weakness but failed to detect OPIDN. More recently, a human case of mild OPIDN was reported to result from ingestion of a massive dose (about 300 mg/kg) in a suicide attempt. Subsequent experiments in adult hens (the currently accepted animal model of choice for studies of OPIDN) showed that doses of CPS in excess of the LD50 in atropine-treated animals inhibited brain neurotoxic esterase (NTE) and produced mild to moderate ataxia. Considering the extensive use of CPS and its demonstrated potential for causing OPIDN at supralethal doses, additional data are needed to enable quantitative estimates to be made of the neuropathic risk of this compound. Previous work has shown that the ability of OP insecticides to cause acute cholinergic toxicity versus OPIDN can be predicted from their relative tendency to inhibit the intended target, acetylcholinesterase (AChE), versus the putative neuropathic target, NTE, in brain tissue. The present study was designed to clarify the magnitude of neuropathic risk associated with CPS exposures by measuring hen brain AChE and NTE inhibition following dosing in vivo and determining the bimolecular rate constant of inhibition (ki) for each enzyme by the active metabolite, CPS oxon (CPO), in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotoxic esterase (NTE) assay: optimized conditions based on detergent-induced shifts in the phenol/4-aminoantipyrine chromophore spectrum.

An assay for neurotoxic esterase (neuropathy target esterase, NTE) was developed by Johnson (1,2) to assess the delayed neurotoxic potential of organophosphorus compounds. NTE activity is calculated from the rate of phenyl valerate hydrolysis resistant to paraoxon and sensitive to mipafox inhibition under specified conditions of inhibitor concentrations, pH, temperature, and incubation times with inhibitors and substrate. The amount of phenol produced is measured colorimetrically after its oxidative coupling with 4-aminoantipyrine to yield 4-N-(1,4-benzoquinoneimine)-antipyrine, a chromophore with a wavelength of maximum absorbance (lambda m) 510 nm and corresponding molar absorptivity (molar extinction coefficient, epsilon) equal to 13,900 M-1cm-1. The assay was improved and simplified later by Johnson (3) without any change in the lambda m or epsilon, even though the chromophore solvent was altered by adding the detergent, sodium dodecyl sulfate (SDS). The present work demonstrates that when the NTE assay is performed according to the improved procedure, with a final [SDS] of 3.0 mg/mL, the lambda m of the chromophore in the assay mixture is shifted from 510 to 490 nm. The same shift in the chromophore lambda m is observed when phenol standards are coupled with 4-aminoantipyrine in solutions containing 3.0 mg/mL SDS. A systematic investigation of the dependence of the lambda m of the chromophore on [SDS] in the assay mixture revealed that the spectral shift increases rapidly at an [SDS] greater than the apparent critical micelle concentration (CMC; estimated to be 0.53 mg/mL under these conditions) and begins to plateau at [SDS] greater than 10 mg/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme cytochemical localization of alkaline phosphatase in cultures of chondrocytes derived from normal and rachitic rats.

Epiphyseal growth plate cartilages were removed from rats which had been maintained on normal laboratory chow or a rachitogenic diet. Chondrocytes were released from the growth plates by collagenase digestion and cultured in tissue chamber slides. After 7, 10 and 12 days of culture, the chondrocytes were removed as intact multilayers and processed for electron microscopical enzyme cytochemical studies. Alkaline phosphatase activity in the cultures was visualized by means of a cerium based capture method. Electron-dense cerium phosphate deposits were localized on the membrane of matrix vesicles and plasma membranes of chondrocytes derived from normal and rachitic animals. The appearance of first crystals within matrix vesicles was characterized by a concomitant decrease in alkaline phosphatase activity in the membrane of these structures. Calcification was initiated at approximately the same time in cultures of chondrocytes derived from normal or rachitic animals. The results suggest that rickets has no serious effects on the capacity of chondrocytes to support matrix calcification in vitro. Additionally, the evidence indicates that alkaline phosphatase-positive matrix vesicles play a significant role in the initiation of this process.

Presence and activity of alkaline phosphatase in two human osteosarcoma cell lines.

The presence and activity of alkaline phosphatase in SAOS-2 and TE-85 human osteosarcoma cells grown in culture were examined at the ultrastructural level. A monoclonal antibody raised against purified human bone osteosarcoma alkaline phosphatase was used to localize the enzyme in cultures of the osteosarcoma cells. Similar cultures were analyzed for alkaline phosphatase activity using an enzyme cytochemical method with cerium as the capture agent. Alkaline phosphatase was immunolocalized at the light microscopic level in an osteogenic sarcoma and ultrastructurally on the SAOS-2 cell membrane and the enclosing membrane of extracellular vesicular structures close to the cells. In contrast, the TE-85 cells were characterized by the absence of all but a few traces of immunolabeling at the cell surface. Enzyme cytochemical studies revealed strong alkaline phosphatase activity on the outer surface of the SAOS-2 cell membrane. Much lower enzyme activity was observed in the TE-85 cells. The results support biochemical data from previous studies and confirm that SAOS-2 cells have a significantly greater concentration of alkaline phosphatase at the plasma membrane.

Heterotopic ossification: a case report and immunohistochemical observations.

This report describes the case of a 69-year-old man with adenocarcinoma of the transverse colon who developed heterotopic ossification in a metastatic axillary lymph node. The areas of pathologic bone formation were characterized by the appearance of osteoblast-like cells at the surfaces of the mineral deposits. Immunostaining for alkaline phosphatase revealed a significant concentration of this enzyme in these cells and, to a lesser degree, on the apical membrane of the glandular cells of the adenocarcinoma adjacent to the ossification centers. Proliferating mesenchymal cells in close proximity to the areas of osteogenesis also showed significant immunolabeling. We conclude that metastatic colonic carcinoma can promote heterotopic ossification, and that alkaline phosphatase is intimately associated with bone formation under these pathologic conditions.

Light microscopic localization of alkaline phosphatase in fetal bovine bone using immunoperoxidase and immunogold-silver staining procedures.

We localized alkaline phosphatase in the metaphyses of fetal bovine tibial bone by use of avidin-biotin-immunoperoxidase and immunogold-silver staining procedures. Low melting-point, paraffin-embedded sections of periodate lysine-paraformaldehyde-fixed undecalcified bone were used for immunostaining. We suggest that the combination of intact embryonic bone with this fixative and the immunohistochemical procedures used in this study may have helped to preserve antigenicity and thus to improve the efficiency of immunolabeling. Similar patterns of alkaline phosphatase localization were produced by the immunoperoxidase and immunogold-silver staining methods. The latter, although free of immunoreagents such as diaminobenzidine, must be monitored closely to avoid nonspecific staining during the silver enhancement procedure. Both methods revealed a concentration of the enzyme in osteoblasts and in areas of osteoid that lined the bone trabeculae. The results support the findings of earlier enzyme cytochemical studies in which osteoblasts were shown to have significant alkaline phosphatase activity.

Topical phenylephrine for the treatment of middle ear effusion.

One hundred fifty-two children were enrolled in a randomized, controlled clinical trial of the efficacy of phenylephrine hydrochloride nose drops or nasal spray in hastening the resolution of middle ear effusion. Children with persistent effusion were recruited for the study during a return visit two weeks after an episode of acute otitis media. Forty-six patients (30%) dropped out of the study, many because they failed to tolerate the medication, especially the nose drops. Another 27 (18%) had to be excluded because of intercurrent illness or systemic drug therapy. Among those children completing the study, rates of clinical and tympanometric cure during the following four weeks were similar in the drug and placebo groups. In view of the absence of documented clinical efficacy and the practical difficulties inherent in their administration, topical decongestants appear to have a limited role, if any, in treating children with persistent effusion.