Obstructive airway disease associated with occupational sodium hydroxide inhalation.
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Sodium hydroxide (NaOH) is well known for its corrosive properties and its ability to generate heat on contact with water. The respiratory effects of industrial exposure to NaOH have, however, never been reported. A 63 year old man worked daily for 20 years cleaning large industrial jam containers by boiling lye (NaOH) solution without using respiratory protective equipment. Physical examination, chest x ray film, pulmonary function tests, and arterial blood gases were all compatible with severe obstructive airway disease with significant air trapping. It is probable that this massive and prolonged occupational exposure to the corrosive effect of NaOH mists induced irritation and burns to the respiratory system, eventually leading to severe obstructive airway disease. (+info)
PURPOSE: Aldehyde dehydrogenase 3A1 (ALDH3A1) is the most abundant soluble protein component in the mouse cornea, produced mainly by corneal epithelial cells. High levels of ALDH3A1 in cornea contribute to maintenance of a stable an d transparent corneal structure. Alkali burn is a common damage to the corneal surface, which produces an alkaline hydrolysis of matrix proteins and induces an inflammatory reaction. Our study was intended to detect changes in ALDH3A1 expression after corneal alkaline burn. METHODS: To address this issue we employed RTQ-PCR to monitor the transcriptional change of ALDH3A1 after alkali burn. We used zymography to test enzyme activity changes of ALDH3A1 in the alkali burn cornea; And SDS-PAGE and mass spectrometry technology were used to verify protein content changes and to identify ALDH3A1 protein. RESULTS: Using zymography, ALDH3A1 enzymic activity was observed to decrease immediately after corneal alkali burn and the levels recovered following healing. Proteins extracted from alkali burned corneas, when run on SDS-PAGE, showed the same sized band (about 54 kDa, which is the molecular weight of ALDH3A1) but in much smaller quantity, compared to normal corneas. This result was further verified by mass spectrometry fingerprinting of the in-gel lysis product. An immediate decrease of ALDH3A1 transcription after alkali burning of the cornea was also found using RTQ-PCR. This level of transcription was gradually restored during healing. CONCLUSIONS: Alkali burn of the corneal surface caused a rapid decrease of ALDH3A1 in the corneal at both the RNA and protein levels, which leads to the loses of the protective component of the corneal surface and makes it vulnerable to further damage. The ALDH3A1 level in the cornea gradually recovered during the healing process. Use of an anti-oxidation reagent as a treatment ingredient for alkali burn of the corneal surface could compensate for the decrease of anti-oxidation protection potential caused by ALDH3A1 loss. (+info)
Decontamination of surgical instruments from prion proteins: in vitro studies on the detachment, destabilization and degradation of PrPSc bound to steel surfaces.
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Effective reprocessing of surgical instruments ensuring elimination of inadvertent contamination with infectious agents causing transmissible spongiform encephalopathies (TSEs) is essential for the prevention of iatrogenic transmission of Creutzfeldt-Jakob disease (CJD) or its new variant (vCJD) from asymptomatic carriers. In a search for effective yet instrument-friendly and routinely applicable reprocessing procedures, we used an in vitro carrier assay to assess the decontamination activity exerted by different reagents on pathological prion protein (PrP(Sc)), the biochemical marker for TSE infectivity, attached to steel surfaces. In this assay, steel wires were contaminated with 263K scrapie brain homogenate and reprocessed for decontamination by exposure to several different test reagents. Residual contamination with PrP(Sc) and its protease-resistant core PrP27-30, still present after reprocessing on the wire surface or in the cleaning solution, was monitored by sensitive Western blot detection without or after proteinase K digestion. Using this approach, various reagents and processing conditions were screened for both their efficacy of decontamination and their active principles, such as detachment, destabilization or degradation of surface-bound prion protein. This revealed that, under appropriate conditions, relatively mild reagents such as 0.2 % SDS/0.3 % NaOH (pH 12.8), a commercially available alkaline cleaner (pH 11.9-12.2), a disinfectant containing 0.2 % peracetic acid and low concentrations of NaOH (pH 8.9) or 5 % SDS (pH 7.1) exert potent decontaminating activities on PrP(Sc)/PrP27-30 attached to steel surfaces. For in vivo validation, wires reprocessed in these reagents have been implanted into reporter animals in ongoing experiments. (+info)
Reaction of phosphorylated and O-glycosylated peptides by chemically targeted identification at ambient temperature.
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Conditions for carrying out chemically targeted identification of peptides containing phosphorylated or glycosylated serine residues have been investigated. Ba(OH)2 was used at ambient temperature to catalyze the beta-elimination reaction at 25 degrees C. Nucleophilic addition of 2-aminoethanethiol was performed in both parallel and tandem experiments. The method was demonstrated by the reaction of beta-casein tryptic digest phosphopeptides and an O-glycosylated peptide. Contrary to an earlier report by others, the glycopeptide was found to react with essentially the same kinetics as phosphopeptides. Conversion of four phosphoserines in residues 15, 17, 18, and 19 from bovine beta-casein N-terminal tryptic phosphopeptides were followed by monitoring the time course of the addition reaction. The chemistry proceeded rapidly at room temperature with a half-reaction time of 15 min. No side-reaction products were observed; however, care was taken to minimize all counter ions that either precipitate barium or neutralize the base. Digestion of the converted peptides with lysine endopeptidase identified all five phosphoserines in the beta-casein tryptic digest. Alternatively, preincubation with base followed by nucleophilic addition of the thiol was found to work satisfactorily. The use of the water-soluble hydrochloride of 2-aminoethanethiol allowed beta-elimination, nucleophilic addition, and desalting to be carried out on a micro C18 reverse phase pipette tip. (+info)
Inhibitory effect of rapamycin on corneal neovascularization in vitro and in vivo.
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PURPOSE: To examine the effect of rapamycin on the proliferation and the migration of human umbilical vein endothelial cells (HUVECs) and on the corneal neovascularization in the corneal alkaline burn murine model. METHODS: HUVEC proliferation, migration, and apoptosis were examined after treatment with rapamycin. The effect of rapamycin on the mRNA expression of FK506 binding protein (FKBP)-12 and mammalian target of rapamycin (mTOR) was also evaluated in vitro. Corneal neovascularization was induced in vivo by an alkaline burn of the cornea with 1 N NaOH on BALB/c mice. Rapamycin was given intraperitoneally at 2 mg/kg body weight once a day for 12 days after the corneal alkaline burn. Growth factors and cytokines related with neovascularization and inflammation were evaluated in the corneal tissue and the peripheral blood by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The corneal neovascularization was evaluated by a slit lamp biomicroscopy. RESULTS: Rapamycin at the concentration of 1000 ng/mL for >48 hours' exposure significantly inhibited the growth of HUVECs. The double chamber assay showed that rapamycin dramatically inhibited the migration of HUVECs at concentrations of 10 and 100 ng/mL and that these concentrations did not affect endothelial cell growth. When TUNEL assays were performed, the number of apoptotic cells increased 1.9-, 2.1-, and 2.6-fold compared with the control at 10, 100, and 1000 ng/mL, respectively, of rapamycin at 48 hours of exposure. RT-PCR showed that the expression of mTOR was suppressed in the HUVECs after rapamycin treatment; however, FKBP-12 expression was not affected. Among the angiogenic factors, gene expression of substance P and hypoxia inducible factor (HIF)-1 alpha was inhibited by rapamycin earlier (1-3 days), with vascular endothelial growth factor (VEGFR)-1 gene expression being suppressed for the first 7 days in the corneal tissue. The protein level of substance P and vascular endothelial growth factor (VEGF) was significantly decreased--more in mice treated with rapamycin than the control mice--as shown by ELISA assay of peripheral blood. Furthermore, rapamycin significantly inhibited corneal neovascularization in the alkaline-burned cornea. CONCLUSIONS: Rapamycin strongly inhibited HUVEC migration at doses that did not cause cytotoxicity and apoptosis in this in vitro model. Rapamycin also suppressed corneal neovascularization, possibly by inhibiting proinflammatory cytokines, as shown by the in vivo study. Therefore, rapamycin may be useful as an angiogenic regulator in the treatment of corneal diseases that manifest with neovascularization. (+info)
High reproducibility using sodium hydroxide-stripped long oligonucleotide DNA microarrays.
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Recently, long oligonucleotide (60- to 70-mer) microarrays for two-color experiments have been developed and are gaining widespread use. In addition, when there is limited availability of mRNA from tissue sources, RNA amplification can and is being used to produce sufficient quantities of cRNA for microarray hybridization. Taking advantage of the selective degradation of RNA under alkaline conditions, we have developed a method to "strip" glass-based oligonucleotide microarrays that use fluorescent RNA in the hybridization, while leaving the DNA oligonucleotide probes intact and usable for a second experiment. Replicate microarray experiments conducted using stripped arrays showed high reproducibility, however, we found that arrays could only be stripped and reused once without compromising data quality. The intraclass correlation (ICC) between a virgin array and a stripped array hybridized with the same sample showed a range of 0.90-0.98, which is comparable to the ICC of two virgin arrays hybridized with the same sample. Using this method, once-stripped oligonucleotide microarrays are usable, reliable, and help to reduce costs. (+info)
Characterization of complexes formed between TSG-6 and inter-alpha-inhibitor that act as intermediates in the covalent transfer of heavy chains onto hyaluronan.
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The high molecular mass glycosaminoglycan hyaluronan (HA) can become modified by the covalent attachment of heavy chains (HCs) derived from the serum protein inter-alpha-inhibitor (IalphaI), which is composed of three subunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfate moiety. The formation of HC.HA is likely to play an important role in the stabilization of HA-rich extracellular matrices in the context of inflammatory disease (e.g. arthritis) and ovulation. Here, we have characterized the complexes formed in vitro between purified human IalphaI and recombinant human TSG-6 (an inflammation-associated protein implicated previously in this process) and show that these complexes (i.e. TSG-6 x HC1 and TSG-6 x HC2) act as intermediates in the formation of HC x HA. This is likely to involve two transesterification reactions in which an ester bond linking an HC to chondroitin sulfate in intact IalphaI is transferred first onto TSG-6 and then onto HA. The formation of TSG-6 x HC1 and TSG-6 x C2 complexes was accompanied by the production of bikunin x HC2 and bikunin x HC1 by-products, respectively, which were observed to break down, releasing free bikunin and HCs. Both TSG-6 x HC formation and the subsequent HC transfer are metal ion-dependent processes; these reactions have a requirement for either Mg2+ or Mn2+ and are inhibited by Co2+. TSG-6, which is released upon the transfer of HCs from TSG-6 onto HA, was shown to combine with IalphaI to form new TSG-6 x HC complexes and thus be recycled. The finding that TSG-6 acts as cofactor and catalyst in the production of HC x HA complexes has important implications for our understanding of inflammatory and inflammation-like processes. (+info)
3-D ultrastructural distribution of collagen in human placental villi at term in relation to vascular tree.
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In order to understand the 3-D distribution of collagen in relation to vascularization, chorionic villi of human placentae, belonging to normal pregnancies at term, were studied by scanning electron microscopy (SEM) after alkali maceration techniques, and by transmission electron microscopy (TEM). The villous tree appeared made of an uninterrupted structure of collagen fibres. The collagen fibres connected the chorionic villi axis with their basal plates and organised differently according to the various levels of villous branching. The collagen of stem villi showed copious fibres. The external fibres (facing the villous surface) were arranged mainly longitudinally. The central core of the villi (inner fibres) were arranged concentrically around the wall of the fetal vessels. Both external and internal fibres formed stratified lamellae or small parallel bundles. The inner core of stem villi showed small holes housing capillary spaces. Mature intermediate and terminal villi showed a scarce amount of collagen arranged in thin concentric layer within the villous core, surrounding numerous dilated capillary and sinusoid spaces.These observations demonstrated that the extracellular matrix of human chorionic villi is highly compartmentalised and shows a variable structural 3-D distribution depending on the branching level of the villous tree, such a distribution ensures the most favourable microenvironment for feto-maternal exchanges and it is likely able to provide a modulated support to the developing chorionic fetal vessels and trophoblastic layer as well. (+info)