Role of NADPH Oxidase 4 in Corneal Endothelial Cells Is Mediated by Endoplasmic Reticulum Stress and Autophagy.

Human corneal-endothelial cells (hCEnCs) are located on the inner layer of the cornea. Injury to CEnCs leads to permanent corneal edema, requiring corneal transplantation. NADPH oxidase 4 (NOX4) has been reported to be implicated in the pathogenesis of CEnCs diseases. Thus, we investigated the role of NOX4 in CEnCs in this study. In an animal study, siRNA for NOX4 (siNOX4) or plasmid for NOX4 (pNOX4) was introduced into the corneal endothelium of rats by electroporation, using a square-wave electroporator (ECM830, Havard apparatus) to decrease or increase the expression of NOX4, respectively, and the rat corneas were cryoinjured through contact with a metal rod of 3 mm diameter frozen in liquid nitrogen for 10 min. The immunofluorescence staining of NOX4 and 8-OHdG showed that the levels of NOX4 and 8-OHdG were decreased in the siNOX4 group compared to the siControl, and increased in the pNOX4 group compared to the pControl at one week after treatment. Without cryoinjury, corneal opacity was more severe, and the density of CEnCs was lower, in pNOX4-treated rats compared to pControl. After cryoinjury, the corneas were more transparent, and the CEnC density was higher, in siNOX4-treated rats. The hCEnCs were cultured and transfected with siNOX4 and pNOX4. The silencing of NOX4 in hCEnCs resulted in a normal cell shape, higher viability, and higher proliferation rate than those transfected with the siControl, while NOX4 overexpression had the opposite effect. NOX4 overexpression increased the number of senescent cells and intracellular oxidative stress levels. NOX4 overexpression increased ATF4 and ATF6 levels, and nuclear translocation of XBP-1, which is the endoplasmic reticulum (ER) stress marker, while the silencing of NOX4 had the opposite effect. Additionally, the mitochondrial membrane potential was hyperpolarized by the silencing of NOX4, and depolarized by NOX4 overexpression. The LC3II levels, a marker of autophagy, were decreased by the silencing of NOX4, and increased by NOX4 overexpression. In conclusion, NOX4 plays a pivotal role in the wound-healing and senescence of hCEnCs, by modulating oxidative stress, ER stress, and autophagy. The regulation of NOX4 may be a potential therapeutic strategy for regulating the homeostasis of CEnCs, and treating corneal-endothelial diseases.

Expression of osteogenic markers after administration of selective estrogen receptor modulators during implant placement in the osteoporotic rat maxilla.

PURPOSE: This study examined the effects of raloxifene during bone formation around the dental implant in the ovariectomy-induced osteoporotic rat maxilla. METHODS: Fifty-four female 10-week-old Sprague-Dawley rats were divided into three groups (n = 18 each); sham-operated (control), ovariectomized (OVX), and ovariectomized and raloxifene-administered (RAL). Eight weeks after ovariectomy, both upper first molars were extracted, and implants were placed 4 weeks post-extraction. The RAL group was given 1 mg/kg of raloxifene per day while the other groups received a vehicle. Six rats in each group were sacrificed at days 4, 7, and 14 and submitted for quantitative reverse transcription polymerase chain reaction and immunohistochemical staining, for evaluation of osteogenic genes expressions. RESULTS: The alkaline phosphatase expression was upregulated in the RAL group compared to the OVX group at day 4. The osteocalcin expression was significantly higher between the RAL group and the OVX group at day 7. Immunohistochemical staining revealed increased expression during the initial bone-forming process and indicated more active bone formation in the RAL group than in the OVX group. CONCLUSION: Raloxifene administration enhanced the osteogenic genes and proteins expression in the bone around the implant. Further studies are required to establish the long-term clinical effects of raloxifene administration.

Intermittent Parathyroid Hormone Improves Bone Formation Around Titanium Implants in Osteoporotic Rat Maxillae.

PURPOSE: Parathyroid hormone (PTH) plays an important role in the treatment of osteoporosis due to its anabolic effect. In this study, PTH was administered intermittently to rats with ovariectomy-induced osteoporosis, titanium implants were placed into the rat maxillae, and the response of surrounding bone was evaluated. MATERIALS AND METHODS: A total of 30 female 8-week-old Sprague-Dawley rats were either ovariectomized to induce osteoporosis or sham operated. After 8 weeks, the upper right first molar was extracted and after a 4-week healing period an implant was placed. The animals were then divided into three groups: the PTH group (n = 10), which had been ovariectomized and received postimplant PTH; the OVX group (n = 10), which had been ovariectomized but did not receive postimplant PTH; and the control group (n = 10), which had been sham operated only (n = 10). Following implant placement, the rats in the PTH group received intermittent doses (three times a week) of PTH (30 µg/kg) subcutaneously in the dorsum. All the rats were sacrificed 4 weeks after implantation and specimens of the peri-implant maxillary bone were harvested, including the implant. Samples were evaluated by histomorphometric analysis and three-dimensional microcomputed tomography. RESULTS: Histomorphometric results showed that the mean bone area per tissue area (BA/TA) was 54.16% ± 2.2% in the PTH group and 45.24% ± 6.3% in the OVX group. The percentage of bone-to-implant contact (BIC) was 45.58% ± 9.4% in the PTH group and 32.00% ± 10.9% in the OVX group. Mean BA/TA and mean BIC values in the PTH group were higher than those in the OVX group; however, the differences were not statistically significant (P > .05). Microstructural data also showed differences between the groups. Bone volume was greater and trabecular bone was thicker in the PTH group than in the OVX group and more trabeculae were found in the PTH group. Bone mineral density was also higher in the PTH group. However, statistical analysis failed to show a significant difference between these two groups in any parameters other than trabecular thickness (P = .023). CONCLUSION: Despite the limitations of this study, intermittent PTH administration in humans may be helpful in accelerating new bone formation around implants. PTH treatment could improve clinical outcomes when dental implants are placed in jaws with low-quality bone.

Biosynthesis of rubradirin as an ansamycin antibiotic from Streptomyces achromogenes var. rubradiris NRRL3061.

The four overlapping cosmids from the rubradirin producer, Streptomyces achromogenes var rubradiris NRRL 3061, have 58 ORFs within a 105.6 kb fragment. These ORFs harbored essential genes responsible for the formation and attachment of four distinct moieties, along with the genes associated with regulatory, resistance, and transport functions. The PKS (rubA) and glycosyltransferase (rubG2) genes were disrupted in order to demonstrate a complete elimination of rubradirin production. The rubradirin biosynthetic pathway was proposed based on the putative functions of the gene products, the functional identification of sugar genes, and the mutant strains.

Proteome analysis of antibody-expressing CHO cells in response to hyperosmotic pressure.

To better understand intracellular responses to hyperosmotic pressure of recombinant Chinese hamster ovary (rCHO) cells expressing an antibody, we have taken a proteomics approach. Using two-dimensional electrophoresis and mass spectrometry, a proteome profile of rCHO cells comprising 23 identified proteins was established. On the basis of this proteome profile, we found three proteins of which expression levels were significantly changed at 450 mOsm/kg. Compared to the results at 300 mOsm/kg, two glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase, were found to be up-regulated, probably leading to an increased metabolic energy for antibody synthesis. The elevation of specific glucose consumption rate at 450 mOsm/kg agreed with the up-regulation of these glycolytic enzymes. On the other hand, tubulin expression was down-regulated, reflecting a depressed cell growth rate at 450 mOsm/kg. Taken together, this study shows the potential of the proteomics approach in understanding intracellular and physiological changes in cells and seeking a better insight into possible environmental or genetic manipulation approaches for increasing foreign protein production in rCHO cells.

Probing lysine acetylation with a modification-specific marker ion using high-performance liquid chromatography/electrospray-mass spectrometry with collision-induced dissociation.

Posttranslational acetylation of proteins regulates many diverse functions, including DNA recognition, protein-protein interaction, and protein stability. The identification of enzymes that regulate protein acetylation has revealed broader use of this modification than was previously suspected. In this study, we describe a method for identifying protein acetylation at lysine residues by analysis of digested protein using HPLC/ESI-MS with a new modification-specific marker ion. Collision-induced dissociation with capillary or nano-LC/ESI-TOF-MS was used to obtain a fragment ion useful as a marker for acetylated lysine. Although the acetylated lysine immonium ion at m/z 143.1 has been used as a marker ion for detecting acetylated lysine, it can be confused with internal fragment ion in some peptides, producing false positive results. We have found a novel marker ion at m/z 126.1, which is a further fragment ion induced by the loss of NH3 from the acetylated lysine immonium ions at m/z 143.1. This novel marker ion was found to be more specific and approximately 9 times more sensitive than the immonium ion at m/z 143.1. In addition, no interfering ions for acetylated peptides were found in the extracted ion chromatogram at m/z 126.1. The utility of this method was demonstrated with acetylated cytochrome c as a model compound. After the modification was probed by the new marker ion, the acetylated lysine site was determined by the CID-MS spectrum. This method was applied to identify histone H4 acetylation in HeLa cells treated with trichostatin A. Three protein bands separated by acid-urea-Triton gel electrophoresis were confirmed as tetra, tri, and diacetylated histone H4 at lysines 5, 8, 12, and 16. This method may be useful for assaying for lysine acetylation, which is an important regulatory process for a range of biological functions.