The transmembrane domain of the rice small protein OsS1Fa1 is responsible for subcellular localization and drought tolerance.

OsS1Fa1, a homologue of spinach S1Fa, is a small protein in rice that contains four distinct conserved motifs and participates in drought tolerance. However, the biological functions of these conserved motifs have not been characterized to date. Therefore, we investigated the roles of these conserved domains in the localization and cellular function of OsS1Fa1. We analysed the subcellular localization of OsS1Fa1 using confocal laser scanning microscopy (CLSM), following particle bombardment and bacterial infiltration. An E. coli in vivo reconstituted sumoylation assay was conducted to investigate sumoylation of OsS1Fa1. We characterized the function of the transmembrane domain of OsS1Fa1 in drought tolerance using transgenic Arabidopsis plants. Fluorescence analysis showed that OsS1Fa1 localized to the nuclear and cytoplasmic membranes. Mutation and cell fractionation analyses revealed that the membrane localization domain determined the subcellular localization of OsS1Fa1. The rice homologue OsS1Fa2 and Arabidopsis orthologs AtS1Fa1, AtS1Fa2, and AtS1Fa3 also exhibited similar localization patterns as OsS1Fa1. Sumoylation analysis demonstrated that OsS1Fa1 was conjugated with the small ubiquitin-related modifier (SUMO). Transgenic analysis showed that overexpression of OsS1Fa1(TMm1), a mutant form of the transmembrane domain of OsS1Fa1, in Arabidopsis did not enhance drought stress tolerance, whereas OsS1Fa1 overexpression improved the drought tolerance of transgenic Arabidopsis. Our data indicate that rice and Arabidopsis S1Fa1 proteins localize in the nuclear and cytoplasmic membranes, and that transmembrane domain determines subcellular localization and plays an important role in drought stress tolerance.

Characterization of rhodanine derivatives as potential disease-modifying drugs for experimental mouse osteoarthritis.

OBJECTIVE: This study was performed to characterize selected rhodanine derivatives as potential preclinical disease-modifying drugs for experimental osteoarthritis (OA) in mice. METHODS: Three rhodanine derivatives, designated rhodanine (R)-501, R-502, and R-503, were selected as candidate OA disease-modifying drugs. Their effects were evaluated by intra-articular (IA) injection in OA mouse models induced by DMM (destabilization of the medial meniscus) or adenoviral overexpression in joint tissues of hypoxia-inducible factor (HIF)-2α or zinc importer ZIP8. The regulatory mechanisms impacted by the rhodanine derivatives were examined in primary-culture chondrocytes and fibroblast-like synoviocytes (FLS). RESULTS: All three rhodanine derivatives inhibited OA development caused by DMM or overexpression of HIF-2α or ZIP8. Compared to vehicle-treated group, for example, IA injection of R-501 in DMM-operated mice reduced median OARSI grade from 3.78 (IQR 3.00-5.00) to 1.89 (IQR 0.94-2.00, P = 0.0001). R-502 and R-503 also reduced from 3.67 (IQR 2.11-4.56) to 2.00 (IQR 1.00-2.00, P = 0.0030) and 2.00 (IQR 1.83-2.67, P = 0.0378), respectively. Mechanistically, the rhodanine derivatives inhibited the nuclear localization and transcriptional activity of HIF-2α in chondrocytes and FLS. They did not bind to Zn2+ or modulate Zn2+ homeostasis in chondrocytes or FLS; instead, they inhibited the nuclear localization and transcriptional activity of the Zn2+-dependent transcription factor, MTF1. HIF-2α, ZIP8, and interleukin-1ß could upregulate matrix-degrading enzymes in chondrocytes and FLS, and the rhodanine derivatives inhibited these effects. CONCLUSION: IA administration of rhodanine derivatives significantly reduced OA pathogenesis in various mouse models, demonstrating that these derivatives have disease-modifying therapeutic potential against OA pathogenesis.

Improved design for a low temperature scanning tunneling microscope with an in situ tip treatment stage.

The Low Temperature Scanning Tunneling Microscope (LT-STM) is an extremely valuable tool not only in surface science but also in condensed matter physics. For years, numerous new ideas have been adopted to perfect LT-STM performances-Ultra-Low Vibration (ULV) laboratory and the rigid STM head design are among them. Here, we present three improvements for the design of the ULV laboratory and the LT-STM: tip treatment stage, sample cleaving stage, and vibration isolation system. The improved tip treatment stage enables us to perform field emission for the purpose of tip treatment in situ without exchanging samples, while our enhanced sample cleaving stage allows us to cleave samples at low temperature in a vacuum without optical access by a simple pressing motion. Our newly designed vibration isolation system provides efficient space usage while maintaining vibration isolation capability. These improvements enhance the quality of spectroscopic imaging experiments that can last for many days and provide increased data yield, which we expect can be indispensable elements in future LT-STM designs.

Reciprocal regulation by hypoxia-inducible factor-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in osteoarthritis.

OBJECTIVE: Hypoxia-inducible factor-2α (HIF-2α) transcriptionally upregulates Nampt in articular chondrocytes. NAMPT, which exhibits nicotinamide phosphoribosyltransferase activity, in turn causes osteoarthritis (OA) in mice by stimulating the expression of matrix-degrading enzymes. Here, we sought to elucidate whether HIF-2α activates the NAMPT-NAD(+)-SIRT axis in chondrocytes and thereby contributes to the pathogenesis of OA. METHODS: Assays of NAD levels, SIRT activity, reporter gene activity, mRNA, and protein levels were conducted in primary cultured mouse articular chondrocytes. Experimental OA in mice was induced by intra-articular (IA) injection of adenovirus expressing HIF-2α (Ad-Epas1) or NAMPT (Ad-Nampt). The functions of SIRT in OA were examined by IA co-injection of SIRT inhibitors or adenovirus expressing individual SIRT isoforms or shRNA targeting specific SIRT isoforms. RESULTS: HIF-2α activated the NAMPT-NAD(+)-SIRT axis in chondrocytes by upregulating NAMPT, which stimulated NAD(+) synthesis and thereby activated SIRT family members. The activated NAMPT-SIRT pathway, in turn, promoted HIF-2α protein stability by negatively regulating its hydroxylation and 26S proteasome-mediated degradation, resulting in increased HIF-2α transcriptional activity. Among SIRT family members (SIRT1-7), SIRT2 and SIRT4 were positively associated with HIF-2α stability and transcriptional activity in chondrocytes. This reciprocal regulation was required for the expression of catabolic matrix metalloproteinases (MMP3, MMP12, and MMP13) and OA cartilage destruction caused by IA injection of Ad-Epas1 Ad-Nampt. CONCLUSION: The reciprocal regulation of HIF-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in OA cartilage destruction caused by HIF-2α or NAMPT.

Electron beam irradiated ITO films as highly transparent p-type electrodes for GaN-based LEDs.

We have investigated the effect of electron beam irradiation on the electrical and optical properties of ITO film prepared by magnetron sputtering method at room temperature. Electron beam irradiation to the ITO films resulted in a significant decrease in sheet resistance from 1.28 x 10(-3) omega cm to 2.55 x 10(-4) omega cm and in a great increase in optical band gap from 3.72 eV to 4.16 eV, followed by improved crystallization and high transparency of 97.1% at a wavelength of 485 nm. The overall change in electrical, optical and structural properties of ITO films is related to annealing effect and energy transfer of electron by electron beam irradiation. We also fabricated GaN-based light-emitting diodes (LEDs) by using the ITO p-type electrode with/without electron beam irradiation. The results show that the LEDs having ITO p-electrode with electron beam irradiation produced higher output power due to the low absorption of light in the p-type electrode.

Reduction of gate leakage current on AlGaN/GaN high electron mobility transistors by electron-beam irradiation.

This study examined the effect of electron-beam (E-beam) irradiation on the AIGaN/GaN HEMTs for the reduction of gate leakage. After E-beam irradiation, the gate leakage current significantly decreased from 2.68 x 10(-8) A to 4.69 x 10(-9) A at a drain voltage of 10 V. The maximum drain current density of the AIGaN/GaN HEMTs with E-beam irradiation increased 14%, and the threshold voltage exhibited a negative shift, when compared to that of the AIGaN/GaN HEMTs before E-beam irradiation. These results strongly suggest that the reduction of gate leakage current resulted from neutralization nitrogen vacancies and removing of oxygen impurities.

Improved electrical and optical properties of ITO/Ag/ITO films by using electron beam irradiation and their application to ultraviolet light-emitting diode as highly transparent p-type electrodes.

We have investigated the effect of insertion of a Ag layer in ITO film as well as electron beam irradiation to the multilayer films on the electrical and optical properties of the ITO-based multilayer deposited by magnetron sputtering method at room temperature. Inserting a very thin Ag layer between ITO layers resulted in a significant decrease in sheet resistance and increased the optical band gap of the ITO/Ag/ITO multilayer to 4.35 eV, followed by a high transparency of approximately 80% at a wavelength of 375 nm. We have also fabricated ultraviolet light-emitting diodes (LED) by using the ITO/Ag/ITO p-type electrode with/without electron beam irradiation. The results show that the UV-LEDs having ITO/Ag/ITO p-electrode with electron beam irradiation produced 19% higher optical output power due to the low absorption of light in the p-type electrode.

Light-extraction enhancement of vertical-injection GaN-based light-emitting diodes fabricated with highly integrated surface textures.

We report on the fabrication of high-efficiency vertical-injection GaN-based light-emitting diodes (LEDs) fabricated with integrated surface textures. An optical ray-tracing simulation shows that the high integration of surface textures can effectively enhance the light-extraction efficiency. The integrated surface textures are fabricated on the top surface of LEDs by generating hexagonal cones on the periodically corrugated surfaces of n-GaN. Compared to reference LEDs without textures, LEDs fabricated with integrated surface textures show an enhancement of the output power by a factor of 2.59, which is in agreement with the calculated results.

The protection of the retina from ischemic injury by the free radical scavenger EGb 761 and zinc in the cat retina.

We investigated the effects of a free radical scavenger (EGb 761) and zinc in experimentally induced ischemic injury in the cat retina. Total retinal ischemia for 90 min was produced in the left eyes of 40 cats by raising intraocular pressure. In group 1, 10 cats were used as control. The free radical scavenger (EGb 761, 100 mg/kg) in group 2 (10 cats) and zinc chloride (250 microg/kg) in group 3 (10 cats) were administered intravenously at the end of ischemia. In group 4, both EGb 761 (100 mg/kg) and zinc chloride (250 microg/kg) were injected into 10 cats. ERG and a histologic study were performed 1 h, 1 day, 3 days, 1 week and 2 weeks after ischemia. The amplitude of the ERG b-wave was 62.73+/-0.32, 84.31+/-6.10, 83.65+/-12.23 and 102.4+/-14.27%, and the summed amplitude of oscillatory potentials was 66.16+/-16.42, 99.44+/-14.92, 95.45+/-6.42 and 99.62+/-12.32% in each group 2 weeks after ischemia. There was no significant effect in animals that received zinc alone (group 3) by the end of 1 week but some additive effect in combining EGb 761 and zinc chloride (group 4) 1 h after ischemia. These findings suggest that the free radical scavenger EGb 761 may efficiently protect the retina from ischemic injury and zinc may have an additive effect when combined with a radical scavenger.

Ultrastructure of surgically excised subfoveal neovascular membranes.

We studied the ultrastructural features of four consecutive subfoveal neovascular membranes (SFNM) associated with age-related macular degeneration. Cellular components of the membranes included retinal pigment epithelial (RPE) cells, endothelium-lined vascular channels, macrophages, myofibroblasts, fibrocytes, glial cells, erythrocytes, and lymphocytes. Extracellular interstitial constituents included collagen fibrils, basal laminar deposits, fibrin and young elastic fibrils. These findings show that SFNMs consist of various cells originating from surrounding tissues and vessels. Among these RPE cells and macrophages are the main cellular components and in conjunction with various extracellular matrix, especially collagen, may play an important role in the formation and maintenance of the membranes.

Degradation of phagosomes and diurnal changes of lysosomes in rabbit retinal pigment epithelium.

Diurnal changes of lysosomes including ultrastructural changes of phagosomes and acid phosphatase reactions in phagosomes, as well as diurnal biochemical changes in cathepsin D activity, were studied in the retinal pigment epithelium (RPE) of the rabbit. The rabbit was maintained on a natural light-dark cycle over seven days in fall and was sacrificed at various times during the day and night. The number of lysosomes or phagosomes in the RPE was the highest at 1.5 hours after exposure to sunlight (8:00 AM), and thereafter decreased with time. Three types of phagosomes were observed and acid phosphatase reactions were different in each type of phagosome; the fresh phagosomes were negative or positive, lamellar bodies positive, and dense bodies partially positive. The biochemical activity of cathepsin D was the highest at 8:00 AM, and this was consistent with the time of peak in phagocytic activity in the RPE. This report shows that phagocytic activity in the RPE occurred in the early stage after exposure to sunlight, and that fresh phagosomes were sequentially degraded to lamellar or dense bodies. Cathepsin D activity also increased, and this was consistent with the phagocytic activity in the RPE.

Safety of intravitreally injected ciprofloxacin in phakic rabbit eyes.

This study was designed to determine the maximal safe drug concentration of intravitreal ciprofloxacin in phakic rabbit eyes. Twenty-two eyes of New Zealand pigmented rabbits received midvitreal ciprofloxacin of 100, 200, 400, 600 or 800 micrograms in BSS Plus, or BSS Plus only. Retinal toxicity was dose-dependent as determined with electroretinography, light microscopy, and transmission electron microscopy. At a dose of greater than 400 micrograms, disorganization of the outer segments was a main pathological finding in transmission electron microscopy. We evaluated retinal function by measuring the electroretinograms for a graded series of flash intensities and by fitting electroretinogram b-wave amplitudes to the Naka-Rushton equation. At a dose of greater than 600 micrograms, Rmax was significantly decreased and log K was significantly increased. N-value tended to decrease. A decrease of b-wave amplitudes caused by retinal toxicity could be detected very sensitively with lower luminance stimuli. Determination of retinal toxicity with lower luminance electroretinography revealed a significant decrease of b-wave amplitudes at a dose of greater than 400 micrograms. We concluded that a safe dose of intravitreal ciprofloxacin in phakic rabbit eyes was 200 micrograms in phakic eyes.

Efficacy of intravitreal hemocoagulase for control of bleeding in rabbit experimental model of vitrectomy.

Hemorrhage during vitrectomy for diabetic retinopathy or in the recently traumatized eye can complicate the surgical procedures and might cause termination and failure of vitrectomy in some cases. The effect of the hemocoagulase, Botropase, on the hemostasis of intraocular bleeding was evaluated in a rabbit model by cutting central retinal vessels in the medullary ray. Addition of the hemocoagulase (1 NIH unit/100 ml) to BSS Plus significantly reduced bleeding time. Immediately after vitrectomy with the use of this hemocoagulase, the average of the maximum amplitudes of the b-wave in electroretinography was normal, although the sensitivity of the electroretinogram was reduced by 0.1 log unit in the experimental eyes which were infused with hemocoagulase solution. The sensitivity showed no significant difference after the second postoperative day. The experimental eyes showed no abnormal findings histologically. Infusate containing Botropase appeared to be a useful adjunct for the control of intraocular bleeding during vitreous surgery.

Effects of Escherichia coli endotoxin on structure and permeability of myocardial capillaries.

A study was carried out to investigate changes in myocardial capillaries induced by endotoxin, in order to clarify the pathogenesis of myocardial damage in endotoxemia. Wistar rats were injected intraperitoneally with 100 mg/kg Escherichia coli lipopolysaccharide and then sacrificed at 1, 2, 3,4, 5, 6, 8, and 24 h after injection. The myocardium was observed by electron microscopy with histochemistry using horseradish peroxidase and immunocytochemistry for Na+, K(+)-ATPase/TPase. The earliest evident endothelial alterations were swelling, increased numbers of pinocytotic vesicles, and formation of cytoplasmic projections. Interstitial edema and focal detachment of the endothelial cells from the basement membrane occurred with time. Vascular permeability was increased after endotoxin injection. Activity of Na+, K(+)-ATPase was reduced on the plasma membrane of the endothelial cells. It is concluded that endotoxin induces structural and enzymatic changes in the myocardial capillary endothelium and an increase of capillary permeability.

Myelo-optic neuropathy caused by aconitine in rabbit model.

A single dose of aconitine (0.6 mg/kg), the principal constituent of aconite, was administered intraperitoneally in order to evaluate its toxic effects on the visual system of a rabbit model. The typical pattern in which the alteration in the visual evoked potential occurred after an aconitine injection consisted of a delay in the onset and peak latency, and a reduction in the amplitude. Histopathologically, there was damage to the myelin sheath of the visual pathway, spinal cord and peripheral nerves. These findings suggest that a toxic dose of aconitine may cause myelo-optic neuropathy in rabbits.