The Role of 2% Rebamipide Eye Drops Related to Conjunctival Differentiation in Superoxide Dismutase-1 (Sod1) Knockout Mice.

Purpose: The superoxide dismutase-1 knockout (Sod1-/-) mouse is an age-related dry eye mouse model. We evaluated the role of 2% rebamipide ophthalmic solution on the conjunctiva and ocular surface alterations in Sod1-/- mice. Methods: Rebamipide eye drops (2%) were instilled in six 50-week-old male Sod1-/- mice and six C57BL/6 strain wild-type (WT) male mice four times a day for 2 weeks. Aqueous tear secretion quantity and tear film breakup time measurements as well as vital stainings were performed. Immunohistochemistry staining of the conjunctiva was performed using SAM pointed domain-containing ETS transcription factor (SPDEF), transglutaminase-1, and involucrin antibodies. Quantitative RT-PCR was carried out to study mRNA expression of the same markers. Results: The mean tear quantities showed no significant changes in both mice strains after treatment (P = 0.24). The mean tear film breakup time (P = 0.003) and vital staining scores significantly improved in the Sod1-/- mice after treatment. Treatment with 2% rebamipide eye drops significantly decreased the corneal fluorescein (P = 0.0093) and Rose Bengal (P = 0.002) staining scores in the Sod1-/- mice. We showed a notable increase in SPDEF and a marked decrease in transglutaminase-1 and involucrin immunohistochemistry stainings, together with a significant increase in SPDEF (P = 0.0003) and a significant decline in transglutaminase-1 (P = 0.0072) and involucrin (P = 0.009) mRNA expression after treatment in the Sod1-/- mice. Conclusions: Topical use of 2% rebamipide drops was observed to improve conjunctival epithelial differentiation and suppress keratinization in the Sod1-/- mice.

[People with stomas - issues and responses in critical periods].

People with stomas who have either been cured of cancer or are living with cancer have achieved good interrelationships among the three issues of"establishing self-care","dealing with stoma complications", and"accepting stomas", and they are maintaining stable physical and mental states.However, self-care may become difficult due to stoma complications and adverse events caused by chemotherapy and radiation therapy in the cancer treatment period, and in advanced phases of cancer serious stoma complications may occur due to deterioration of general condition and advancing cancer. Therefore, there is a risk that those stable physical and mental states will collapse.In order to deal with this critical state, in the cancer treatment period, stoma skin care is used for handling skin problems around the stoma, and for adverse events such as hand-and-foot syndrome, braces that are easy to operate are chosen from among various types of stoma braces in order to cover impediments.During advanced phases of cancer, care is conducted with the main priority placed on physical stability in order to ensure that the three major complications of stoma varicose veins, stoma prolapse, and parastomal hernia do not worsen and significantly affect general condition and daily life.Stoma outpatient treatment that provides lifelong support for such issues, and the existence of skin- and excretion-care certified nurses who provided highly specialized selfcare support, are extremely important for cancer survivors with stomas.

A novel antibody microarray format using non-covalent antibody immobilization with chemiluminescent detection.

To date, protein and antibody microarrays have been used in reverse-phase and sandwich-based methods in order to detect known proteins such as biomarkers in samples. Our group developed "libraries" of antibodies against unknown proteins, referred to as mKIAA proteins, and we attempted to discover candidate novel biomarkers by protein expression profiling.To profile mKIAA protein expression using these antibodies, we established an antibody microarray system using chemiluminescent detection. A number of techniques for protein-antibody microarrays have been reported; however, no entirely suitable protocol for crude protein samples has been established. To address this issue, we immobilized purified antibodies on hydrophilic surface polymer slides (Maxisorp, Nunc). Although our system is based on the direct labeling of crude protein samples, we achieved sufficient sensitivity (detection limit: 50 pg mL(-1)) and low backgrounds. This sensitivity is on a level with the sandwich immunoassay-based antibody array system. Using our protocol, we developed an antibody microarray spotted with 960 anti-mKIAA antibodies (total: 3888 spots for quadruplicate assessments), and we carried out protein expression profiling of mKIAA proteins. In this study, we generated an expression profile of 960 mKIAA proteins and compared the present results with those obtained via cDNA microarray.

A chip-based miniaturized format for protein-expression profiling: the exploitation of comprehensively produced antibodies.

Numerous antibodies have been developed and validated in recent years, and show promise for use in novel functional protein assays. Such assays would be an alternative to pre-existing comprehensive assays, such as DNA microarrays. Antibody microarrays are thought to represent those functional protein assays. While a variety of attempts have been made to apply DNA microarray technology to antibody microarrays, a fully optimized protocol has not been established. We have been conducting a project to comprehensively produce antibodies against mouse KIAA ("KI" stands for "Kazusa DNA Research Institute" and "AA" are reference characters) proteins. Using our library of antibodies, we established a novel antibody microarray format that utilizes surface plasmon resonance (SPR) technology. A label-free real-time measurement of protein expression in crude cell lysates was achieved by direct readout of the bindings using SPR. Further refinement of the antibody microarray format enabled us to detect a smaller quantity of target proteins in the lysate without the bulk effect. In this review, we first summarize available antibody array formats and then describe the above-mentioned format utilizing updated SPR technology.

Label-free detection of proteins in crude cell lysate with antibody arrays by a surface plasmon resonance imaging technique.

We established a label-free method of measuring proteins in crude cell lysate using antibody arrays and surface plasmon resonance (SPR) imaging. The refractivity of the running buffer was adjusted with that of the lysate to overcome the bulk effect. The chemistries of the fabricated arrays were investigated to reduce nonspecific adsorption on the array surface. We found that the hydrophilicity of the poly(ethylene glycol) moiety and lower electrostatic charge on the surface provided a specific measurement of antigen-antibody interaction. We validated the system by measuring the expression of eight proteins in the mouse brain and comparing the results to those by conventional Western blotting. The detection limit of the antibody array was approximately 30 ng/mL in crude cell lysate, on the same order as that of previous SPR research. The system enabled quick, label-free, and high-throughput analysis of abundant proteins with minimal sample volume ( approximately 200 muL). It is expected that our SPR antibody array will be applicable for direct protein expression profiling of cell lysate, as well as for cell phenotyping, food analysis, discovery of new biomarkers, and immunological disease diagnostics.

Species-specific patterns of sexual dimorphism in the expression of fruitless protein, a neural musculinizing factor in Drosophila.

In Drosophila melanogaster, male-specific forms of the fruitless (fru) gene product, mFru protein, function as a neural sex-determination factors that directs the development of at least two male characteristics, namely courtship and mating behavior and the formation of the muscle of Lawrence (MOL). In D. melanogaster, the male-specific expression of Fru protein in motoneurons is responsible for the male-limited induction of the MOL by such neurons. Although no Drosophila species whose females have the MOL are known, there are many Drosophila species whose males lack the MOL. We performed immunohistochemical staining of the central nervous system (CNS) from 9 Drosophila species to determine whether the mFru expression profile is different between MOL-present and MOL-absent species. In 8 of the 9 species, Fru protein expression in the CNS is strictly male-specific, regardless of the presence or absence of the MOL. The sole exception is D. suzukii, in which females express the Fru protein though less extensively than males do: Fru expression in the CNS of female D. suzukii is restricted to the lamina and ventral ganglia. Expression of Fru protein in the lamina is observed in males of D. virilis and in both sexes of D. suzukii, but not in males and females of the 7 other species. These results indicate that sexually dimorphic expression of the Fru protein has been subjected to species-specific modulation during evolution.

A novel approach to protein expression profiling using antibody microarrays combined with surface plasmon resonance technology.

We have previously described our systems for the high-throughput production of antibodies against mouse KIAA proteins and their validation (Proteomics 2004, 4, 1412-1416). Using our "libraries" of antibodies, we established a novel antibody microarray system in which surface plasmon resonance (SPR) technology is utilized for signal detection. Up to 400 real-time antibody-target bindings could be measured simultaneously within a single hour. This rapid detection was achieved by direct readout of the bindings using SPR technology. To evaluate our system, we assessed the reproducibility on crude protein samples and obtained satisfactorily reproducible results, exhibiting correlation values >0.92. Using this SPR-based antibody microarray system, we examined mKIAA protein expression in five different adult mouse tissues and identified the specific tissue expression patterns of several mKIAA proteins.

Targeted expression of Ip3 sponge and Ip3 dsRNA impaires sugar taste sensation in Drosophila.

We evaluated the role of IP(3) in sugar taste reception in Drosophila melanogaster by inactivating the IP(3) signaling using genetic tools. We used the "IP(3) sponge," composed of the modified ligand-binding domain from the mouse IP(3) receptor, which was designed to absorb IP(3) in competition with native IP(3) receptors. Another tool was a transgene that generates double-stranded RNA against IP(3) receptor mRNA. Both inhibitors diminished the sensitivity of flies to trehalose and sucrose, as estimated by behavioral assays and electrophysiological recordings from the sugar receptor cells. The result indicates that IP(3) signaling is indispensable for sugar reception in Drosophila.

Male-specific expression of the fruitless protein is not common to all Drosophila species.

Sex-specific behavioral patterns must be a result of sexual differences in the structure and/or function of the central nervous system (CNS). Male Drosophila melanogaster mutants for the fruitless (fru) locus exhibit enhanced male-to-male courtship. The fru mutant males are accompanied by malformation of the male-specific muscle of Lawrence (MOL), which, in wild-type males, is induced by male motoneurons innervating it. These two phenotypes are the consequences of impaired sex determination of CNS neurons. In D. melanogaster, although the fru mRNAs are transcribed in the CNS of both the male and female, the Fru protein is only translated in the male CNS. This male-specific translation of Fru was also observed in D. simulans, D. yakuba, D. pseudoobscura and D. virilis; however, in D. suzukii, the Fru protein expression was detected even in the female CNS.

Expression analysis of the lingerer gene in the larval central nervous system of Drosophila melanogaster.

The lingerer (lig) gene is necessary for initiation and termination of copulatory behavior in Drosophila melanogaster. The lig gene encodes cytoplasmic proteins, and is expressed in the central nervous system (CNS) during the late third-instar larval stage when the lig function is required for normal copulation to occur after adult eclosion. To characterize the lig-expressing cells in the late third-instar larval CNS, we have isolated a genomic fragment containing the promoter/enhancer region of the lig gene, and established transgenic lines in which expression of reporter genes is controlled by the lig promoter/enhancer. In the larval brain, reporter genes were expressed in all of the glial cells and in clusters of neurons that projected contralaterally. In the larval ventral ganglion, reporter genes were expressed in subperineurial glia, peripheral exit glia, and a number of interneurons, but not in motor neurons. In the cloned promoter/enhancer region, we have found the sequence motif for binding of the REPO protein, a transcription factor essential for the differentiation and maintenance of glial cells. The lig gene is thus one of the candidate target genes for the REPO transcription factor.

Implication of Rho-associated kinase in the elevation of extracellular dopamine levels and its related behaviors induced by methamphetamine in rats.

A growing body of evidence suggests that several protein kinases are involved in the expression of pharmacological actions induced by a psychostimulant methamphetamine. The present study was designed to investigate the role of the Rho/Rho-associated kinase (ROCK)-dependent pathway in the expression of the increase in extracellular levels of dopamine in the nucleus accumbens and its related behaviors induced by methamphetamine in rats. Methamphetamine (1 mg/kg, subcutaneously) produced a substantial increase in extracellular levels of dopamine in the nucleus accumbens, with a progressive augmentation of dopamine-related behaviors including rearing and sniffing. Methamphetamine also induced the decrease in levels of its major metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA). Both the increase in extracellular levels of dopamine and the induction of dopamine-related behaviors by methamphetamine were significantly suppressed by pretreatment with an intranucleus accumbens injection of a selective ROCK inhibitor Y-27632. In contrast, Y-27632 had no effect on the decrease in levels of DOPAC and HVA induced by methamphetamine. Under these conditions, there were no changes in protein levels of membrane-bound RhoA in the nucleus accumbens following methamphetamine treatment. It is of interest to note that the microinjection of Y-27632 into the nucleus accumbens failed to suppress the increases in extracellular levels of dopamine, DOPAC, and HVA in the nucleus accumbens induced by subcutaneous injection of a prototype of micro -opioid receptor agonist morphine (10 mg/kg). Furthermore, perfusion of a selective blocker of voltage-dependent Na+ channels, tetrodotoxin (TTx) into the rat nucleus accumbens did not affect the increase in extracellular levels of dopamine in the rat nucleus accumbens by methamphetamine, whereas the morphine-induced dopamine elevation was eliminated by this application of TTx. The extracellular level of dopamine in the nucleus accumbens was also increased by perfusion of a selective dopamine re-uptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy]-4-(3-phenylpropyl)piperazine (GBR-12909) in the nucleus accumbens. This effect was not affected by pretreatment with intranucleus accumbens injection of Y-27632. These findings provide first evidence that Rho/ROCK pathway in the nucleus accumbens may contribute to the increase in extracellular levels of dopamine in the nucleus accumbens evoked by a single subcutaneous injection of methamphetamine. In contrast, this pathway is not essential for the increased level of dopamine in this region induced by morphine, providing further evidence for the different mechanisms of dopamine release by methamphetamine and morphine in rats.

Pathology of the adult central nervous system induced by genetic inhibition of programmed cell death in Drosophila pupae.

In the spinster (spin) mutant of Drosophila melanogaster, the extent of programmed cell death (PCD) in the abdominal ganglion 6 h after puparium formation (APF) is significantly reduced. The shortening of the abdominal ganglion, which is normally completed 48 h APF, does not occur. After eclosion, neurodegeneration accompanied by accumulation of autofluorescent materials is manifested in the central nervous system (CNS) of the spin mutant. The materials accumulated in the spin-mutant CNS contain a substance that is immunopositive to an antibody against GM2 ganglioside. Halving the dosage of three cell death genes, rpr, grim, and hid, blocks shortening of the abdominal ganglion and induces neurodegeneration accompanied by accumulation of autofluorescent materials in the adult CNS. These observations suggest that the primary action of the spin mutation is to reduce the extent of PCD 6 h APF, which concomitantly leads to a failure in shortening of the abdominal ganglion and to neurodegeneration of the adult CNS. Arch.

A putative sigma1 receptor antagonist NE-100 attenuates the discriminative stimulus effects of ketamine in rats.

Ketamine, one of the dissociative anaesthetic agents, has been shown to produce psychotomimetic effects. It has been well documented that activation of sigma receptors is responsible for the pathogenesis of some psychiatric disorders. In the present study, the effects of NE-100, a putative sigma(1) receptor antagonist, was investigated in rats trained to discriminate between ketamine (5 mg/kg, i.p.) from saline under a fixed-ratio 10 food-reinforced procedure. Here we report for the first time that NE-100 (1 mg/kg) produced a shift to the right in the dose-response curve for ketamine's discriminative stimulus effects. These results suggest that the sigma(1) receptor is, at least in part, involved in the discriminative stimulus effects of ketamine.