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author:("Sato, shigeji")
1.  Prognostic Judgment at Post-Surgery by Biochemical Parameters in Beef Cattle with Left Displaced Abomasum 
ABSTRACT
We examined whether the postoperative prognosis of beef cattle with left displaced abomasum (LDA) can be estimated from changes in laboratory parameters. Preoperatively, beef cattle with LDA showed increases in plasma glucose with decreased serum insulin in the glucose tolerance test compared to non-LDA cattle. Postoperatively, the cattle with LDA were retrospectively divided into two groups, good and bad prognoses. Although plasma glucose concentrations significantly increased either pre- or postoperatively, no difference was noted between the good and bad prognosis groups. Serum insulin concentrations in the bad prognosis group significantly decreased, compared to those in the good prognosis group. These findings suggest that beef cattle with LDA elicit disturbed glucose metabolite pre- and postoperatively, and serum insulin levels may predict their prognoses after surgery.
doi:10.1292/jvms.14-0187
PMCID: PMC4221180  PMID: 24998331
beef cattle; laboratory test; left displaced abomasum; prognosis
2.  Characteristics of Fluid Composition of Left Displaced Abomasum in Beef Cattle Fed High-Starch Diets 
ABSTRACT
To clarify the pathophysiology of left displaced abomasum (LDA), beef cattle fed high-starch diets were examined. The abomasal pH in beef cattle with LDA was lower than that in non-LDA reference animals (data from beef cattle at an abattoir), suggesting that it facilitated acidity. Bacteriological examinations of the abomasal fluid in cattle with LDA revealed the presence of Pseudomonas spp., Clostridium spp. and Candida spp., presumably reflecting the accelerated influx of ruminal fluid into the abomasum. Biochemical analyses of serum revealed that LDA cattle had higher lactic acid and lower vitamin A and E levels than non-LDA reference animals. These results indicate that beef cattle with LDA may suffer from vitamin A and E deficiencies due to maldigestion of starch and the high acidity of abomasal fluid.
doi:10.1292/jvms.13-0601
PMCID: PMC4155201  PMID: 24813464
abomasal fluid; beef cattle; left displaced abomasum; starch diets; vitamin A
3.  Effects of a Bacteria-Based Probiotic on Ruminal pH, Volatile Fatty Acids and Bacterial Flora of Holstein Calves 
ABSTRACT
Twelve ruminally cannulated Holstein calves (age, 12 ± 3 weeks) were used to identify the effect of a probiotic comprised of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum on ruminal components. The calves were adapted to a diet containing a 50% high-concentrate (standard diet) for 1 week, and then, the probiotic was given once daily for 5 days (day 1–5) at 1.5 or 3.0 g/100 kg body weight to groups of four calves each. Four additional calves fed the standard diet without probiotic served as the corresponding control. Ruminal pH was measured continuously throughout the 15-day experimental period. Ruminal fluid was collected via a fistula at a defined time predose and on days 7 and 14 to assess volatile fatty acid (VFA), lactic acid and ammonia-nitrogen concentrations, as well as the bacterial community. The probiotic at either dose improved the reduced 24-hr mean ruminal pH in calves. The circadian patterns of the 1 hr mean ruminal pH were identical between the probiotic doses. In both probiotic groups, ruminal lactic acid concentrations remained significantly lower than that of the control. Probiotic did not affect ruminal VFA concentrations. L. plantarum and C. butyricum were not detected in the rumen of calves given the high-dose probiotic, whereas Enterococcus spp. remained unchanged. These results suggest that calves given a probiotic had stable ruminal pH levels (6.6–6.8), presumably due to the effects of the probiotic on stabilizing rumen-predominant bacteria, which consume greater lactate in the rumen.
doi:10.1292/jvms.14-0028
PMCID: PMC4108772  PMID: 24614603
calf; probiotic; ruminal bacteria; ruminal pH
4.  Six1 is a key regulator of the developmental and evolutionary architecture of sensory neurons in craniates 
BMC Biology  2014;12:40.
Background
Various senses and sensory nerve architectures of animals have evolved during adaptation to exploit diverse environments. In craniates, the trunk sensory system has evolved from simple mechanosensory neurons inside the spinal cord (intramedullary), called Rohon-Beard (RB) cells, to multimodal sensory neurons of dorsal root ganglia (DRG) outside the spinal cord (extramedullary). The fish and amphibian trunk sensory systems switch from RB cells to DRG during development, while amniotes rely exclusively on the DRG system. The mechanisms underlying the ontogenic switching and its link to phylogenetic transition remain unknown.
Results
In Xenopus, Six1 overexpression promoted precocious apoptosis of RB cells and emergence of extramedullary sensory neurons, whereas Six1 knockdown delayed the reduction in RB cell number. Genetic ablation of Six1 and Six4 in mice led to the appearance of intramedullary sensory neuron-like cells as a result of medial migration of neural crest cells into the spinal cord and production of immature DRG neurons and fused DRG. Restoration of SIX1 expression in the neural crest-linage partially rescued the phenotype, indicating the cell autonomous requirements of SIX1 for normal extramedullary sensory neurogenesis. Mouse Six1 enhancer that mediates the expression in DRG neurons activated transcription in Xenopus RB cells earlier than endogenous six1 expression, suggesting earlier onset of mouse SIX1 expression than Xenopus during sensory development.
Conclusions
The results indicated the critical role of Six1 in transition of RB cells to DRG neurons during Xenopus development and establishment of exclusive DRG system of mice. The study provided evidence that early appearance of SIX1 expression, which correlated with mouse Six1 enhancer, is essential for the formation of DRG-dominant system in mice, suggesting that heterochronic changes in Six1 enhancer sequence play an important role in alteration of trunk sensory architecture and contribute to the evolution of the trunk sensory system.
doi:10.1186/1741-7007-12-40
PMCID: PMC4084797  PMID: 24885223
Dorsal root ganglia; Enhancer; Evolution; Neural crest cell; Rohon-Beard cell; Sensory neuron; Six genes
5.  Immune-Stimulatory Effects of a Bacteria-Based Probiotic on Peripheral Leukocyte Subpopulations and Cytokine mRNA Expression Levels in Scouring Holstein Calves 
ABSTRACT
Subpopulations of peripheral leukocytes and cytokine mRNA expression levels were evaluated in scouring and healthy Holstein calves (age 10 ± 5 days; n=42) treated with a probiotic consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum. The calves were assigned to the scouring or healthy group and then subdivided into pathogen-positive treated (n=8), pathogen-positive control (n=8), pathogen-negative treated (n=6), pathogen-negative control (n=6), healthy treated (n=6) and healthy control (n=8) groups. A single dose of the probiotic (3.0 g/100 kg body weight) was given to each calf in the treatment groups for 5 days. Blood samples were collected on the first day of scour occurrence (day 0) and on day 7. In the scouring calves, smaller peripheral leukocyte subpopulations and cytokine mRNA expression levels were noted on day 0. The numbers of CD3+ T cells; CD4+, CD8+ and WC1+ γδ T cell subsets; and CD14+, CD21+ and CD282+ (TLR2) cells were significantly increased in the scouring and healthy treated calves on day 7. Furthermore, interleukin-6, tumor necrosis factor-alpha and interferon-gamma mRNA expression was elevated in the peripheral leukocytes of the scouring and healthy treated calves on day 7. The scouring calves given the probiotic recovered on day 7. A significantly smaller number of peripheral leukocytes and lower cytokine mRNA expression level might be induced by scouring in calves. Repeated probiotic administration might stimulate cellular immunity and encourage recovery from scouring in pre-weaning Holstein calves.
doi:10.1292/jvms.13-0534
PMCID: PMC4073336  PMID: 24451928
cytokine; leukocyte; probiotic; scouring calf; toll-like receptor 2
6.  Histological study on the treatment of vascular malformations resistant to pulsed dye laser 
Laser Therapy  2013;22(3):181-186.
Background and Aims: The pulsed dye laser (PDL) is recognized both as an effective treatment for vascular malformations and the as first treatment of choice for these lesions. However, PDL irradiation has poor efficacy in some patients, particularly the elderly. The present study histologically assessed such patients to try to elucidate the reason.
Materials and Methods: A pulsed dye laser was used in 3 subjects in whom previous laser treatment was ineffective. Three-millimeter punch biopsies were obtained before laser treatment, 1 week and 3 months after the laser treatment. Each specimen was stained with toluidine blue and examined under light microscopy followed by electron microscopy with oolong tea extract (OTE) staining.
Results: Microscopy revealed an increase in the vasculature at baseline and an increased number of dermal fibroblasts. One week post-irradiation, inflammatory cell infiltration was observed together with extensive interstitial perivascular edema. At 1 week and 3 months after laser irradiation, normal structures were observed for both blood vessels and capillary endothelial cells. Mild changes were noted in other interstitial features, but findings obtained 3 months after irradiation were almost similar to those before irradiation.
Conclusions: The lower efficacy of PDL treatment in the elderly was possibly due to the markedly low amount of red blood cells in our subjects' blood vessels, a major chromophore for the PDL, was markedly low. It is possible that age-related denaturation of dermal matrix collagen plays some role in maintaining the vasculature in the interstitium with edema, and inflammatory cell infiltration could lead to the cellular release of some cytokines which favor reconstruction of the vasculature.
doi:10.5978/islsm.13-OR-13
PMCID: PMC3813995  PMID: 24204091
7.  Effects of a Bacteria-Based Probiotic on Subpopulations of Peripheral Leukocytes and Their Cytokine mRNA Expression in Calves 
ABSTRACT
Eight Holstein calves (10 ± 3 weeks) were used to examine the interaction between a bacteria-based probiotic agent (probiotic) and the function of peripheral blood mononuclear cells (PBMCs). The probiotic, consisting of Lactobacillus plantarum, Enterococcus faecium and Clostridium butyricum, was administered orally at 3.0 g/100 kg body weight to calves once daily for 5 consecutive days. Calves given the vehicle alone with no probiotic served as the control. In the treatment group, increases in numbers of CD282+ (TLR2) monocytes, CD3+ T cells and CD4+, CD8+ and WC1+ γδ T cell subsets were noted on day 7 post-placement compared to predose day and the control group. Expression of interleukin (IL)-6, interferon-gamma (INF-γ) and tumor necrosis factor-alpha (TNF-α) was elevated in peripheral leukocytes on days 7 and 14. These results suggest that peripheral blood leukocytes in healthy calves may be stimulated via the gastrointestinal microbiota, which was increased by the oral probiotic treatment, with overall stability of the rumen bacterial flora. The 5-day repeated administration of a bacteria-based probiotic may enhance cellular immune function in weaned calves.
doi:10.1292/jvms.13-0370
PMCID: PMC3982805  PMID: 24131856
calf; cytokine; leukocyte; probiotic; toll-like receptor 2
8.  High CO2 Triggers Preferential Root Growth of Arabidopsis thaliana Via Two Distinct Systems Under Low pH and Low N Stresses 
Plant and Cell Physiology  2014;55(2):269-280.
Biomass allocation between shoots and roots is an important strategy used by plants to optimize growth in various environments. Root to shoot mass ratios typically increase in response to high CO2, a trend particularly evident under abiotic stress. We investigated this preferential root growth (PRG) in Arabidopsis thaliana plants cultivated under low pH/high CO2 or low nitrogen (N)/high CO2 conditions. Previous studies have suggested that changes in plant hormone, carbon (C) and N status may be related to PRG. We therefore examined the mechanisms underlying PRG by genetically modifying cytokinin (CK) levels, C and N status, and sugar signaling, performing sugar application experiments and determining primary metabolites, plant hormones and expression of related genes. Both low pH/high CO2 and low N/high CO2 stresses induced increases in lateral root (LR) number and led to high C/N ratios; however, under low pH/high CO2 conditions, large quantities of C were accumulated, whereas under low N/high CO2 conditions, N was severely depleted. Analyses of a CK-deficient mutant and a starchless mutant, in conjunction with sugar application experiments, revealed that these stresses induce PRG via different mechanisms. Metabolite and hormone profile analysis indicated that under low pH/high CO2 conditions, excess C accumulation may enhance LR number through the dual actions of increased auxin and decreased CKs.
doi:10.1093/pcp/pcu001
PMCID: PMC3913443  PMID: 24401956
Auxin; Cytokinin; High CO2; Low nitrogen; Low pH; Root to shoot ratio
9.  FGFR1-Frs2/3 Signalling Maintains Sensory Progenitors during Inner Ear Hair Cell Formation 
PLoS Genetics  2014;10(1):e1004118.
Inner ear mechanosensory hair cells transduce sound and balance information. Auditory hair cells emerge from a Sox2-positive sensory patch in the inner ear epithelium, which is progressively restricted during development. This restriction depends on the action of signaling molecules. Fibroblast growth factor (FGF) signalling is important during sensory specification: attenuation of Fgfr1 disrupts cochlear hair cell formation; however, the underlying mechanisms remain unknown. Here we report that in the absence of FGFR1 signaling, the expression of Sox2 within the sensory patch is not maintained. Despite the down-regulation of the prosensory domain markers, p27Kip1, Hey2, and Hes5, progenitors can still exit the cell cycle to form the zone of non-proliferating cells (ZNPC), however the number of cells that form sensory cells is reduced. Analysis of a mutant Fgfr1 allele, unable to bind to the adaptor protein, Frs2/3, indicates that Sox2 maintenance can be regulated by MAP kinase. We suggest that FGF signaling, through the activation of MAP kinase, is necessary for the maintenance of sensory progenitors and commits precursors to sensory cell differentiation in the mammalian cochlea.
Author Summary
The ability of our brain to perceive sound depends on its conversion into electrical impulses within the cochlea of the inner ear. The cochlea has dedicated specialized cells, called inner ear hair cells, which register sound energy. Environmental effects, genetic disorders or just the passage of time can damage these cells, and the damage impairs our ability to hear. If we could understand how these cells develop, we might be able to exploit this knowledge to generate new hair cells. In this study we address an old problem: how do signals from the fibroblast growth factor (FGF) family control hair cell number? We used mice in which one of the receptors for FGF (Fgfr1) is mutated and found that the expression of a stem cell protein, Sox2 is not maintained. Sox2 generally acts to keep precursors in the cochlea in a pre-hair cell state. However, in mutant mice Sox2 expression is transient, diminishing the ability of precursors to commit to a hair cell fate. These findings suggest that it may be possible to amplify the number of hair cell progenitors in culture by tuning FGF activity, providing a route to replace damaged inner ear hair cells.
doi:10.1371/journal.pgen.1004118
PMCID: PMC3900395  PMID: 24465223
10.  Effects of Elevated CO2 on Levels of Primary Metabolites and Transcripts of Genes Encoding Respiratory Enzymes and Their Diurnal Patterns in Arabidopsis thaliana: Possible Relationships with Respiratory Rates 
Plant and Cell Physiology  2014;55(2):341-357.
Elevated CO2 affects plant growth and photosynthesis, which results in changes in plant respiration. However, the mechanisms underlying the responses of plant respiration to elevated CO2 are poorly understood. In this study, we measured diurnal changes in the transcript levels of genes encoding respiratory enzymes, the maximal activities of the enzymes and primary metabolite levels in shoots of Arabidopsis thaliana grown under moderate or elevated CO2 conditions (390 or 780 parts per million by volume CO2, respectively). We examined the relationships between these changes and respiratory rates. Under elevated CO2, the transcript levels of several genes encoding respiratory enzymes increased at the end of the light period, but these increases did not result in changes in the maximal activities of the corresponding enzymes. The levels of some primary metabolites such as starch and sugar phosphates increased under elevated CO2, particularly at the end of the light period. The O2 uptake rate at the end of the dark period was higher under elevated CO2 than under moderate CO2, but higher under moderate CO2 than under elevated CO2 at the end of the light period. These results indicate that the changes in O2 uptake rates are not directly related to changes in maximal enzyme activities and primary metabolite levels. Instead, elevated CO2 may affect anabolic processes that consume respiratory ATP, thereby affecting O2 uptake rates.
doi:10.1093/pcp/pct185
PMCID: PMC3913440  PMID: 24319073
Arabidopsis thaliana; Elevated CO2; Primary metabolites; Respiration; Transcription
11.  Characterization of Metabolic States of Arabidopsis thaliana Under Diverse Carbon and Nitrogen Nutrient Conditions via Targeted Metabolomic Analysis 
Plant and Cell Physiology  2014;55(2):306-319.
Plant growth and metabolism are regulated in response to various environmental factors. To investigate modulations in plant metabolism by the combined action of elevated atmospheric CO2 concentration and other nutritional factors, we performed targeted metabolomic analysis using Arabidopsis thaliana plants grown under 24 different conditions where the CO2 concentration, amounts and species of nitrogen source, and light intensity were modified. Our results indicate that both the biosynthesis of diverse metabolites and growth are promoted in proportion to the CO2 concentration at a wide range of CO2 levels, from ambient concentrations to an extremely high concentration (3,600 p.p.m.) of CO2. This suggests that A. thaliana has the potential to utilize effectively very high concentrations of CO2. On the other hand, ammonium (but not nitrate) supplied as an additional nitrogen source induced drastic alterations in metabolite composition, including increases in the contents of glucose, starch and several amino acids, and reductions in the tricarboxylic acid (TCA) cycle-related organic acid content under any CO2 conditions. Hierarchical clustering analysis using the metabolite profiles revealed that ammonium is a prominent factor determining metabolic status, while the CO2 concentration is not. However, ammonium-induced metabolic alterations were differently modified by high concentrations of CO2. Hence, our results imply that increases in CO2 concentration may differently influence plant metabolism depending on the nitrogen nutrient conditions.
doi:10.1093/pcp/pct192
PMCID: PMC3913442  PMID: 24343996
Ammonium response; Arabidopsis thaliana; CO2 response; Nitrogen response; Targeted metabolomic analysis
12.  Investigation of Various Essential Factors for Optimum Infrared Thermography 
ABSTRACT
We investigated various essential factors for optimum infrared thermography for cattle clinics. The effect of various factors on the detection of surface temperature was investigated in an experimental room with a fixed ambient temperature using a square positioned on a wall. Various factors of animal objects were examined using cattle to determine the relationships among presence of hair, body surface temperature, surface temperature of the eyeball, the highest temperature of the eye circle, rectum temperature and ambient temperature. Also, the surface temperature of the flank at different time points after eating was examined. The best conditions of thermography for cattle clinics were determined and were as follows: (1) The distance between a thermal camera and an object should be fixed, and the camera should be set within a 45-degree angle with respect to the objects using the optimum focal length. (2) Factors that affect the camera temperature, such as extreme cold or heat, direct sunshine, high humidity and wind, should be avoided. (3) For the comparison of thermographs, imaging should be performed under identical conditions. If this is not achievable, hairless parts should be used.
doi:10.1292/jvms.13-0133
PMCID: PMC3942937  PMID: 23759714
cattle; environment; infrared thermography
13.  Ultrastructural assessment of cellulite morphology: clues to a therapeutic strategy? 
Laser Therapy  2013;22(2):131-136.
Introduction and Aims: Cellulite is a problematic condition affecting mostly women, characterized by a bumpy or nodular skin surface. Recent approaches with laser treatment have offered some promise. The present study sought to identify possible targets for laser treatment or light therapy through an ultrastructural investigation of the condition.
Subjects and Methods: Study subjects comprised 7 healthy Japanese female volunteers (Age range 37–46 yr, average 38.4) with cellulite, graded on the 4-point Nurnberger-Muller cellulite severity scale. Four patients were at grade 2 and 3 at grade 3. Three millimeter punch biopsies were obtained and routinely processed for light and transmission electron microscopy.
Results: Microphotography of specimens from cellulite patients demonstrated the presence of fibrotic septa which divided up larger clusters of adipose tissue into smaller packets, with the septa acting as a tethering system, thus producing the typical dimpling pattern. Ultrastructural findings showed proliferation of collagen and elastic fibers down into the cellulite tissue with compression of capillaries and congestion of arterioles, resulting in poor blood flow.
Conclusions: The histological and ultrastructural findings of cellulite clearly distinguish the condition from simple fat deposition. The remodeling of the fat layer into lobulated packets of lipocytes sequestered by fibrotic septa with a high proportion of elastic fibers would suggest the use of a fiber-based interstitial laser-assisted lipolysis system at an appropriate wavelength which might offer benefits through disruption of the septae through a photomechanical effect and lipolysis of the sequestered lipocytes. This could be followed by a course of near-infrared phototherapy to accelerate clearance of freed lipid and debris and reestablish the vascular system.
doi:10.5978/islsm.13-OR-12
PMCID: PMC3806062  PMID: 24155558
14.  Neural correlates of adaptive social responses to real-life frustrating situations: a functional MRI study 
BMC Neuroscience  2013;14:29.
Background
Frustrating situations are encountered daily, and it is necessary to respond in an adaptive fashion. A psychological definition states that adaptive social behaviors are “self-performing” and “contain a solution.” The present study investigated the neural correlates of adaptive social responses to frustrating situations by assessing the dimension of causal attribution. Based on attribution theory, internal causality refers to one’s aptitudes that cause natural responses in real-life situations, whereas external causality refers to environmental factors, such as experimental conditions, causing such responses. To investigate the issue, we developed a novel approach that assesses causal attribution under experimental conditions. During fMRI scanning, subjects were required to engage in virtual frustrating situations and play the role of protagonists by verbalizing social responses, which were socially adaptive or non-adaptive. After fMRI scanning, the subjects reported their causal attribution index of the psychological reaction to the experimental condition. We performed a correlation analysis between the causal attribution index and brain activity. We hypothesized that the brain region whose activation would have a positive and negative correlation with the self-reported index of the causal attributions would be regarded as neural correlates of internal and external causal attribution of social responses, respectively.
Results
We found a significant negative correlation between external causal attribution and neural responses in the right anterior temporal lobe for adaptive social behaviors.
Conclusion
This region is involved in the integration of emotional and social information. These results suggest that, particularly in adaptive social behavior, the social demands of frustrating situations, which involve external causality, may be integrated by a neural response in the right anterior temporal lobe.
doi:10.1186/1471-2202-14-29
PMCID: PMC3605341  PMID: 23497355
Adaptive social behavior; Causal attribution; Anterior temporal lobe; Integration
15.  Early Vitreous Hemorrhage after Vitrectomy with Preoperative Intravitreal Bevacizumab for Proliferative Diabetic Retinopathy 
Purpose:
To evaluate the effect of preoperative intravitreal bevacizumab (IVB) on surgical procedures, visual prognosis, and postoperative complications, especially postoperative vitreous hemorrhage, in cases with proliferative diabetic retinopathy (PDR).
Materials and Methods:
Seventy-one eyes of 54 consecutive patients (23 eyes of 18 women, 48 eyes of 36 men) were investigated in this study. Twenty-five eyes received IVB one to 30 days before the vitrectomy (Bevacizumab Group) and the other 46 eyes had the vitrectomy alone (Control Group). The surgical procedures, best-corrected visual acuities at baseline, 1, 3, and 6 months after the vitrectomy, and postoperative complications in the Bevacizumab Group were compared to the Control Group.
Results:
The patients were significantly younger in the Bevacizumab Group compared to the Control Group (P = 0.008). The incidence of preoperative vitreous hemorrhage, tractional retinal detachment, and iris neovascularization was significantly higher in the Bevacizumab Group than in the Control Group (P = 0.017, 0.041, and 0.018, respectively). The surgical procedures performed and the visual acuity at all time points was not significantly different between groups (P > 0.05, all comparisons). The incidence of early (≤4 weeks) postoperative vitreous hemorrhage was significantly higher in the Bevacizumab Group (27%) than in the Control Group (7%; P = 0.027) although the rate of late (>4 weeks) postoperative vitreous hemorrhage was not significantly different between groups (P > 0.05).
Conclusion:
Vitrectomy with preoperative IVB may have no detrimental effect on surgical procedures and achieves the surgical outcomes for repair of PDR equal to vitrectomy alone despite the obvious selection bias of the patients in this study. However, special monitoring is highly recommended for early postoperative vitreous hemorrhage because bevacizumab in the vitreous may be washed out during vitrectomy.
doi:10.4103/0974-9233.106387
PMCID: PMC3617529  PMID: 23580852
Bevacizumab; Postoperative Vitreous Haemorrhage; Proliferative Diabetic Retinopathy; Vitrectomy; Vascular Endothelial Growth Factor
16.  Low Fluence Q-Switched Nd: YAG Laser Toning and Q-Switched Ruby Laser in the Treatment of Melasma:A Comparative Split-Face Ultrastructural Study 
Laser Therapy  2012;21(1):15-21.
Background: Melasma still presents as a difficult entity to treat, especially in the Asian skin phe-notype. Recently laser toning with the Q-switched Nd:YAG has attracted attention. The present study investigated the efficacy of Q-switched Nd:YAG laser toning for melasma, with a histopathological comparison with the Q-switched ruby laser.
Subjects and Methods: Eight Japanese females (41–57 yr, mean 52.5 yr) with Fitzpatrick skin type III and bilateral melasma participated in the study. One half of each subject's face (randomly chosen) was treated with Q-switched 1064 nm Nd:YAG laser toning (pulse width 5–20 ns; spot size, 6 mm diameter; fluence, 3.0 J/cm2, 5–7 passes, once/week, 4 weeks: QS:YAG group), and the contralateral half with a single treatment using a Q-switched ruby laser (694.5 nm, pulse width 20 ns, spot size 4 mm diameter; fluence 4.0 J/cm2, 1 pass with approximately 20% overlap: QS:Ruby group). Skin biopsies were taken immediately after the 4th Nd:YAG session and the single ruby session, and histopathological comparison was performed with light- and transmission electron microscopy (TEM).
Results: Improvement in melasma pigmentation was seen in both the QS:YAG- and QS:Ruby-treat-ed sides, and this was well-maintained in the QS:YAG group. Ultrastructurally, melanin granules were destroyed in both groups, but there was considerably more morphological epidermal and dermal damage in the QS:Ruby specimens compared with minimal epidermal disruption and cellular damage in the QS:YAG specimens.
Conclusions: Q-switched 1064 nm Nd:YAG laser toning offered superior results in the treatment of melasma in the Japanese skin type compared with the Q-switched ruby laser, both ultrastructurally with less immediately post-treatment cellular damage and macroscopically, and a longer recurrence-free interval.
doi:10.5978/islsm.12-OR-03
PMCID: PMC3944591  PMID: 24610976
Q-switched laser; laser toning; selective photothermolysis; subcellular selective photothermolysis; transmission electron microscopy
17.  Heparan Sulfate Regulates Intraretinal Axon Pathfinding by Retinal Ganglion Cells 
Retinal ganglion cell (RGC) axon projection toward the optic nerve head requires the expression of heparan sulfate (HS) in the neural retina, suggesting that HS in the retina functions as an essential modulator of Netrin-1– and Slit-mediated intraretinal RGC axon guidance.
Purpose.
Heparan sulfate (HS) is abundantly expressed in the developing neural retina; however, its role in the intraretinal axon guidance of retinal ganglion cells (RGCs) remains unclear. In this study, the authors examined whether HS was essential for the axon guidance of RGCs toward the optic nerve head.
Methods.
The authors conditionally ablated the gene encoding the exostosin-1 (Ext1) enzyme, using the dickkopf homolog 3 (Dkk3)-Cre transgene, which disrupted HS expression in the mouse retina during directed pathfinding by RGC axons toward the optic nerve head. In situ hybridization, immunohistochemistry, DiI tracing, binding assay, and retinal explant assays were performed to evaluate the phenotypes of the mutants and the roles of HS in intraretinal axon guidance.
Results.
Despite no gross abnormality in RGC distribution, the mutant RGC axons exhibited severe intraretinal guidance errors, including optic nerve hypoplasia, ectopic axon penetration through the full thickness of the neural retina and into the subretinal space, and disturbance of the centrifugal projection of RGC axons toward the optic nerve head. These abnormal phenotypes shared similarities with the RGC axon misguidance caused by mutations of genes encoding Netrin-1 and Slit-1/2. Explant assays revealed that the mutant RGCs exhibited disturbed Netrin-1–dependent axon outgrowth and Slit-2–dependent repulsion.
Conclusions.
The present study demonstrated that RGC axon projection toward the optic nerve head requires the expression of HS in the neural retina, suggesting that HS in the retina functions as an essential modulator of Netrin-1 and Slit-mediated intraretinal RGC axon guidance.
doi:10.1167/iovs.11-7559
PMCID: PMC3176022  PMID: 21743013
18.  Epitope Analysis of Cerebrospinal Fluid IgG in Japanese Multiple Sclerosis Patients Using Phage Display Method 
To investigate the antigen recognized by cerebrospinal fluid (CSF) high affinity IgG in patients with multiple sclerosis (MS), the phage display method was applied to the CSF from 15 MS and 10 control patients. Peptide sequences recognized by MS and control CSF IgG were individual specific, and no common motif was found. Peptide sequences frequently showed homology to various kinds of amino acid sequences of ubiquitous viruses such as epstein barr virus (EBV) and herpes simplex virus (HSV), although the frequency was not specific to MS patients. MS CSF IgG may recognize various types of ubiquitous viral antigen and may be increased by a bystander response.
doi:10.1155/2011/353417
PMCID: PMC3216389  PMID: 22132333
19.  Analysis of Transcriptional Regulatory Pathways of Photoreceptor Genes by Expression Profiling of the Otx2-Deficient Retina 
PLoS ONE  2011;6(5):e19685.
In the vertebrate retina, the Otx2 transcription factor plays a crucial role in the cell fate determination of both rod and cone photoreceptors. We previously reported that Otx2 conditional knockout (CKO) mice exhibited a total absence of rods and cones in the retina due to their cell fate conversion to amacrine-like cells. In order to investigate the entire transcriptome of the Otx2 CKO retina, we compared expression profile of Otx2 CKO and wild-type retinas at P1 and P12 using microarray. We observed that expression of 101- and 1049-probe sets significantly decreased in the Otx2 CKO retina at P1 and P12, respectively, whereas, expression of 3- and 4149-probe sets increased at P1 and P12, respectively. We found that expression of genes encoding transcription factors involved in photoreceptor development, including Crx, Nrl, Nr2e3, Esrrb, and NeuroD, was markedly down-regulated in the Otx2 CKO at both P1 and P12. Furthermore, we identified three human retinal disease loci mapped in close proximity to certain down-regulated genes in the Otx2 CKO retina including Ccdc126, Tnfsf13 and Pitpnm1, suggesting that these genes are possibly responsible for these diseases. These transcriptome data sets of the Otx2 CKO retina provide a resource on developing rods and cones to further understand the molecular mechanisms underlying photoreceptor development, function and disease.
doi:10.1371/journal.pone.0019685
PMCID: PMC3094341  PMID: 21602925
20.  Comparative transcriptomic characterization of aluminum, sodium chloride, cadmium and copper rhizotoxicities in Arabidopsis thaliana 
BMC Plant Biology  2009;9:32.
Background
Rhizotoxic ions in problem soils inhibit nutrient and water acquisition by roots, which in turn leads to reduced crop yields. Previous studies on the effects of rhizotoxic ions on root growth and physiological functions suggested that some mechanisms were common to all rhizotoxins, while others were more specific. To understand this complex system, we performed comparative transcriptomic analysis with various rhizotoxic ions, followed by bioinformatics analysis, in the model plant Arabidopsis thaliana.
Results
Roots of Arabidopsis were treated with the major rhizotoxic stressors, aluminum (Al) ions, cadmium (Cd) ions, copper (Cu) ions and sodium (NaCl) chloride, and the gene expression responses were analyzed by DNA array technology. The top 2.5% of genes whose expression was most increased by each stressor were compared with identify common and specific gene expression responses induced by these stressors. A number of genes encoding glutathione-S-transferases, peroxidases, Ca-binding proteins and a trehalose-synthesizing enzyme were induced by all stressors. In contrast, gene ontological categorization identified sets of genes uniquely induced by each stressor, with distinct patterns of biological processes and molecular function. These contained known resistance genes for each stressor, such as AtALMT1 (encoding Al-activated malate transporter) in the Al-specific group and DREB (encoding dehydration responsive element binding protein) in the NaCl-specific group. These gene groups are likely to reflect the common and differential cellular responses and the induction of defense systems in response to each ion. We also identified co-expressed gene groups specific to rhizotoxic ions, which might aid further detailed investigation of the response mechanisms.
Conclusion
In order to understand the complex responses of roots to rhizotoxic ions, we performed comparative transcriptomic analysis followed by bioinformatics characterization. Our analyses revealed that both general and specific genes were induced in Arabidopsis roots exposed to various rhizotoxic ions. Several defense systems, such as the production of reactive oxygen species and disturbance of Ca homeostasis, were triggered by all stressors, while specific defense genes were also induced by individual stressors. Similar studies in different plant species could help to clarify the resistance mechanisms at the molecular level to provide information that can be utilized for marker-assisted selection.
doi:10.1186/1471-2229-9-32
PMCID: PMC2666732  PMID: 19309492
21.  Time-resolved metabolomics reveals metabolic modulation in rice foliage 
BMC Systems Biology  2008;2:51.
Background
To elucidate the interaction of dynamics among modules that constitute biological systems, comprehensive datasets obtained from "omics" technologies have been used. In recent plant metabolomics approaches, the reconstruction of metabolic correlation networks has been attempted using statistical techniques. However, the results were unsatisfactory and effective data-mining techniques that apply appropriate comprehensive datasets are needed.
Results
Using capillary electrophoresis mass spectrometry (CE-MS) and capillary electrophoresis diode-array detection (CE-DAD), we analyzed the dynamic changes in the level of 56 basic metabolites in plant foliage (Oryza sativa L. ssp. japonica) at hourly intervals over a 24-hr period. Unsupervised clustering of comprehensive metabolic profiles using Kohonen's self-organizing map (SOM) allowed classification of the biochemical pathways activated by the light and dark cycle. The carbon and nitrogen (C/N) metabolism in both periods was also visualized as a phenotypic linkage map that connects network modules on the basis of traditional metabolic pathways rather than pairwise correlations among metabolites. The regulatory networks of C/N assimilation/dissimilation at each time point were consistent with previous works on plant metabolism. In response to environmental stress, glutathione and spermidine fluctuated synchronously with their regulatory targets. Adenine nucleosides and nicotinamide coenzymes were regulated by phosphorylation and dephosphorylation. We also demonstrated that SOM analysis was applicable to the estimation of unidentifiable metabolites in metabolome analysis. Hierarchical clustering of a correlation coefficient matrix could help identify the bottleneck enzymes that regulate metabolic networks.
Conclusion
Our results showed that our SOM analysis with appropriate metabolic time-courses effectively revealed the synchronous dynamics among metabolic modules and elucidated the underlying biochemical functions. The application of discrimination of unidentified metabolites and the identification of bottleneck enzymatic steps even to non-targeted comprehensive analysis promise to facilitate an understanding of large-scale interactions among components in biological systems.
doi:10.1186/1752-0509-2-51
PMCID: PMC2442833  PMID: 18564421
22.  Functional Roles of Otx2 Transcription Factor in Postnatal Mouse Retinal Development▿ † 
Molecular and Cellular Biology  2007;27(23):8318-8329.
We previously reported that Otx2 is essential for photoreceptor cell fate determination; however, the functional role of Otx2 in postnatal retinal development is still unclear although it has been reported to be expressed in retinal bipolar cells and photoreceptors at postnatal stages. In this study, we first examined the roles of Otx2 in the terminal differentiation of photoreceptors by analyzing Otx2; Crx double-knockout mice. In Otx2+/−; Crx−/− retinas, photoreceptor degeneration and downregulation of photoreceptor-specific genes were much more prominent than in Crx−/− retinas, suggesting that Otx2 has a role in the terminal differentiation of the photoreceptors. Moreover, bipolar cells decreased in the Otx2+/−; Crx−/− retina, suggesting that Otx2 is also involved in retinal bipolar-cell development. To further investigate the role of Otx2 in bipolar-cell development, we generated a postnatal bipolar-cell-specific Otx2 conditional-knockout mouse line. Immunohistochemical analysis of this line showed that the expression of protein kinase C, a marker of mature bipolar cells, was significantly downregulated in the retina. Electroretinograms revealed that the electrophysiological function of retinal bipolar cells was impaired as a result of Otx2 ablation. These data suggest that Otx2 plays a functional role in the maturation of retinal photoreceptor and bipolar cells.
doi:10.1128/MCB.01209-07
PMCID: PMC2169187  PMID: 17908793
23.  Evaluation of Metabolic Alteration in Transgenic Rice Overexpressing Dihydroflavonol-4-reductase 
Annals of Botany  2006;98(4):819-825.
• Background and Aims Previous studies have shown that transgenic rice plants overexpressing YK1, which possesses dihydroflavonol-4-reductase (DFR) activity, showed biotic and abiotic stress tolerance. High throughput profiles of metabolites have also been shown in such transgenic plants by Fourier transform ion cyclotron mass spectrometry. In this study, capillary electrophoresis mass spectrometry analysis (CE/MS) was employed to identify precise metabolites such as organic acids, amino acids and sugars.
• Methods Using CE/MS, we analysed several metabolites of glycolysis, the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway. In addition, the concentrations of sugars and ion were quantified.
• Key Results In YK1 (DFR)-overexpressing plants, the concentrations of cis-aconitate, isocitrate and 2-oxoglutarate were higher in leaves, whereas those of fructose-1,6-bisphosphate and glyceraldehyde-3-phosphate were lower in roots. In seeds, the amounts of free amino acids and metals were altered, whereas sugars in seeds were kept constant. In YK1 calli, an approx. 3-fold increase in glutathione was observed, whereas the activities of glutathione peroxidase and glutathione reductase were concomitantly increased.
• Conclusions The overexpression of YK1 (DFR) was associated with slight changes in the amounts of several metabolites analysed in whole plants, whilst glutathione derivatives were substantially increased in suspension-cultured cells.
doi:10.1093/aob/mcl162
PMCID: PMC2806160  PMID: 16849376
Metabolome; dihydroflavonol-4-reductase; capillary electrophoresis; mass spectrometry; rice; Oryza sativa
24.  Cooperation of Six and Eya in Activation of Their Target Genes through Nuclear Translocation of Eya 
Molecular and Cellular Biology  1999;19(10):6815-6824.
Drosophila sine oculis and eyes absent genes synergize in compound-eye formation. The murine homologues of these genes, Six and Eya, respectively, show overlapping expression patterns during development. We hypothesized that Six and Eya proteins cooperate to regulate their target genes. Cotransfection assays were performed with various combinations of Six and Eya to assess their effects on a potential natural target, myogenin promoter, and on a synthetic promoter, the thymidine kinase gene promoter fused to multimerized Six4 binding sites. A clear synergistic activation of these promoters was observed in certain combinations of Six and Eya. To investigate the molecular basis for the cooperation, we first examined the intracellular distribution of Six and Eya proteins in transfected COS7 cells. Coexpression of Six2, Six4, or Six5 induced nuclear translocation of Eya1, Eya2, and Eya3, which were otherwise distributed in the cytoplasm. In contrast, coexpression of Six3 did not result in nuclear localization of any Eya proteins. Six and Eya proteins were coimmunoprecipitated from nuclear extracts prepared from cotransfected COS7 cells and from rat liver. Six domain and homeodomain, two evolutionarily conserved domains among various Six proteins, were necessary and sufficient for the nuclear translocation of Eya. In contrast, the Eya domain, a conserved domain among Eya proteins, was not sufficient for the translocation. A specific interaction between the Six domain and homeodomain of Six4 and Eya2 was observed by yeast two-hybrid analysis. Our results suggest that transcription regulation of certain target genes by Six proteins requires cooperative interaction with Eya proteins: complex formation through direct interaction and nuclear translocation of Eya proteins. This implies that the synergistic action of Six and Eya is conserved in the mouse and is mediated through cooperative activation of their target genes.
PMCID: PMC84678  PMID: 10490620
25.  A Protein Critical for a Theiler’s Virus-Induced Immune System-Mediated Demyelinating Disease Has a Cell Type-Specific Antiapoptotic Effect and a Key Role in Virus Persistence 
Journal of Virology  1998;72(11):8605-8612.
TO subgroup strains of Theiler’s murine encephalomyelitis virus (TMEV) induce a persistent central nervous system infection and demyelinating disease in mice. This disease serves as an experimental model of multiple sclerosis (MS) because the two diseases have similar inflammatory white matter pathologies and because the immune system appears to mediate demyelination in both processes. We previously reported (H. H. Chen, W. P. Wong, L. Zhang, P. L. Ward, and R. P. Roos, Nat. Med. 1:927–931, 1995) that TO subgroup strains use an alternative initiation codon (in addition to the AUG used to synthesize the picornavirus polyprotein from one long open reading frame) to translate L*, a novel protein that is out of frame with the polyprotein and which plays a key role in the demyelinating disease. We now demonstrate that L* has antiapoptotic activity in macrophage cells and is critical for virus persistence. The antiapoptotic action of L* as well as the differential translation of L* and virion capsid proteins may foster virus persistence in macrophages and interfere with virus clearance. The regulation of apoptotic activity in inflammatory cells may be important in the pathogenesis of TMEV-induced demyelinating disease as well as MS.
PMCID: PMC110271  PMID: 9765399

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