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1.  Azathioprine is essential following cyclosporine for patients with steroid-refractory ulcerative colitis 
AIM: To evaluate long-term prognosis following cyclosporine treatment by examining the rate of surgery avoidance among cyclosporine responders.
METHODS: We retrospectively reviewed clinical records for 29 patients diagnosed with severe steroid-refractory ulcerative colitis in our hospital from August 1997 to August 2008 and treated with cyclosporine by continuous intravenous infusion. All patients were treated with intravenous corticosteroids for more than 5 d prior to cyclosporine therapy. Administration was continued for up to 21 d under serum monitoring to maintain cyclosporine levels between 400 and 600 ng/mL. Clinical activity was assessed before and after cyclosporine therapy using the clinical activity index score, with a reduction of ≥ 5 considered to indicate a response. Among responders, we defined cases not requiring surgery for more than 5 years as exhibiting long-term efficacy of cyclosporine. Factors considered to be possibly predictive of long-term efficacy of cyclosporine were sex, age, disease duration, clinical activity index score, C-reactive protein level, hemoglobin level, disease extent, endoscopic findings, and clinical course.
RESULTS: Cyclosporine was not discontinued due to side effects in any patient. Nineteen (65.5%) of 29 patients were considered responders. A statistically significant (P = 0.004) inverse association was observed between an endoscopic finding of “mucosal bleeding” and responsive cases. Fifteen (9 males, 6 females) of these 19 patients were followed for 5 years or more, of whom 9 (60%) exhibited long-term efficacy of cyclosporine. Of the 10 non-responders, 9 (90%) underwent surgery within 6 mo of cyclosporine therapy. None of the following factors had a significant impact on the long-term efficacy of cyclosporine: sex, age, duration of disease, clinical activity index score, C-reactive protein level, hemoglobin level, extent of disease, endoscopic findings, or clinical course. In contrast, a significant association was observed for maintenance therapy with azathioprine after cyclosporine therapy (P = 0.0014).
CONCLUSION: Maintenance therapy with azathioprine might improve the long-term efficacy of continuously infused cyclosporine for severe steroid-refractory ulcerative colitis patients.
doi:10.3748/wjg.v21.i1.254
PMCID: PMC4284343  PMID: 25574099
Ulcerative colitis; Cyclosporine; Maintenance therapy; Azathioprine; Long-term prognosis
2.  Retinol Dehydrogenase (RDH12) Protects Photoreceptors from Light-induced Degeneration in Mice*S 
The Journal of biological chemistry  2006;281(49):37697-37704.
RDH12 has been suggested to be one of the retinol dehydrogenases (RDH) involved in the vitamin A recycling system (visual cycle) in the eye. Loss of function mutations in the RDH12 gene were recently reported to be associated with autosomal recessive childhood-onset severe retinal dystrophy. Here we show that RDH12 localizes to the photoreceptor inner segments and that deletion of this gene in mice slows the kinetics of all-trans-retinal reduction, delaying dark adaptation. However, accelerated 11-cis-retinal production and increased susceptibility to light-induced photoreceptor apoptosis were also observed in Rdh12−/− mice, suggesting that RDH12 plays a unique, nonredundant role in the photoreceptor inner segments to regulate the flow of retinoids in the eye. Thus, severe visual impairments of individuals with null mutations in RDH12 may likely be caused by light damage1.
doi:10.1074/jbc.M608375200
PMCID: PMC4124513  PMID: 17032653
3.  Effects of Potent Inhibitors of the Retinoid Cycle on Visual Function and Photoreceptor Protection from Light Damage in MiceS 
Molecular pharmacology  2006;70(4):1220-1229.
Regeneration of the chromophore 11-cis-retinal is essential for the generation of light-sensitive visual pigments in the vertebrate retina. A deficiency in 11-cis-retinal production leads to congenital blindness in humans; however, a buildup of the photoisomerized chromophore can also be detrimental. Such is the case when the photoisomerized all-trans-retinal is produced but cannot be efficiently cleared from the internal membrane of the outer segment discs. Sustained increase of all-trans-retinal can lead to the formation of toxic condensation products in the eye. Thus, there is a need for potent, selective inhibitors that can regulate the flux of retinoids through the metabolism pathway termed the visual (retinoid) cycle. Here we systematically study the effects of the most potent inhibitor of this cycle, retinylamine (Ret-NH2), on visual function in mice. Prolonged, sustainable, but reversible suppression of the visual function was observed by Ret-NH2 as a result of its storage in a prodrug form, N-retinylamides. Direct comparison of other inhibitors such as fenretinide and 13-cis-retinoic acid showed multiple advantages of Ret-NH2 and its amides, including a higher potency, specificity, and lower transcription activation. Our results also revealed that mice treated with Ret-NH2 were completely resistant to the light-induced retina damage. As an experimental tool, Ret-NH2 allows the replacement of the native chromophore with synthetic analogs in wild-type mice to better understand the function of the chromophore in the activation of rhodopsin and its metabolism through the retinoid cycle.
doi:10.1124/mol.106.026823
PMCID: PMC4106413  PMID: 16837623
4.  Expression of B7-H3, a Potential Factor of Tumor Immune Evasion in Combination with the Number of Regulatory T Cells, Affects Against Recurrence-Free Survival in Breast Cancer Patients 
Annals of Surgical Oncology  2014;21(Suppl 4):546-554.
Background
In the tumor microenvironment, factors inhibiting the targeting of cancer cells by activated T cells have recently been noted. B7-H3 belongs to the B7 superfamily of immune regulatory ligands and plays an important role in the adaptive immune response of co-inhibitory/stimulatory factors in regulating T cells. However, the degree to which B7-H3 directly affects tumor immune evasion mechanisms remains unclear, particularly in patients with breast cancer. Regulatory T cells (Tregs) are known as a key player in the inhibition of immune mechanisms. The present study demonstrated that expression of B7-H3 on tumor cells and the number of Tregs in the tumor microenvironment independently affected prognosis in breast cancer patients.
Methods
We immunohistochemically investigated the presence of B7-H3 and forkhead box P3 (Foxp3)-positive Tregs in pathological specimens from 90 patients with breast cancer.
Results
Positive B7-H3 expression was associated with shorter recurrence-free survival (RFS) (p = 0.014). A higher percentage of Foxp3-positive cells also correlated with shorter RFS (p = 0.039). Multivariate analysis showed B7-H3 as an independent factor on RFS. Foxp3 expression in tumor-infiltrating lymphocytes (TILs) correlated significantly with larger tumor size (>2 cm), expression of human epidermal growth factor receptor 2 (HER2), and higher nuclear grade (p = 0.003, p < 0.001, p = 0.001, respectively). No correlation was identified between expression of B7-H3 and the percentage of Foxp3-positive TILs.
Conclusions
B7-H3 and Foxp3 can be regarded as markers of poor prognosis in breast cancer. These expressions were not correlated, suggesting that B7-H3 expression plays an independent role in tumor immune evasion, regardless of Tregs.
doi:10.1245/s10434-014-3564-2
PMCID: PMC4236607  PMID: 24562936
5.  Systems pharmacology identifies drug targets for Stargardt disease–associated retinal degeneration 
The Journal of Clinical Investigation  2013;123(12):5119-5134.
A systems pharmacological approach that capitalizes on the characterization of intracellular signaling networks can transform our understanding of human diseases and lead to therapy development. Here, we applied this strategy to identify pharmacological targets for the treatment of Stargardt disease, a severe juvenile form of macular degeneration. Diverse GPCRs have previously been implicated in neuronal cell survival, and crosstalk between GPCR signaling pathways represents an unexplored avenue for pharmacological intervention. We focused on this receptor family for potential therapeutic interventions in macular disease. Complete transcriptomes of mouse and human samples were analyzed to assess the expression of GPCRs in the retina. Focusing on adrenergic (AR) and serotonin (5-HT) receptors, we found that adrenoceptor α 2C (Adra2c) and serotonin receptor 2a (Htr2a) were the most highly expressed. Using a mouse model of Stargardt disease, we found that pharmacological interventions that targeted both GPCR signaling pathways and adenylate cyclases (ACs) improved photoreceptor cell survival, preserved photoreceptor function, and attenuated the accumulation of pathological fluorescent deposits in the retina. These findings demonstrate a strategy for the identification of new drug candidates and FDA-approved drugs for the treatment of monogenic and complex diseases.
doi:10.1172/JCI69076
PMCID: PMC3859412  PMID: 24231350
6.  Retinal Photodamage Mediated by All-trans-retinal† 
Photochemistry and photobiology  2012;88(6):1309-1319.
Accumulation of all-trans-retinal (all-trans-RAL), reactive vitamin A aldehyde, is one of the key factors in initiating retinal photodamage. This photodamage is characterized by progressive retinal cell death evoked by light exposure in both an acute and chronic fashion. Photo-activated rhodopsin releases all-trans-RAL which is subsequently transported by ATP–binding cassette transporter 4 and reduced to all-trans-retinol by all-trans-retinol dehydrogenases located in photoreceptor cells. Any interruptions in the clearing of all-trans-RAL in the photoreceptors can cause an accumulation of this reactive aldehyde and its toxic condensation products. This accumulation may result in the manifestation of retinal dystrophy including human retinal degenerative diseases such as Stargardt’s disease and age-related macular degeneration. Here, we discuss the mechanisms of all-trans-RAL clearance in photoreceptor cells by sequential enzymatic reactions, the visual (retinoid) cycle, and potential molecular pathways of retinal photodamage. We also review recent imaging technologies to monitor retinal health status as well as novel therapeutic strategies preventing all-trans-RAL-associated retinal photodamage.
doi:10.1111/j.1751-1097.2012.01143.x
PMCID: PMC3424387  PMID: 22428905
7.  Importance of appropriate pharmaceutical management in pregnant women with ulcerative colitis 
BMC Research Notes  2013;6:210.
Background
Ulcerative colitis (UC) often occurs in women of childbearing age. Compared to Western countries, however, few studies have investigated the impact of UC on the progress of pregnancy in Asian populations.
Methods
We retrospectively examined 91 pregnancies in 64 patients with UC experienced at our hospital and related institutions from 1991 to 2011, focusing on the relationship between the progression of UC during pregnancy, progress of the pregnancy itself, and the treatment of UC.
Results
In 80 of 91 pregnancies the patient had already been diagnosed with UC at the time she became pregnant, of whom 31 (38.8%) experienced exacerbation during pregnancy. Regarding severity, moderate or severe active-stage disease during pregnancy was seen in 13.7% of those who had been in remission at the onset of pregnancy versus 58.6% of those who had been in the active stage at onset (OR 8.9: 95%CI 3.0~26.4; P<0.01). The incidence of miscarriage or abortion was 9.8% in pregnancies in which UC was in remission at onset versus 31% in those in which it was in the active stage at onset (OR 4.1: 95%CI 1.2~13.9; P=0.02). Among patients, 62.5% were receiving pharmaceutical treatment at onset of pregnancy. Exacerbation during pregnancy occurred in 26.5% of the group who continued to receive the same treatment during pregnancy versus 56.3% of those with a dose decrease or discontinuation after onset (OR 3.6: 95%CI 1.0~12.4; P=0.04).
Conclusions
UC patients wishing to conceive should do so when in remission and continue appropriate pharmaceutical treatment during pregnancy.
doi:10.1186/1756-0500-6-210
PMCID: PMC3680032  PMID: 23706098
Ulcerative colitis; Pregnancy; Clinical course; Treatment
8.  Retinal cone and rod photoreceptor cells exhibit differential susceptibility to light–induced damage 
Journal of Neurochemistry  2012;121(1):146-156.
All-trans-retinal and its condensation-products can cause retinal degeneration in a light–dependent manner and contribute to the pathogenesis of human macular diseases such as Stargardt’s disease and age–related macular degeneration (AMD). Although these toxic retinoid by–products originate from rod and cone photoreceptor cells, the contribution of each cell type to light–induced retinal degeneration is unknown. Here the primary objective was to learn whether rods or cones are more susceptible to light–induced, all–trans–retinal–mediated damage. Previously, we reported that mice lacking enzymes that clear all–trans–retinal from the retina, ATP–binding cassette transporter 4 (ABCA4) and retinol dehydrogenase 8 (RDH8), manifested light-induced retinal dystrophy. We first examined early-stage-AMD patients and found retinal degenerative changes in rod-rich rather than cone-rich regions of the macula. We then evaluated transgenic mice with rod–only and cone–like–only retinas in addition to progenies of such mice inbred with Rdh8−/− Abca4−/− mice. Of all these strains, Rdh8−/− Abca4−/− mice with a mixed rod–cone population showed the most severe retinal degeneration under regular cyclic light conditions. Intense light exposure induced acute retinal damage in Rdh8−/− Abca4−/− and rod–only mice but not cone–like–only mice. These findings suggest that progression of retinal degeneration in Rdh8-/- Abca4-/- mice is affected by differential vulnerability of rods and cones to light.
doi:10.1111/j.1471-4159.2012.07647.x
PMCID: PMC3303932  PMID: 22220722
visual cycle; photoreceptor; retinoid; retina; Stargardt’s disease; age-related macular degeneration
9.  Mandatory Palliative Care Education for Surgical Residents: Initial Focus on Teaching Pain Management 
Background
Knowledge concerning palliative care and the associated skills, including effective pain control, is essential for surgeons who treat cancer patients in daily practice. This study focuses on a palliative care training course that has been mandatorily conducted for all surgical residents of our hospital since 2009.
Methods
We evaluated the effectiveness of our mandatory palliative care training course by conducting a retrospective study of the patients' medical records and participants' questionnaire results and discussed the importance of palliative care education for surgical residents.
Results
All 12 surgical residents who participated in the course in 2009 had graduated 4–9 years back. They were assigned to look after a total of 92 cases (average, 7.66 cases per resident) during the course. The purpose of care in most cases (92.3%) was to mitigate pain. Introducing analgesic adjuvants such as gabapentin or amitriptyline accounted for the largest part of initial interventions (23.9%) aimed at controlling cancer pain, followed by changes in route of administration or doses of prior opioid analgesics (21.7%). Interventions with opioid analgesics were conducted most frequently (47.7%). The overall pain improvement rate was 89.1%. We used a questionnaire after the course to evaluate its effectiveness.
Conclusions
The surgical residents stated that it was a meaningful course through which they gained practical knowledge on palliative care and that the experience would change their approach to home care.
doi:10.1093/jjco/hys205
PMCID: PMC3559015  PMID: 23275645
palliative care; surgeon; postgraduate training
10.  Primary amines protect against retinal degeneration in mouse models of retinopathies 
Nature chemical biology  2011;8(2):170-178.
Vertebrate vision is initiated by photoisomerization of the visual pigment chromophore, 11-cis-retinal, and is maintained by continuous regeneration of this retinoid through a series of reactions termed the retinoid cycle. However, toxic side reaction products, especially those involving reactive aldehyde groups of the photoisomered product, all-trans-retinal, can cause severe retinal pathology. Here we lowered peak concentrations of free all-trans-retinal with primary amine-containing FDA-approved drugs that did not inhibit chromophore regeneration in mouse models of retinal degeneration. Schiff base adducts between all-trans-retinal and these amines were identified by mass spectrometry. Adducts were observed in mouse eyes only when an experimental drug protected the retina from degeneration in both short-term and long-term treatment experiments. This study demonstrates a molecular basis of all-trans-retinal-induced retinal pathology and identifies an assemblage of FDA-approved compounds with protective effects against this pathology in a mouse model that displays features of Stargardt’s and age-related retinal degeneration.
doi:10.1038/nchembio.759
PMCID: PMC3518042  PMID: 22198730
Photoreceptor cells; A2E; RPE; retina; Stargardt’s disease; age-related macular degeneration; retinal degeneration; retinal condensation products
11.  Presynaptic Selectivity of a Ligand for Serotonin 1A Receptors Revealed by In Vivo PET Assays of Rat Brain 
PLoS ONE  2012;7(8):e42589.
A novel investigational antidepressant with high affinity for the serotonin transporter and the serotonin 1A (5-HT1A) receptor, called Wf-516 (structural formula: (2S)-1-[4-(3,4-dichlorophenyl)piperidin-1-yl]-3-[2-(5-methyl-1,3,4-oxadiazol-2-yl)benzo[b]furan-4-yloxy]propan-2-ol monohydrochloride), has been found to exert a rapid therapeutic effect, although the mechanistic basis for this potential advantage remains undetermined. We comparatively investigated the pharmacokinetics and pharmacodynamics of Wf-516 and pindolol by positron emission tomographic (PET) and autoradiographic assays of rat brains in order to elucidate their molecular interactions with presynaptic and postsynaptic 5-HT1A receptors. In contrast to the full receptor occupancy by pindolol in PET measurements, the binding of Wf-516 to 5-HT1A receptors displayed limited capacity, with relatively high receptor occupancy being achieved in regions predominantly containing presynaptic receptors. This selectivity was further proven by PET scans of neurotoxicant-treated rats deficient in presynaptic 5-HT1A receptors. In addition, [35S]guanosine 5′-O-[γ-thio]triphosphate autoradiography indicated a partial agonistic ability of Wf-516 for 5-HT1A receptors. This finding has lent support to reports that diverse partial agonists for 5-HT1A receptors exert high sensitivity for presynaptic components. Thus, the present PET data suggest a relatively high capacity of presynaptic binding sites for partial agonists. Since our in vitro and ex vivo autoradiographies failed to illustrate these distinct features of Wf-516, in vivo PET imaging is considered to be, thus far, the sole method capable of pharmacokinetically demonstrating the unique actions of Wf-516 and similar new-generation antidepressants.
doi:10.1371/journal.pone.0042589
PMCID: PMC3413639  PMID: 22880045
12.  LRF is an essential downstream target of GATA1 in erythroid development and regulates BIM-dependent apoptosis 
Developmental cell  2009;17(4):527-540.
SUMMARY
GATA-1-dependent transcription is essential for erythroid differentiation and maturation. Suppression of programmed cell death is also thought to be critical for this process; however, the link between these two features of erythropoiesis has remained elusive. Here, we show that the POZ-Krüppel family transcription factor, LRF (also known as Zbtb7a/Pokemon), is a direct target of GATA1 and plays an essential anti-apoptotic role during terminal erythroid differentiation. We find that loss of Lrf leads to lethal anemia in embryos, due to increased apoptosis of late stage erythroblasts. This programmed cell death is Arf- and p53-independent and is instead mediated by up-regulation of the pro-apoptotic factor Bim. We identify Lrf as a direct repressor of Bim transcription. In strong support of this mechanism, genetic Bim-loss delays the lethality of Lrf-deficient embryos and rescues their anemia-phenotype. Thus, our data defines a key transcriptional cascade for effective erythropoiesis, whereby GATA-1 suppresses BIM-mediated apoptosis via LRF.
doi:10.1016/j.devcel.2009.09.005
PMCID: PMC3134301  PMID: 19853566
13.  The LRF transcription factor regulates mature B cell development and the germinal center response in mice 
The Journal of Clinical Investigation  2011;121(7):2583-2598.
B cells play a central role in immune system function. Deregulation of normal B cell maturation can lead to the development of autoimmune syndromes as well as B cell malignancies. Elucidation of the molecular features of normal B cell development is important for the development of new target therapies for autoimmune diseases and B cell malignancies. Employing B cell–specific conditional knockout mice, we have demonstrated here that the transcription factor leukemia/lymphoma-related factor (LRF) forms an obligate dimer in B cells and regulates mature B cell lineage fate and humoral immune responses via distinctive mechanisms. Moreover, LRF inactivation in transformed B cells attenuated their growth rate. These studies identify what we believe to be a new key factor for mature B cell development and provide a rationale for targeting LRF dimers for the treatment of autoimmune diseases and B cell malignancies.
doi:10.1172/JCI45682
PMCID: PMC3223838  PMID: 21646720
14.  Noninvasive multi–photon fluorescence microscopy resolves retinol and retinal–condensation products in mouse eyes 
Nature medicine  2010;16(12):1444-1449.
Multi–photon excitation fluorescence microscopy (MPM) can image certain molecular processes in vivo. In the eye, fluorescent retinyl esters in sub–cellular structures called retinosomes mediate regeneration of the visual chromophore, 11–cis–retinal, by the visual cycle. But harmful fluorescent condensation products were also identified previously. We report that in wild type mice, excitation with λ ~730 nm identified retinosomes in the retinal pigment epithelium, whereas excitation with λ ~910 nm revealed at least one additional retinal fluorophore. The latter fluorescence was absent in eyes of genetically modified mice lacking a functional visual cycle, but accentuated in eyes of older WT mice and mice with defective clearance of all–trans–retinal, an intermediate in the visual cycle. MPM, a noninvasive imaging modality that facilitates concurrent monitoring of retinosomes along with potentially harmful products in aging eyes, has the potential to detect early molecular changes due to age–related macular degeneration and other defects in retinoid metabolism.
doi:10.1038/nm.2260
PMCID: PMC3057900  PMID: 21076393
Photoreceptor cells; fluorescence microscopy; retinosomes; retinyl esters; A2E; RPE; retina; two–photon fluorescence excitation microscopy; three–photon fluorescence excitation microscopy
15.  Glycosylation of the West Nile Virus Envelope Protein Increases In Vivo and In Vitro Viral Multiplication in Birds 
Many West Nile (WN) virus isolates associated with significant outbreaks possess a glycosylation site on the envelope (E) protein. E-protein glycosylated variants of New York (NY) strains of WN virus are more neuroinvasive in mice than the non-glycosylated variants. To determine how E protein glycosylation affects the interactions between WN virus and avian hosts, we inoculated young chicks with NY strains of WN virus containing either glycosylated or non-glycosylated variants of the E protein. The glycosylated variants were more virulent and had higher viremic levels than the non-glycosylated variants. The glycosylation status of the variant did not affect viral multiplication and dissemination in mosquitoes in vivo. Glycosylated variants showed more heat-stable propagation than non-glycosylated variants in mammalian (BHK) and avian (QT6) cells but not in mosquito (C6/36) cells. Thus, E-protein glycosylation may be a requirement for efficient transmission of WN virus from avian hosts to mosquito vectors.
doi:10.4269/ajtmh.2010.09-0262
PMCID: PMC2844573  PMID: 20348522
16.  IgG-class anti-PF4/heparin antibodies and symptomatic DVT in orthopedic surgery patients receiving different anti-thromboembolic prophylaxis therapeutics 
Background
Heparin-induced thrombocytopenia (HIT) is a thromboembolic complication that can occur with unfractionated heparin (UFH) or low molecular weight heparin (LMWH). Our objective was to determine and compare the incidence of IgG-class HIT antibodies in patients undergoing total hip arthroplasty (THA) or total knee arthroplasty (TKA) with different antithrombotic prophylaxis therapies and their contributions to the occurrence of venous thromboembolism (VTE).
Methods
A prospective observational study was performed for 374 Japanese patients undergoing THA or TKA to determine the incidence of VTE. IgG-class anti-PF4/heparin antibodies were measured using IgG-specific EIA before and after the operation.
Results
In the clinical outcome, the incidence of symptomatic deep vein thrombosis (DVT) was 15.0% (56/374, TKA; 35, THA; 21) and pulmonary emboli (PE) were not observed. The total seroconversion incidence of IgG-class PF4/heparin antibodies was 19.8% (74/374). The seroconversion incidence of IgG-class PF4/heparin antibodies was higher in patients receiving UFH (32.7%) compared to those receiving LMWH (9.5%) or fondaparinux (14.8%). Furthermore, the seroconversion incidence was significantly higher in patients undergoing TKA compared to those undergoing THA. Based on multivariate analysis, seroconversion of the IgG-class PF4/heparin antibodies was independent a risk factor for symptomatic DVT.
Conclusion
Our findings show that the seroconversion of IgG-class anti-PF4/heparin antibodies differed with various anti-thrombotic prophylaxis therapeutics and was associated with the risk of DVT in a subset of patients undergoing total joint arthroplasty (TKA and THA).
doi:10.1186/1471-2474-12-22
PMCID: PMC3224260  PMID: 21261941
17.  Regulation of B versus T lymphoid lineage fate decision by the proto-oncogene LRF 
Science (New York, N.Y.)  2007;316(5826):860-866.
Hematopoietic stem cells in the bone marrow give rise to lymphoid progenitors, which subsequently differentiate into B and T lymphocytes. Here we show that the proto-oncogene LRF plays an essential role in the B versus T lymphoid cell fate decision. We demonstrate that LRF is key for instructing early lymphoid progenitors to develop into B lineage cells by repressing T cell-instructive signals produced by the cell fate signal protein, Notch. We propose a new model for lymphoid lineage commitment, in which LRF acts as a master regulator of B versus T lineage fate decision.
doi:10.1126/science.1140881
PMCID: PMC2978506  PMID: 17495164
18.  Loss of cone photoreceptors caused by chromophore depletion is partially prevented by the artificial chromophore pro-drug, 9-cis-retinyl acetate 
Human Molecular Genetics  2009;18(12):2277-2287.
Inactivating mutations in the retinoid isomerase (RPE65) or lecithin:retinol acyltransferase (LRAT) genes cause Leber congenital amaurosis (LCA), a severe visual impairment in humans. Both enzymes participate in the retinoid (visual) cycle, the enzymatic pathway that continuously generates 11-cis-retinal, the chromophore of visual pigments in rod and cone photoreceptor cells needed for vision. We investigated human RPE65–LCA patients and mice with visual cycle abnormalities to determine the impact of chronic chromophore deprivation on cones. Young patients with RPE65 mutations showed foveal cone loss along with shortened inner and outer segments of remaining cones; cone cell loss also was dramatic in young mice lacking Rpe65 or Lrat gene function. To selectively evaluate cone pathophysiology, we eliminated the rod contribution to electroretinographic (ERG) responses by generating double knockout mice lacking Lrat or Rpe65 together with an inactivated rod-specific G protein transducin gene (Gnat1−/−). Cone ERG responses were absent in Gnat1−/−Lrat−/− mice which also showed progressive degeneration of cones. Cone ERG responses in Gnat1−/−Rpe65−/− mice were markedly reduced and declined over weeks. Treatment of these mice with the artificial chromophore pro-drug, 9-cis-retinyl acetate, partially protected inferior retinal cones as evidenced by improved ERGs and retinal histochemistry. Gnat1−/− mice chronically treated with retinylamine, a selective inhibitor of RPE65, also showed a decline in the number of cones that was ameliorated by 9-cis-retinyl acetate. These results suggest that chronic lack of chromophore leads to progressive loss of cones in mice and humans. Therapy for LCA patients should be geared toward early adequate delivery of chromophore to cone photoreceptors.
doi:10.1093/hmg/ddp163
PMCID: PMC2685761  PMID: 19339306
19.  Modulation of molecular interactions and function by rhodopsin palmitylation† 
Biochemistry  2009;48(20):4294-4304.
Rhodopsin is palmitylated at two cysteine residues in its carboxyl terminal region. We have looked at the effects of palmitylation on the molecular interactions formed by rhodopsin using single-molecule force spectroscopy and the function of rhodopsin using both in vitro and in vivo approaches. A knockin mouse model expressing palmitate-deficient rhodopsin was used for live animal in vivo studies and to obtain native tissue samples for in vitro assays. We specifically looked at the effects palmitylation has on the chromophore-binding pocket, interactions of rhodopsin with transducin, and molecular interactions stabilizing the receptor structure. The structure of rhodopsin is largely unperturbed by the absence of palmitate linkage. The binding pocket for the chromophore 11-cis-retinal is minimally altered as palmitate-deficient rhodopsin exhibited the same absorbance spectrum as wild-type rhodopsin. Similarly, the rate of release of all-trans-retinal after light activation was the same both in the presence and absence of palmitylation. Significant differences were observed in the rate of transducin activation by rhodopsin and in the force required to unfold the last stable structural segment in rhodopsin at its carboxyl terminal region. A 1.3-fold reduction in the rate of transducin activation by rhodopsin was observed in the absence of palmitylation. Single-molecule force spectroscopy revealed a 2.1-fold reduction in normalized force required to unfold the carboxyl terminal end of rhodopsin. The absence of palmitylation in rhodopsin therefore destabilizes the molecular interactions formed in the carboxyl terminal end of the receptor, which appears to hinder the activation of transducin by light-activated rhodopsin.
doi:10.1021/bi900417b
PMCID: PMC2710298  PMID: 19348429
21.  Limited Roles of Rdh8, Rdh12, and Abca4 in all-trans-Retinal Clearance in Mouse Retina 
Purpose
Although the retinoid cycle is essential for vision, all-trans-retinal and the side products of this cycle are toxic. Delayed clearance of all-trans-retinal causes accumulation of its condensation products, A2E, and all-trans-retinal dimer (RALdi), both associated with human macular degeneration. The protective roles were examined of the all-trans-RDHs, Rdh8 and Rdh12, and the ATP-binding cassette transporter Abca4, retinoid cycle enzymes involved in all-trans-retinal clearance.
Methods
Mice genetically engineered to lack Rdh8, Rdh12, and Abca4, either singly or in various combinations, were investigated because all-trans-retinal clearance is achieved by all-trans-RDHs and Abca4. Knockout mice were evaluated by spectral-domain optical coherence tomography (SD-OCT), electroretinography, retinal morphology, and visual retinoid profiling with HPLC and MS. ARPE19 cells were examined to evaluate A2E and RALdi oxidation and toxicity induced by exposure to UV and blue light.
Results
Rdh8−/−Abca4−/− and Rdh8−/−Rdh12−/−Abca4−/− mice displayed slowly progressive, severe retinal degeneration under room light conditions. Intense light-induced acute retinal degeneration was detected by SD-OCT in Rdh8−/−Rdh12−/− Abca4−/− mice. Amounts of A2E in the RPE correlated with diminished all-trans-retinal clearance, and the highest A2E amounts were found in Rdh8−/−Rdh12−/−Abca4−/− mice. However oxidized A2E was not found in any of these mice, and A2E oxidation was not induced by blue light and UV illumination of A2E-loaded ARPE19 cells. Of interest, addition of all-trans-retinal did activate retinoic acid receptors in cultured cells.
Conclusions
Rdh8, Rdh12, and Abca4 all protect the retina and reduce A2E production by facilitating all-trans-retinal clearance. Delayed all-trans-retinal clearance contributes more than A2E oxidation to light-induced cellular toxicity.
doi:10.1167/iovs.09-3944
PMCID: PMC2792889  PMID: 19553623
22.  Evaluation of Potential Therapies for a Mouse Model of Human Age-Related Macular Degeneration Caused by Delayed all-trans-Retinal Clearance 
Purpose
Evaluate the efficacy of potential therapeutics in Rdh8−/− Abca4−/−mice, a rodent model of human age-related macular degeneration (AMD).
Methods
Therapeutic efficacy of several antioxidant agents (ascorbic acid, α-lipoic acid, α-tocopherol, Mn(III)-tetrakis(4-benzoic acid)-porphyrin, and butylated hydroxytoluene), an immunosuppressive agent with antivascular endothelial growth factor (VEGF) activity (sirolimus, also known as rapamycin), a retinoid cycle inhibitor (retinylamine), and an artificial chromophore (9-cis-retinyl acetate) were evaluated side by side in a recently described murine model of AMD, the Rdh8−/− Abca4−/−mouse. This animal exhibits a retinopathy caused by delayed all-trans-retinal clearance resulting from the absence of both ATP-binding cassette transporter 4 (Abca4) and retinol dehydrogenase 8 (Rdh8) activities. Drug efficacy was evaluated by retinal histologic analyses and electroretinograms (ERGs).
Results
All tested agents partially prevented atrophic changes in the Rdh8−/− Abca4−/−retina with retinylamine demonstrating the greatest efficacy. A significant reduction of complement deposition on Bruch’s membrane was observed in sirolimus-treated mice, although the severity of retinal degeneration was similar to that observed in antioxidant- and 9-cis-retinyl acetate–treated mice. Sirolimus treatment of 6-month-old Rdh8−/− Abca4−/−mice for 4 months prevented choroidal neovascularization without changing retinal VEGF levels.
Conclusions
Mechanism-based therapy with retinylamine markedly attenuated degenerative retinopathy in Rdh8−/− Abca4−/−mice. Further understanding of pathogenic mechanisms involved in AMD is needed to develop more effective therapeutics.
doi:10.1167/iovs.09-3581
PMCID: PMC2782428  PMID: 19494204
23.  Evaluation of 9-cis-Retinyl Acetate Therapy in Rpe65−/− Mice 
PURPOSE
Mice lacking retinal pigment epithelium–specific 65-kDa protein (RPE65) develop retinopathy and blindness resembling Leber congenital amaurosis. Effects of 9-cis-retinyl acetate (9-cis-R-Ac) on visual function and retinopathy progression were tested in Rpe65−/− mice.
METHODS
Young C57Bl/6 mice were given 9-cis-R-Ac in each of four different oil-based vehicle solutions by gastric gavage to identify the vehicle most suitable for drug delivery by measuring retinoid levels in plasma. Then doses of 9-cis-R-Ac ranging from 1 to 100 mg/kg were administered to 5- to 12-week-old Rpe65−/− mice by different treatment regimens, including single doses and either intermittent or daily doses for various periods up to 8 weeks. Retinoid effects on visual function were evaluated by electroretinography, retinoid analyses, histologic methods, and vision-dependent behavioral testing.
RESULTS
Soybean oil vehicle provided the highest 9-cis-R-Ac metabolite levels in plasma. Single doses of 9-cis-R-Ac (6.25–50 mg/kg) provided significant dose-dependent improvement in electroretinographic responses. Well-tolerated daily doses (1–12.5 mg/kg) for 2 weeks induced remarkable improvement of retinal function. Significant dose-dependent improvement of electroretinographic responses was observed 6 days after administration of 9-cis-R-Ac daily for 3 days at 1 to 12.5 mg/kg. Mice given either daily or intermittent 9-cis-R-Ac treatment at 1 and 4 mg/kg and evaluated 8 weeks later displayed dose-dependent improvement of retinal function and morphology, whereas retinal function deteriorated in control animals. Treated mice also performed better than control animals in vision-dependent behavioral tests.
CONCLUSIONS
Treatment with 9-cis-R-Ac improves visual function and preserves retinal morphology in Rpe65−/− mice.
doi:10.1167/iovs.09-3700
PMCID: PMC2764364  PMID: 19407008
24.  Transmission of Specific Genotype Streptomycin Resistant Strains of Mycobacterium tuberculosis in the Tokyo Metropolitan Area in Japan 
Background
From 2003 through to 2004, an outbreak of tuberculosis was identified at a university campus in Yokohama City, located in the southern part of the Tokyo Metropolitan Area (TMA). All Mycobacterium tuberculosis (M. tuberculosis) strains detected with regards to this outbreak turned out to be Streptomycin resistant with matched patterns of 14 IS6110 bands of Restriction Fragment Length Polymorphism (RFLP). The M. tuberculosis bacilli, which had the matched IS6110 band patterns with resistance to Streptomycin to those of bacilli isolated in the outbreak, were also concurrently detected through either the population-based or the hospital-based DNA fingerprinting surveillance of M. tuberculosis either in Shinjuku City or in Kawasaki City respectively.
The aim of the present study is to describe the spread of the specific genotype strains of M. tuberculosis in the TMA as observed in the above incident, and to identify the possible transmission routes of the strains among people living in urban settings in Japan.
Methods
We applied Variable Numbers of Tandem Repeats (VNTR) analysis to all M. tuberculosis isolates which were resistant to Streptomycin with a matched IS6110-RFLP band pattern (M-strains). They were isolated either from cases related to the tuberculosis outbreak that happened at a university, or through DNA fingerprinting surveillance of M. tuberculosis both in Shinjuku City and in Kawasaki City. For VNTR analysis, 12MIRU loci, 4ETR loci, seven loci by Supply, four loci by Murase (QUB15, Mtub24, VNTR2372, VNTR3336) were selected.
Results
Out of a total of 664 isolates collected during the study period, 46 isolates (6.9%) were identified as M-strains. There was a tendency that there was a higher proportion of those patients whose isolates belonged to M4-substrains, with four copies of tandem repeat at the ETR-C locus, to have visited some of the internet-cafés in the TMA than those whose isolates belonged to M5-substrains, with five copies at the ETR-C locus, although statistically not significant (38.1% vs. 10.0%, Exact p = 0.150).
Conclusion
Although firm conclusions could not be reached through the present study, it suggested that we have to take into consideration that tuberculosis can be transmitted in congregated facilities like internet cafés where tuberculosis high-risk people and general people share common spaces.
doi:10.1186/1471-2334-9-138
PMCID: PMC2749854  PMID: 19709409
25.  Utilization of a Biodegradable Mulch Sheet Produced from Poly(Lactic Acid)/Ecoflex®/Modified Starch in Mandarin Orange Groves 
We have developed a mulch sheet made by inflation molding of PLA, Ecoflex® and modified starch, which all have different biodegradabilities. A field test of use as an agricultural mulch sheet for mandarin oranges was carried out over two years. The mechanical properties of the mulch sheet were weakened with time during the field test, but the quality of the mandarin oranges increased, a result of the controlled degradation of the sheet. The most degradable modified starch degraded first, allowing control of the moisture on the soil. Accelerator mass spectroscopy was used for evaluation of the biomass carbon ratio. The biomass carbon ratio decreased by degradation of the biobased materials, PLA and modified starch in the mulch sheet.
doi:10.3390/ijms10083599
PMCID: PMC2758133  PMID: 19812715
poly(lactic acid); modified starch; mulch sheet; mandarin orange; biodegradable materials; biomass carbon ratio

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