Investigation and functional analysis of microRNAs in lens and identification of cataract-related genes

研究種目：基盤研究（Ｃ） 研究期間：2008～2010 課題番号：2 0 5 9 2 0

Neuroprotective effect of peroxiredoxin 6 against hypoxia-induced retinal ganglion cell damage

Abstract Background The ability to respond to changes in the extra-intracellular environment is prerequisite for cell survival. Cellular responses to the environment include elevating defense systems, such as the antioxidant defense system. Hypoxia-evoked reactive oxygen species (ROS)-driven oxidative stress is an underlying mechanism of retinal ganglion cell (RGC) death that leads to blinding disorders. The protein peroxiredoxin 6 (PRDX6) plays a pleiotropic role in negatively regulating death signaling in response to stressors, and thereby stabilizes cellular homeostasis. Results We have shown that RGCs exposed to hypoxia (1%) or hypoxia mimetic cobalt chloride display reduced expression of PRDX6 with higher ROS expression and activation of NF-κB. These cells undergo apoptosis, while cells with over-expression of PRDX6 demonstrate resistance against hypoxia-driven RGC death. The RGCs exposed to hypoxia either with 1% oxygen or cobalt chloride (0-400 μM), revealed ~30%-70% apoptotic cell death after 48 and 72 h of exposure. Western analysis and real-time PCR showed elevated expression of PRDX6 during hypoxia at 24 h, while PRDX6 protein and mRNA expression declined from 48 h onwards following hypoxia exposure. Concomitant with this, RGCs showed increased ROS expression and activation of NF-κB with IkB phosphorylation/degradation, as examined with H2DCF-DA and transactivation assays. These hypoxia-induced adverse reactions could be reversed by over-expression of PRDX6. Conclusion Because an abundance of PRDX6 in cells was able to attenuate hypoxia-induced RGC death, the protein could possibly be developed as a novel therapeutic agent acting to postpone RGC injury and delay the progression of glaucoma and other disorders caused by the increased-ROS-generated death signaling related to hypoxia.Peer Reviewe

A frameshift deletion mutation in the cardiac myosin-binding protein C gene associated with dilated phase of hypertrophic cardiomyopathy and dilated cardiomyopathy

SummaryObjectivesA few studies reported that some mutations in the cardiac myosin-binding protein C (MyBPC) gene were associated with dilated phase of hypertrophic cardiomyopathy (D-HCM) resembling dilated cardiomyopathy (DCM). We studied 5 unrelated cardiomyopathy probands caused by an identical mutation in the MyBPC gene. The results of clinical and genetic investigations in these patients are presented in this paper.MethodsWe analyzed MyBPC gene in DCM patients as well as patients with HCM.ResultsAn R945fs/105 mutation, 2-base deletion at nucleotides 18,535 and 18,536, was identified in 4 of the 176 HCM probands and in 1 of the 54 DCM probands. Genetic analysis in relatives of those probands revealed another one member with this mutation. A total of 6 subjects had R945fs/105 mutation. The mean age of these six patients at diagnosis was 61 years. At initial evaluation, three of them were diagnosed as having HCM with normal left ventricular (LV) systolic function. The other two patients already had D-HCM. The remaining one patient was diagnosed as having DCM because of reduced LV systolic function (ejection fraction=31%) without increased LV wall thickness. During follow-up (7.6 years), all three patients with impaired LV systolic function were admitted for treatment of heart failure and/or sustained ventricular tachycardia. Finally, one patient with the diagnosis of D-HCM died of heart failure.ConclusionsThe patients with this mutation may develop LV systolic dysfunction and suffer from cardiovascular events through mid-life and beyond

LEDGF: survival of embryonic chick retinal photoreceptor cells

PURPOSE. Lens epithelium-derived growth factor (LEDGF) is a novel adhesive, survival, and growth factor for lens epithelial cells, keratinocytes, fibroblasts, and cos7 cells. In the presence of LEDGF, these cells acquire resistance to environmental stresses, and in the absence of LEDGF they die. The effects of LEDGF on survival of embryonic chick retinal photoreceptor cells under serum starvation and heat stress were studied. METHODS. The expression pattern of LEDGF in embryonic chick retinal photoreceptor cells was investigated with protein blot analysis and immunohistochemistry using antibodies (Abs) to LEDGF. Retinal cells were cultured in serum-free medium for up to 6 days in the presence of varying amounts of LEDGF at 37°or 41°C. The photoreceptor cells were immunostained with Abs to arrestin and counted to evaluate the photoreceptor cell viability. Heat shock proteins in the cultured cells were quantified by protein blot analysis with Ab probes and semiquantitative reverse transcription-polymerase chain reaction analysis. RESULTS. LEDGF was found predominantly in the nucleus of neuroretinal cells, including photoreceptor cells. In the presence of LEDGF, photoreceptor cells manifested increased resistance to serum starvation and thermal stress and survived for a longer period. The levels of heat shock protein 90 were elevated in those cells. Most retinal cells died in the absence of LEDGF

A phase 2 basket trial of combination therapy with trastuzumab and pertuzumab in patients with solid cancers harboring human epidermal growth factor receptor 2 amplification (JUPITER trial)

Introduction: Human epidermal growth factor receptor 2 (HER2) gene amplification and mutations have emerged as oncogenic drivers and therapeutic targets not limited to breast and gastric cancers, but also in a variety of cancers. However, even if an actionable gene alteration is found, the incidence of HER2 amplification in these cancers is less than 5%. It is too difficult to conduct a conventional randomized, controlled trial in a rare fraction. Therefore, we have designed a organ-agnostic basket study, which covers a variety of solid cancers harboring HER2 amplification, in 1 study protocol. Methods/Design: This trial is a multicenter, single-arm, basket phase 2 study in Japan. Patients with solid cancers harboring HER2 amplification that have progressed with standard treatment, or rare cancers for which there is no standard treatment, will be eligible. Target cancers include bile duct, urothelial, uterine, ovarian, and other solid cancers where HER2 amplification is detected by comprehensive genomic profiling using next-generation sequencing technology. A total of 38 patients will be treated with combination therapy with trastuzumab and pertuzumab every 3 weeks until disease progression, unmanageable toxicity, death, or patient refusal. The primary endpoint is the objective response rate, and secondary endpoints are progression-free survival, overall survival, and duration of response. Discussion: The aim of this trial is to evaluate the safety and efficacy of combination therapy with trastuzumab and pertuzumab in patients with locally advanced or metastatic, solid cancers harboring HER2 amplification. Instead of focusing on 1 organ type, our trial design uses a basket study focusing on HER2 amplification, regardless of the site or origin of the cancer. The results of our study will advance clinical and scientific knowledge concerning the treatment of locally advanced, rare solid cancers harboring HER2 amplification, using the combination of trastuzumab and pertuzumab. Trial registration: This trial was registered in Japan Registry of Clinical Trials (jCRT) on February 25, 2019, as jRCT2031180150

Susceptibility of muridae cell lines to ecotropic murine leukemia virus and the cationic amino acid transporter 1 viral receptor sequences: implications for evolution of the viral receptor

Ecotropic murine leukemia viruses (Eco-MLVs) infect mouse and rat, but not other mammalian cells, and gain access for infection through binding the cationic amino acid transporter 1 (CAT1). Glycosylation of the rat and hamster CAT1s inhibits Eco-MLV infection, and treatment of rat and hamster cells with a glycosylation inhibitor, tunicamycin, enhances Eco-MLV infection. Although the mouse CAT1 is also glycosylated, it does not inhibit Eco-MLV infection. Comparison of amino acid sequences between the rat and mouse CAT1s shows amino acid insertions in the rat protein near the Eco-MLV-binding motif. In addition to the insertion present in the rat CAT1, the hamster CAT1 has additional amino acid insertions. In contrast, tunicamycin treatment of mink and human cells does not elevate the infection, because their CAT1s do not have the Eco-MLV-binding motif. To define the evolutionary pathway of the Eco-MLV receptor, we analyzed CAT1 sequences and susceptibility to Eco-MLV infection of other several murinae animals, including the southern vole (Microtus rossiaemeridionalis), large Japanese field mouse (Apodemus speciosus), and Eurasian harvest mouse ( Micromys minutus). Eco-MLV infection was enhanced by tunicamycin in these cells, and their CAT1 sequences have the insertions like the hamster CAT1. Phylogenetic analysis of mammalian CAT1s suggested that the ancestral CAT1 does not have the Eco-MLV-binding motif, like the human CAT1, and the mouse CAT1 is thought to be generated by the amino acid deletions in the third extracellular loop of CAT1

Cellular distribution of lens epithelium-derived growth factor (LEDGF) in the rat eye: loss of LEDGF from nuclei of differentiating cells

Lens epithelium-derived growth factor (LEDGF) enhances the survival and growth of cells. To understand LEDGF's spatial localization and its putative function(s) during proliferation and differentiation, we localized LEDGF during terminal differentiation in whole rat lenses, lens epithelial cell (LEC) explants stimulated with FGF-2, and insulin, iris, human LECs with lentoids. In addition, intracellular localization of LEDGF was performed in other ocular tissues: ciliary body, retina, and cornea. We found the immunopositivity of nuclear LEDGF decreased in LECs of the equatorial region. In contrast, immunopositivity of LEDGF was detected in the cytoplasm of LECs and superficial fiber cells. After treating LEC explants with FGF-2 and insulin, which are known to be differentiating factors for LECs, the nuclei of these cells showed no LEDGF immunopositivity, but explants did express p57 kip2 , a differentiation marker protein. Also, immunopositive LEDGF was not detected in the nuclei of differentiated cells, lentoid body, and corneal epithelial cells. This demonstrated that the loss of LEDGF from the nucleus may be associated with the process of terminal differentiation that might be in some way common with the biochemical mechanisms of apoptosis. The spatial and temporal distribution of LEDGF in the present study also provides a vision for further investigation as to how this protein is involved in cell fate determination.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42235/1/s00418-003-0518-3.pd