Effect of CDK7 inhibitor on MYCN-amplified retinoblastoma.

Retinoblastoma (RB) is a common malignancy that primarily affects pediatric populations. Although a well-known cause of RB is RB1 mutation, MYCN amplification can also lead to the disease, which is a poor prognosis factor. Studies conducted in various tumor types have shown that MYCN inhibition is an effective approach to impede tumor growth. Various indirect approaches have been developed to overcome the difficulty of directly targeting MYCN, such as modulating the super enhancer (SE) upstream of MYCN. The drug used in this study to treat MYCN-amplified RB was THZ1, a CDK7 inhibitor that can effectively suppress transcription by interfering with the activity of SEs. The study findings confirmed the anticancer activity of THZ1 against RB in both in vitro and in vivo experiments. Therapy with THZ1 was found to affect numerous genes in RB according to the RNA-seq analysis. Moreover, the gene expression changes induced by THZ1 treatment were enriched in ribosome, endocytosis, cell cycle, apoptosis, etc. Furthermore, the combined analysis of ChIP-Seq and RNA-seq data suggested a potential role of SEs in regulating the expression of critical transcription factors, such as MYCN, OTX2, and SOX4. Moreover, ChIP-qPCR experiments were conducted to confirm the interaction between MYCN and SEs. In conclusion, THZ1 caused substantial changes in gene transcription in RB, resulting in inhibited cell proliferation, interference with the cell cycle, and increased apoptosis. The efficacy of THZ1 is positively correlated with the degree of MYCN amplification and is likely exerted by interfering with MYCN upstream SEs.

An X-ray inactivated vaccine against Pseudomonas aeruginosa Keratitis in mice.

Pseudomonas aeruginosa (P. aeruginosa) is one of the most prevalent pathogens of bacterial keratitis. Bacterial keratitis is a major cause of blindness worldwide. The rising incidence of multidrug resistance of P. aeruginosa precludes treatment with conventional antibiotics. Herein, we evaluated the protective efficiency and explored the possible underlying mechanism of an X-ray inactivated vaccine (XPa) using a murine P. aeruginosa keratitis model. Mice immunized with XPa exhibit reduced corneal bacterial loads and pathology scores. XPa vaccination induced corneal macrophage polarization toward M2, averting an excessive inflammatory reaction. Furthermore, histological observations indicated that XPa vaccination suppressed corneal fibroblast activation and prevented irreversible visual impairment. The potency of XPa against keratitis highlights its potential utility as an effective and promising vaccine candidate for P. aeruginosa.

EZH2-mediated H3K27me3 is a predictive biomarker and therapeutic target in uveal melanoma.

Although gene mutations and aberrant chromosomes are associated with the pathogenesis and prognosis of uveal melanoma (UM), potential therapeutic targets still need to be explored. We aim to determine the predictive value and potential therapeutic target of EZH2 in uveal melanoma. Eighty-five uveal melanoma samples were recruited in our study, including 19 metastatic and 66 nonmetastatic samples. qRT-PCR, immunohistochemistry staining, and western blotting were applied to detect the expression of EZH2 and H3K27me3. We found that EZH2 (41/85, 48.24%) and H3K27me3 (49/85, 57.65%) were overexpressed in uveal melanoma. The expression of EZH2 was not significantly associated with metastasis. High H3K27me3 expression was correlated with poor patient prognosis. UNC 1999, an EZH2 inhibitor, can downregulate H3K27me3 expression and has the most potency to inhibit OMM1 cell growth by the cell cycle and ferroptosis pathway. These results indicate that H3K27me3 can be a biomarker predicting a poor prognosis of UM. EZH2 is the potential therapeutic target for UM.

Retinal Degeneration: Molecular Mechanisms and Therapeutic Strategies.

Retinal degenerative diseases are the main retinal diseases that threaten vision. Most retinal degenerative diseases are inherited diseases, including autosomal recessive inheritance, autosomal dominant inheritance, X-linked inheritance, and mitochondrial inheritance; therefore, emerging gene therapy strategies may provide an alternative method of treatment. Currently, three viral vectors are usually used in gene therapy studies: adenovirus, lentivirus, and adeno-associated virus. Other gene therapies have their own advantages, such as DNA nanoparticles, antisense oligonucleotides, and gene editing therapies. In addition, retinal degenerative diseases are often accompanied by abnormalities of retinal cells, including photoreceptor and retinal pigment epithelial cells. At present, stem cell transplantation is a promising new treatment for retinal degenerative diseases. Common sources of stem cells include retinal progenitor cells, induced pluripotent stem cells, embryonic stem cells, and mesenchymal stem cells. In addition, retina explant cultures in vitro can be used as an effective platform for screening new therapies for retinal degenerative diseases. Drugs that actually reach the retinal layer are more controlled, more consistent, and less invasive when using retinal explants. Furthermore, studies have shown that the imbalance of the gut microbiota is closely related to the occurrence and development of diabetic retinopathy. Therefore, the progression of diabetic retinopathy may be restrained by adjusting the imbalance of the gut microbiota. The purpose of this review is to discuss and summarize the molecular mechanisms and potential therapeutic strategies of retinal degenerative diseases.

DZNep protects against retinal ganglion cell death in an NMDA-induced mouse model of retinal degeneration.

Epigenetic gene enhancer of zeste homolog-2 (Ezh2) is reported to be associated with ocular neurodegenerative diseases; however, its underlying mechanism is poorly understood. The present study aimed to determine the role of 3-deazaneplanocin A (DZNep), which inhibits the transcription of Ezh2 by reducing the trimethylation of histone 3 lysine 27 (H3K27me3), in a retinal ganglion cell (RGC) degeneration model. Retinal damage was caused by intravitreal injection of N-methyl-D-aspartate (NMDA). DZNep and the vehicle control were intravitreally applied immediately post-NMDA injection. The severity of retinal damage was evaluated by immunofluorescence and terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining, and retinal function was determined by electroretinogram (ERG). The transcriptome was examined by RNA sequencing and quantitative PCR (qPCR). Microglial cells were detected by immunohistochemistry. DZNep significantly prevented the cell death in the ganglion cell layer (GCL) and inner nuclear layer (INL) induced by NMDA. DZNep preserved the ERG b- and a-wave amplitudes and the b/a ratio in NMDA-treated mice. Moreover, RNA sequencing and qPCR revealed that neuroprotective genes were upregulated and played an important role in preserving retinal cells. In addition, DZNep inhibited the NMDA-induced activation of microglial cells. Our results suggest that H3K27me3 controls RGC survival at the transcriptional and epigenetic levels. The absence of H3K27me3 deposition upregulates neuroprotective genes to protect RGCs. Therefore, DZNep, which inhibits Ezh2 activity, could be a novel therapeutic treatment for ocular neurodegenerative diseases.

Heterogeneity of GNAQ/11 mutation inversely correlates with the metastatic rate in uveal melanoma.

PURPOSE: To determine whether the GNAQ/11 mutation correlated with the outcome of patients with uveal melanoma (UM) when genetic heterogeneity was considered. METHODS: We performed a retrospective study of sixty-seven patients with UM. The heterogeneity of GNAQ/11 was examined by using droplet digital PCR. The correlation between metastasis and heterogeneity of the GNAQ/11 mutation was analysed. Disease free survival curves were constructed using the Kaplan-Meier method, and the Wilcoxon log-rank test was used to compare the curves. RESULTS: The GNAQ/11 mutation ratio was varied between each case. Among these patients, 28.35% of them harboured homogeneous mutation of GNAQ/11, 62.69% present heterogeneous mutation and 8.96% didn't present either GNAQ or GNA11 mutation. The tumour with heterogeneous mutation of GNAQ/11 has a higher metastatic rate than that with homogeneous mutation (13/29 vs 1/18, p=0.027). In Kaplan-Meier analysis, metastasis-free survival was not significantly associated with either homogeneous or heterogeneous mutation of GNAQ/11. CONCLUSION: The mutation ratio of GNAQ/11 in UM was quite variable. The tumour with heterogeneous mutation of GNAQ/11 is more likely to develop a poor prognosis than that with homogeneous mutation of GNAQ/11.

Mutations of GNAQ, GNA11, SF3B1, EIF1AX, PLCB4 and CYSLTR in Uveal Melanoma in Chinese Patients.

BACKGROUND: The purpose of this study is to determine the mutation frequencies of key driver genes in uveal melanoma (UM) in Chinese patients and to detect associations between metastasis and the mutation of these genes. METHOD: A total of 85 patients with UM were enrolled in this study, including 18 patients with metastasis and 67 without metastasis. Sanger sequencing covering the mutational hotspot regions of the G protein subunit alpha Q (GNAQ), GNA11, splicing factor 3B subunit 1 (SF3B1), X-linked eukaryotic translation initiation factor 1A (EIF1AX), phospholipase C beta 4 (PLCB4) and cysteinyl leukotriene receptor 2 (CYSLTR2) genes was used to analyse the mutations in Chinese patients. RESULTS: The frequencies of GNAQ and GNA11 mutations in UM were 45% (38/85) and 35% (30/85) respectively. The frequencies of SF3B1 and EIF1AX mutations were 37% (31/85) and 9% (8/85) respectively. Only 2 mutations were detected in exon 4 of GNAQ, and no mutations were detected in exon 4 of GNA11. A novel mutation, c.627G>T (Q209H) in GNA11 was found. The detected mutations affecting SF3B1 were c.1873C>T (R625C), c.1874G>A (R625H) and c.1874G>T (R625L). The association between the mutations in SF3B1 and low risk of metastasis was statistically significant (OR 0.17, 95% CI 0.035-0.819). The mutations affecting EIF1AX were -23G>A (5'-UTR), c.5C>G (P2R), c.23G>A (G8Q), c.25G>C (G9A) and c.38_39GC>CT (R13P). No mutations were found in the PLCB4 and CYSLTR2 genes. Unfortunately, information on BRCA1-associated protein 1 could not be obtained. CONCLUSIONS: These data indicate that mutations in the PLCB4 and CYSLTR2 genes are rare in Chinese UM patients. The mutations in GNAQ, GNA11 and EIF1AX were not associated with metastasis, whereas SF3B1 mutations were correlated with low risk of metastasis and demonstrated a protective effect in UM patients in China.

[Effects of Histone Methyltransferase Inhibitors on the Survival, Apoptosis and Cell Cycle of Raji Cells].

OBJECTIVE: To determine the effects of three histone methylase inhibitors UNC1999, DZNep and GSK343 on the survival, apoptosis and cell cycle of non-hodgkin's lymphoma Raji cells. METHODS: PCR amplified 16 and 18 exons of enhancer of zeste homolog 2 ( EZH2) gene were detected. The expression of EZH2 in normal adult lymphocytes and Raji cells was detected by Western blot. The Raji cells were treated by UNC1999, DZNep and GSK343, followed by CCK-8 assays analyzing cell survival, flow cytometry detecting cell apoptosis and cell cycle, and Western blot detecting the expressions of EZH2 and H3K27 me3. RESULTS: The Sanger sequencing results showed that the Raji cells did not carry Y641 and A677 mutation sites of EZH2. The Western blot results showed high expressions of EZH2 in the Raji cells. The results of CCK-8 showed that UNC1999, DZNep and GSK343 inhibited cell survival, and the weakest effect was from DZNep. The flow cytometric assay showed that UNC1999, DZNep and GSK343 promoted apoptosis of the Raji cells, and the effect of UNC1999 was stronger than that of GSK343 and DZNep. The cell cycle was arrested at phase G 1/G 0 after treatment of the Raji cells with the three inhibitors, with UNC1999 triggering the most significant changes. The Western blot showed that UNC1999 and GSK343 inhibited the histone methylase activity of EZH2 and significantly reduced the expression of H3K27 me3. CONCLUSION: EZH2 inhibitors can inhibit cell survival, promote cell apoptosis and arrest cell cycle at phase G 1/G 0 of Raji cells through reducing the expression of H3K27me3. UNC1999 has a stronger effect than GSK343 and DZNep.

Investigation of indoleamine 2,3-dioxygenase 1 expression in uveal melanoma.

The purpose of this study was to investigate indoleamine 2,3-dioxygenase 1 (IDO1) expression and its implications in uveal melanoma (UM). Bioinformatics analysis was performed on microarray data (GSE22138 and GSE27831) from the Gene Expression Omnibus (GEO) database to evaluate IDO1 expression in mRNA level. Ninety-two cases in the database were divided into the IDO1-high group (46 cases) and IDO1-low group (46 cases). Paraffin embedded tumor sections from 27 patients with UM were studied by immunofluorescence. The mRNA results showed that IDO1 expression was inversely correlated with tumor thickness (9.93 ±â€¯3.33 mm in IDO1-high group vs. 11.56 ±â€¯2.38 mm in IDO1-low group) (p = 0.016) and metastatic rate (30.4% in IDO1-high group vs. 69.6% in IDO1-low group, p < 0.001). The IDO1-high group showed higher immune cell gene expression: CD3D (6.56 ±â€¯1.0 vs. 5.46 ±â€¯0.53, p < 0.0001), CD4 (4.72 ±â€¯0.4 vs. 4.2 ±â€¯0.42, p < 0.0001), and CD68 (6.17 ±â€¯1.23 vs. 5.53 ±â€¯0.77, p = 0.015). Further analysis showed that immune-suppressive T regulatory cell genes (CD3D, CD4, IL2RA and FOXP3) were expressed in 67.4% (31/46) cases in the IDO1-high group and 23.91% (11/46) cases in the IDO1-low group. In addition, IDO1 and interferon gamma (IFNG) mRNA expression were strongly correlated (r = 0.70, p < 0.0001). The correlation analysis of different immune checkpoints showed that IDO1 was positively correlated with CD274(PDL1), but not CTLA4 or PDCD1.The disease-free survival (DFS) in the IDO1-high/IFNG-high group was better than that of the IDO1-low/IFNG-low group. The IDO1 immunostaining result showed that 2 cases in 18 UMs with Bruch's membrane (BM) rupture and 7 out of 9 cases without BM rupture were scored high (Grade 2-3) (p = 0.001). Comparing the immune cells staining results between IDO1-high group and IDO1-low group, higher percentage of patients in the former group had high levels of T cells and macrophages infiltration, but only the difference in macrophage was statistically significant (CD68, 77.78% vs. 27.78%, p = 0.04). The analysis based on GEO data and the result from immunostaining study are consistent with each other. In conclusion, the expression of IDO1 is probably induced by IFNγ from infiltrated immune cells in UM. BM rupture is an important indicator of IDO1 expression level and distribution pattern. The complex role of IDO1 may limit its therapeutic effect in UM.

DZNep inhibits H3K27me3 deposition and delays retinal degeneration in the rd1 mice.

Retinitis pigmentosa (RP) is a group of inherited retinal degenerative diseases causing progressive loss of photoreceptors. Numerous gene mutations are identified to be related with RP, but epigenetic modifications may also be involved in the pathogenesis. Previous studies suggested that both DNA methylation and histone acetylation regulate photoreceptor cell death in RP mouse models. However, the role of histone methylation in RP has never been investigated. In this study, we found that trimethylation of several lysine sites of histone H3, including lysine 27 (H3K27me3), increased in the retinas of rd1 mice. Histone methylation inhibitor DZNep significantly reduced the calpain activity, delayed the photoreceptor loss, and improved ERG response of rd1 retina. RNA-sequencing indicated that DZNep synergistically acts on several molecular pathways that regulate photoreceptor survival in rd1 retina, including PI3K-Akt and photoreceptor differentiation pathways, revealing the therapeutic potential of DZNep for RP treatment. PI3K-Akt pathway and H3K27me3 form a feedback loop in rd1 retina, thus PI3K inhibitor LY294002 reduces phosphorylation of Ezh2 at serine 21 and enhances H3K27me3 deposition, and inhibiting H3K27me3 by DZNep can activate PI3K-Akt pathway by de-repressing gene expression of PI3K subunits Pik3r1 and Pik3r3. These findings suggest that histone methylation, especially H3K27me3 deposition is a novel mechanism and therapeutic target for retinal degenerative diseases, similar to H3K27me3-mediated ataxia-telangiectasia in Atm -/- mouse.

E2f1 mediates high glucose-induced neuronal death in cultured mouse retinal explants.

Diabetic retinopathy (DR) is the most common complication of diabetes and remains one of the major causes of blindness in the world; infants born to diabetic mothers have higher risk of developing retinopathy of prematurity (ROP). While hyperglycemia is a major risk factor, the molecular and cellular mechanisms underlying DR and diabetic ROP are poorly understood. To explore the consequences of retinal cells under high glucose, we cultured wild type or E2f1-/- mouse retinal explants from postnatal day 8 with normal glucose, high osmotic or high glucose media. Explants were also incubated with cobalt chloride (CoCl2) to mimic the hypoxic condition. We showed that, at 7 days post exposure to high glucose, retinal explants displayed elevated cell death, ectopic cell division and intact retinal vascular plexus. Cell death mainly occurred in excitatory neurons, such as ganglion and bipolar cells, which were also ectopically dividing. Many Müller glial cells reentered the cell cycle; some had irregular morphology or migrated to other layers. High glucose inhibited the hyperoxia-induced blood vessel regression of retinal explants. Moreover, inactivation of E2f1 rescued high glucose-induced ectopic division and cell death of retinal neurons, but not ectopic cell division of Müller glial cells and vascular phenotypes. This suggests that high glucose has direct but distinct effects on retinal neurons, glial cells and blood vessels, and that E2f1 mediates its effects on retinal neurons. These findings shed new light onto mechanisms of DR and the fetal retinal abnormalities associated with maternal diabetes, and suggest possible new therapeutic strategies.

Hypoxia-Inducible Factor-1α Target Genes Contribute to Retinal Neuroprotection.

Hypoxia-inducible factor (HIF) is a transcription factor that facilitates cellular adaptation to hypoxia and ischemia. Long-standing evidence suggests that one isotype of HIF, HIF-1α, is involved in the pathogenesis of various solid tumors and cardiac diseases. However, the role of HIF-1α in retina remains poorly understood. HIF-1α has been recognized as neuroprotective in cerebral ischemia in the past two decades. Additionally, an increasing number of studies has shown that HIF-1α and its target genes contribute to retinal neuroprotection. This review will focus on recent advances in the studies of HIF-1α and its target genes that contribute to retinal neuroprotection. A thorough understanding of the function of HIF-1α and its target genes may lead to identification of novel therapeutic targets for treating degenerative retinal diseases including glaucoma, age-related macular degeneration, diabetic retinopathy, and retinal vein occlusions.

Antiviral Activity of a Cloned Peptide RC28 Isolated from the Higher Basidiomycetes Mushroom Rozites caperata in a Mouse Model of HSV-1 Keratitis.

An Escherichia coli-expressed peptide with a molecular weight of 28.26, derived from the complementary DNA of antiviral protein RC28 isolated from the mushroom Rozites caperata (=Cortinarius caperatus), demonstrated potent antiviral activity against herpes simplex virus-1 in Vero cells and in a herpes simplex virus-1 mouse keratitis model. Plaque assays in Vero cells showed that the peptide reduced viral yields by at least 1.2 logs; in the animal model the cloned peptide delayed the occurrence of stromal keratitis and alleviated the severity of the disease. We believe this is the first report of a cloned mushroom peptide with antiviral activity for the prevention and treatment of a viral disease.

Effects of nerve growth factor on nerve regeneration after corneal nerve damage.

The study aims to determine the relation between the effects of mouse nerve growth factor (mNGF) and nerve regeneration after corneal surgery nerve damage. Mechanical nerve injury animal model was established by LASIK (the excimer laser keratomileusis) surgery in 12 Belgian rabbits. mNGF and the balanced salt solution (BBS) were alternatively administered in the left and right eye two times every day for 8 weeks. The morphous and growth of the sub-basal nerve plexus and superficial stroma were observed by in vivo confocal microscopy at the end of weeks 1, 2, 4 and 8 after the surgery. The animal model is successfully established. The morphology and density of corneal nerve have been observed and demonstrated by confocal microscopy. A systematic administration of mNGF can significantly promote the nerve regeneration at the end of weeks 1, 2, 4 and 8, which comparing to the administration of balanced salt solution (P < 0.05). mNGF has effect on sub-basal nerve plexus and superficial stroma after corneal nerve damage which is caused by LASIK. The experimental results suggested that the mNGF may solve the problem of dry eye after LASIK.

A novel MYOC heterozygous mutation identified in a Chinese Uygur pedigree with primary open-angle glaucoma.

PURPOSE: To characterize the clinical features of a Chinese Uygur pedigree with primary open-angle glaucoma (POAG) and to identify mutations in two candidate genes, trabecular meshwork inducible glucocorticoid response (MYOC/TIGR) and human dioxin-inducible cytochrome P450 (CYP1B1). METHODS: Twenty one members from a Chinese Uygur family of four generations were included in the study. All participants underwent complete ophthalmologic examinations. Five were diagnosed as POAG, four as glaucoma suspects, and the rest were asymptomatic. Molecular genetic analysis was performed on all subjects included in the study. All exons of CYP1B1 and MYOC were amplified by polymerase chain reaction (PCR), sequenced and compared with a reference database. The variations detected were evaluated in available family members as well as 102 normal controls. Possible changes in structure and function of the protein induced by amino acid variance were predicted by bioinformatics analysis. RESULTS: Elevated intraocular pressure and late-stage glaucomatous cupping of the optic disc were found in five patients of this family. A novel heterozygous missense mutation c.1151 A>G in exon 3 of MYOC was found in all five patients diagnosed as POAG and four glaucoma suspects, but not in the rest of the family members and 102 normal controls. This mutation caused an amino acid substitution of aspartic acid to glycine at position 384 (p. D384G) of the MYOC protein. This substitution may cause structural and functional changes of the protein based on bioinformatics analysis. No mutations were found in CYP1B1. CONCLUSIONS: Our study suggests that the novel mutation D384G of MYOC is likely responsible for the pathogenesis of POAG in this pedigree.

NOD2/CARD15 gene mutation identified in a Chinese family with Blau syndrome.

PURPOSE: To characterize the clinical features of a Chinese pedigree with Blau syndrome and to identify mutations in the NOD2/CARD15 (nucleotide-binding oligomerization domain containing 2/caspase recruitment domain family, member 15) gene. METHODS: Clinical features of this family were evaluated. Genomic DNA was obtained from blood samples, and all exons of NOD2/CARD15 were amplified by polymerase chain reaction (PCR) and direct DNA sequencing of PCR products was performed for mutations in NOD2/CARD15. RESULTS: Granulomatous arthritis, uveitis, and skin granulomas were found in all affected members. Sequencing analysis demonstrated a heterozygous C>T mutation in exon 4 of NOD2/CARD15 in all patients of this pedigree, which resulted in an amino acid substitution at position 334 (p.R334W). CONCLUSIONS: The R334W mutation in NOD2/CARD15 caused Blau syndrome in a Chinese pedigree. This is the first report of R334W mutation in NOD2/CARD15 in a Chinese pedigree of this disease.

Sequence analysis of MYOC and CYP1B1 in a Chinese pedigree of primary open-angle glaucoma.

PURPOSE: To analyze two candidate genes, trabecular meshwork inducible glucocorticoid response (MYOC/TIGR) and human dioxin-inducible cytochrome P450 (CYP1B1), in a Chinese pedigree of primary open-angle glaucoma. METHODS: In a three-generation family containing 14 members, four of them were patients with primary open-angle glaucoma, one was a glaucoma suspect, and the rest were asymptomatic. All members of the family underwent complete ophthalmologic examinations. Exons of MYOC and CYP1B1 were amplified by polymerase chain reaction, sequenced, and compared with a reference database. RESULTS: Elevated intraocular pressure and impaired visual field were found in all patients. One MYOC heterozygous mutation G367R, in exon 3 was identified in four patients and the suspect, but not in the rest of the family members. Meanwhile, four single nucleotide polymorphisms in MYOC and CYP1B1 genes were found. CONCLUSIONS: Although the G367R mutation of MYOC, which causes primary open-angle glaucoma in the form of autosomal dominant inheritance, has been reported in some other ethnicities, it was found in Chinese pedigree for the first time.

A novel mitochondrial tRNA(Val) T1658C mutation identified in a CPEO family.

PURPOSE: To analyze mitochondrial DNA (mt DNA) gene mutations in a 19-year-old female patient, who presented with chronic progressive external ophthalmoplegia (CPEO), together with her mother and younger sister. METHODS: The diagnosis of mitochondrial myopathy was made based on clinical and biologic analysis. Histochemical methods were used to detect ragged-red fibers (RRFs) and ragged-blue fibers (RBFs) on a muscle biopsy of the patient. All mitochondrial gene DNA fragments of the patient, her mother, and younger sister were amplified by polymerase chain reaction. The products were sequenced and compared with reference databases. RESULTS: A novel T1658C mutation and a known A10006G mutation were identified in the mitochondrial tRNA(Val) gene and the tRNA(Gly) gene, respectively, in the patient, her mother, and younger sister. The T1658C mutation changes the T loop structure of mitochondrial tRNA(Val) and the A10006G mutation disturbs the D loop of mitochondrial tRNA(Gly). CONCLUSIONS: The T1658C and A10006G mutations of mtDNA may be responsible for the pathogenesis of the patient with CPEO.

Neuroprotective effects of C-type natriuretic peptide on rat retinal ganglion cells.

PURPOSE. To evaluate the potential neuroprotective effects of C-type natriuretic peptide (CNP) on rat retinal ganglion cells (RGCs). METHODS. Cultured adult rat retinal cells were treated with vehicle, CNP, or atrial natriuretic peptide (ANP), followed by cytotoxic insults (glutamate, TNFalpha, or withdrawal of trophic factor). RGC survival was analyzed by counting Thy-1-positive cells in each well. For in vivo evaluation, N-methyl-d-aspartate (NMDA) with or without CNP was injected intravitreally into rat eyes. At various time points after injection, retinal cross-sections were analyzed for thickness changes in the retinal layers, and retinal flat mounts were assessed by counting cresyl violet-labeled or TUNEL-positive cells. Expressions of natriuretic peptide receptor-B (NPRB) and apoptosis-related genes in retina, including Bcl-xL, BAX, and micro-calpain, were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS. At 50 and 500 nM, CNP, but not ANP, significantly (P < 0.05) protected against glutamate-insult and trophic factor withdrawal-induced RGC death in vitro. Neither peptide significantly affected TNFalpha-induced cytotoxicity. Intravitreal injection of NMDA (20 nanomoles) significantly (P < 0.05) decreased the thickness of the inner plexiform layer (IPL), induced cell loss, increased the number of TUNEL-positive cells in the RGC layer, and upregulated the expression of Bcl-xL, BAX, and micro-calpain. All these effects were significantly (P < 0.05) alleviated by concomitant injection of CNP (4.5 nmol, 10 microg). The neuroprotective effects of CNP were maintained up to 14 days after CNP injection. CONCLUSIONS. CNP protects rat RGCs against the apoptotic damage induced by insults such as excitatory amino acid, both in vitro and in vivo.

Sequence analysis of MYOC and CYP1B1 in a Chinese pedigree of juvenile glaucoma with goniodysgenesis.

PURPOSE: This study was designed to analyze two candidate genes, myocilin (MYOC) and cytochrome P450 1B1 (CYP1B1), in a Chinese pedigree of juvenile glaucoma with goniodysgenesis. METHODS: In a three-generation family of juvenile glaucoma with goniodysgenesis (13 members), six of them were patients with glaucoma and the rest were asymptomatic. All members of the family underwent complete ophthalmologic examinations. Exons of MYOC and CYP1B1 were amplified by polymerase chain reaction, sequenced, and compared with a reference database. RESULTS: Elevated intraocular pressure (IOP) and visual function impairment was found in all patients, and goniodysgenesis was noticed in five of them (nine eyes) with relatively transparent corneas. One MYOC heterozygous mutation, c.1109 C>T (P370L), in exon 3 was identified in all six patients but not in the asymptomatic family members. Two CYP1B1 single nucleotide polymorphisms (SNPs), g.3947 C>G (R48G) in exon 2 and 372-12 C>T in intron 1, were identified in all six patients and but not in the asymptomatic family members except the proband's grandmother. Three SNPs were identified, 730 + 35 A>G in intron 2 of MYOC and g.8131 G>C (V432L) and g.8184 T>C (D449D) in exon 3 of CYP1B1. CONCLUSIONS: The presence of a P370L mutation of MYOC in all six glaucoma patients suggests a casual association between this mutation and juvenile glaucoma with goniodysgenesis. The possible role of SNPs of CYP1B1 in the pathogenesis of the disease remains to be elucidated.