High glucose induced inflammation is inhibited by copper chelation via rescuing mitochondrial fusion protein 2 in retinal pigment epithelial cells.

Altered trace element homeostasis is associated with diabetic complications, and studies have shown elevated copper levels in the serum of individuals with type 1 & 2 diabetes. Copper chelation has been shown to be beneficial by preventing or reversing diabetic organ damage and developing as a new treatment strategy for treating diabetic complications. Diabetic retinopathy is the major vision-threatening complication of diabetes. Recent studies have reported copper to be elevated in the serum of patients with diabetic retinopathy. Here in this study, we attempt to unravel the role of copper chelator penicillamine in retinal pigment epithelial cells exposed to high glucose (HG) and copper as a model for diabetic retinopathy. We have found that high glucose by itself and along with copper alters the mitochondrial morphology, reduces the expression of the mitochondrial fusion protein 2 (MFN2), and induces endoplasmic reticulum (ER) stress and inflammation. Copper chelation with penicillamine reduced all these changes in mitochondria, thereby rescuing the cells from mitochondrial damage and inflammation.

Structure based design of inhibitory peptides targeting ornithine decarboxylase dimeric interface and in vitro validation in human retinoblastoma Y79 cells.

Polyamine synthesis in human cells is initiated by catalytic action of Ornithine decarboxylase (ODC) on Ornithine. Elevated levels of polyamines are manifested proliferating cancer cells and are found to promote tumour cell adhesion. Di-flouro methyl orninthine is a known inhibitor of ODC, however its usage is limited due its low affinity quick clearance and incompetent cellular uptake, thus posing a need for potential inhibitors. Currently, peptides are substituting drugs, as these are highly selective, specific and potent. Hence, in this study, the interacting interfaces of native homodimeric form of ODC and its heterodimer with Antizyme were probed to design inhibitory peptides targeting ODC. The designed peptides were validated for structural fitness by extensive molecular dynamics simulations and Circular dichroism studies. Finally, these peptides were validated in Y79 retinoblastoma cells for impact on ODC activity, cytotoxicity cell cycle and cell adhesion. On collective analysis, Peptide3 (Pep 3) and Peptide4 (Pep 4) were found to be potentially targeting ODC, as these peptides showed significant decrease in intracellular polyamine levels, cell adhesion and cell cycle perturbation in Y79 cells. Thus, Pep 3 and Pep 4 shall be favourably considered as therapeutic agents for targeting ODC mediated proliferation in retinoblastoma.Communicated by Ramaswamy H. Sarma.

Copper mediates mitochondrial biogenesis in retinal pigment epithelial cells.

Age related macular degeneration (AMD) is a multifactorial disease with genetic, biochemical and environmental risk factors. We observed a significant increase in copper levels in choroid-RPE from donor eyeballs with AMD. Adult retinal pigment epithelial cells (ARPE19 cells) exposed to copper in-vitro showed a 2-fold increase in copper influx transporter CTR1 and copper uptake at 50 µM concentration. Further there was 2-fold increase in cytochrome C oxidase activity and a 2-fold increase in the mRNA expression of NRF 2 with copper treatment. There was a significant increase in mitochondrial biogenesis markers PGC1ß and TFAM which was confirmed by mitochondrial mass and copy number. On the contrary, in AMD choroid-RPE, the CTR1 mRNA was found to be significantly down-regulated compared to its respective controls. SCO1 and PGC1ß mRNA showed an increase in choroid-RPE. Our study proposes copper to play an important role in mitochondrial biogenesis in RPE cells.

Free amino acids hydroxyproline, lysine, and glycine promote differentiation of retinal pericytes to adipocytes: A protective role against proliferative diabetic retinopathy.

AIM: This study was conducted to estimate the aminoacid levels in the vitreous of patients with proliferative diabetic retinopathy, and to correlate it with the adiponectin levels. Secondly to test if these amino acids can alter or induce adiponectin levels and its related factors in retinal cells like pericyte as an in vitro model. METHODS: All human studies were done as per declaration of Helsinki with institutional approval and after obtaining consent from participating individuals. The vitreous amino acids were estimated in PDR (Proliferative diabetic retinopathy) and MH (Macular Hole) as disease control using HPLC. Bovine retinal pericytes (BRP) were cultured in DMEM/F12 medium and treated with 0.5 mM of any one of the individual amino acids (proline, hydroxyproline, phenylalanine, alanine, serine, glycine, lysine, isoleucine or valine) along with 100 nM insulin for 14 days in high glucose (25 mM) condition. The mRNA expression profile of adipogenic markers (such as Pref1, APN, ZAG and PPARγ), angiogenic markers (VEGF, MMP-2 and MMP-9, TGF-ß) and antioxidant markers (Nrf2 and UCP-2) were evaluated by qPCR. Adipogenesis was further confirmed by adipogenesis assay, secretion of adiponectin in medium and triglyceride accumulation by Oil red O staining in Bovine retinal pericytes. RESULTS: Amino acids valine (p < 0.004), isoleucine (p < 0.0007), leucine (p < 0.022), serine (p < 0.0007), glycine (p < 0.001), alanine (p < 0.017), phenylalanine (p < 0.013), and lysine (p < 0.001) were significantly elevated in the vitreous of PDR group (n = 30) when compared to macular hole (n = 20). There was a significant positive correlation between serine (p < 0.021), alanine (p < 0.00016), phenylalanine (p < 0.04), isoleucine (p < 0.023), leucine (p < 0.043), and lysine (p < 0.026) with adiponectin level in the vitreous. The amino acids hydroxyproline, proline, lysine, glycine and alanine induced the triglyceride accumulation and expression of Adiponectin. VEGF and MMP-9 expression was decreased with all the amino acids treated and PEDF was significantly increased with phenylalanine treatment. TGFß mRNA expression showed a significant decrease with proline, alanine, glycine, lysine and isoleucine. The Nrf2 expression was significantly increased in alanine and serine when compared to control. The UCP-2 gene showed a significant increase in proline and lysine treatment. DISCUSSION AND CONCLUSION: Our results suggest that amino acids hydroxyproline, proline, lysine, glycine and alanine which are elevated in the PDR vitreous show a tendency to induce adipogenic effects in retinal pericytes by triggering the accumulation of triglycerides and adiponectin. Hence we hypothesize that these aminoacids when elevated along with insulin and glucose can induce metabolic changes in pericytes. The functional implications of these changes tend to be protective as it increases the antioxidant potential and decreases the angiogenesis markers which are potentially pathogenic.

Insights on ornithine decarboxylase silencing as a potential strategy for targeting retinoblastoma.

Ornithine Decarboxylase (ODC) is a key enzyme involved in polyamine synthesis and is reported to be up regulated in several cancers. However, the effect of ODC gene silencing in retinoblastoma is to be understood for utilization in therapeutic applications. Hence, in this study, a novel siRNA (small interference RNA) targeting ODC was designed and validated in Human Y79 retinoblastoma cells for its effects on intracellular polyamine levels, Matrix Metalloproteinase 2 & 9 activity and Cell cycle. The designed siRNA showed efficient silencing of ODC mRNA expression and protein levels in Y79 cells. It also showed significant reduction of intracellular polyamine levels and altered levels of oncogenic LIN28b expression. By this study, a regulatory loop is proposed, wherein, ODC silencing in Y79 cells to result in decreased polyamine levels, thereby, leading to altered protein levels of Lin28b, MMP-2 and MMP-9, which falls in line with earlier studies in neuroblastoma. Thus, by this study, we propose ODC silencing as a prospective strategy for targeting retinoblastoma.

Effect of adiponectin on expression of vascular endothelial growth factor and pigment epithelium-derived factor: an in vitro study.

AIM: This study was carried out to identify the role of adiponectin (APN) in modulating the expression of vascular endothelial growth factor (VEGF) and pigment epithelial-derived factor (PEDF) in relation to ocular angiogenesis. MATERIALS AND METHODS: Human retinal pigment epithelial cell lines (ARPE-19) were cultured in Dulbeco's minimum essential medium with 10% fetal bovine serum (FBS) and exposed to varying concentrations of recombinant adiponectin (5-5 ng/ml) for 1 h. Analysis of VEGF and PEDF mRNA was done by reverse transcriptase and further quantified by quantitative polymerase chain reaction. VEGF and PEDF protein expression were studied using enzyme-linked immune sorbent assay (ELISA). STATISTICAL ANALYSIS: Unpaired Student's t-test was used to analyze the significance. P < 0.05 was accepted as statistically significant. RESULTS: ARPE cells exposed to APN showed decreased expression of VEGF mRNA, protein whereas PEDF protein is unaltered and PEDF mRNA was increased. CONCLUSION: Our in vitro study on ARPE exposed to APN showed a negative correlation with VEGF levels. Thus indicating the protective role for APN in angiogenesis-related diseases.

Tolerance of ARPE 19 cells to organophosphorus pesticide chlorpyrifos is limited to concentration and time of exposure.

Age related macular degeneration is a blinding disease common in elder adults. The prevalence of age related macular degeneration has been found to be 1.8% in the Indian population. Organophosphates are widely used insecticides with well documented neurological effects, and the persistent nature of these compounds in the body results in long term health effects. Farmers exposed to organophosphorus pesticides in USA had an earlier onset of age related macular degeneration when compared to unexposed controls. A recent study found significant levels of an organophosphate, termed chlorpyrifos, in the blood samples of Indian farmers. Therefore, in understanding the link between age related macular degeneration and chlorpyrifos, the need for investigation is important. Our data show that ARPE-19 (retinal pigment epithelial cells) exhibit a cytoprotective response to chlorpyrifos as measured by viability, mitochondrial membrane potential, superoxide dismutase activity, and increased levels of glutathione peroxidase and reduced glutathione, after 24 h exposure to chlorpyrifos. However, this cytoprotective response was absent in ARPE-19 cells exposed to the same range of concentrations of chlorpyrifos for 48 h. These results have physiological significance, since HPLC analysis showed that effects of chlorpyrifos were mediated through its entry into ARPE-19 cells. HPLC analysis also showed that chlorpyrifos remained stable, as we recovered up to 80% of the chlorpyrifos added to 6 different ocular tissues.

Isthmin is a novel secreted angiogenesis inhibitor that inhibits tumour growth in mice.

Anti-angiogenesis represents a promising therapeutic strategy for the treatment of various malignancies. Isthmin (ISM) is a gene highly expressed in the isthmus of the midbrain-hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kD protein containing a thrombospondin type 1 repeat domain in the central region and an adhesion-associated domain in MUC4 and other proteins (AMOP) domain at the C-terminal. In this work, we demonstrate that ISM is a novel angiogenesis inhibitor. Recombinant mouse ISM inhibited endothelial cell (EC) capillary network formation on Matrigel through its C-terminal AMOP domain. It also suppressed vascular endothelial growth factor (VEGF)-basic fibroblast growth factor (bFGF) induced in vivo angiogenesis in mouse. It mitigated VEGF-stimulated EC proliferation without affecting EC migration. Furthermore, ISM induced EC apoptosis in the presence of VEGF through a caspase-dependent pathway. ISM binds to αvß(5) integrin on EC surface and supports EC adhesion. Overexpression of ISM significantly suppressed mouse B16 melanoma tumour growth through inhibition of tumour angiogenesis without affecting tumour cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmental vessels in the trunk. Our results demonstrate that ISM is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis.

Decorin derived antiangiogenic peptide LRR5 inhibits endothelial cell migration by interfering with VEGF-stimulated NO release.

Excessive angiogenesis plays critical roles in many human diseases including cancer. We have previously shown that human decorin derived 26 amino acids peptide Leucine Rich Repeat 5 (LRR5) inhibits multiple aspects of angiogenesis including vascular endothelial growth factor (VEGF) stimulated migration of endothelial cells (ECs). In this study, we have characterized the molecular mechanism of LRR5 which reveals that its anti-migratory effect on ECs is mediated by inhibiting VEGF-stimulated endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) release. LRR5 carried out this function through signaling pathways that involves PI3 kinase and Akt, but not ERK. This anti-NO release effect is mediated by the C-terminal 13 amino acids of LRR5, correlating with the anti-migratory function of this region.

The first but not the second thrombospondin type 1 repeat of ADAMTS5 functions as an angiogenesis inhibitor.

Angiogenesis is critical for tumour growth and metastasis where factors that regulate this process are potential targets for development of anti-cancer drugs. In this study, we show that the first TSR domain of the extracellular matrix protease ADAMTS5, unlike the second TSR, has anti-angiogenic activities where it inhibits endothelial cell tube formation on Matrigel, reduces cell proliferation and attachment, while promoting cell apoptosis and migration, all in a dose-dependent manner. Furthermore, it influences the architecture of endothelial cells by disrupting actin stress fibres and reducing focal adhesions, likely via suppressing RhoA activation. TSR1 of ADAMTS5 is therefore a novel anti-angiogenic peptide and could serve as a prototype for future development into anti-cancer drugs.

Developing antiangiogenic peptide drugs for angiogenesis-related diseases.

Angiogenesis is regulated by stimulators and inhibitors and involve multiple biological processes including endothelial cell proliferation, migration, cell-cell and cell-matrix adhesion, assembly into tube structures as well as apoptosis. Designing and developing peptides for therapeutic application to inhibit angiogenesis is an important area in antiangiogenic drug development. Small peptides have advantages over proteins for therapeutic application, due to their stability, solubility, increased bio-availability and lack of immune response in the host cell. Endogenous protein angiogenesis stimulators and inhibitors hold vital information for designing antiangiogenic peptides for drug development. These proteins function through their interaction with extracellular matrix molecules, cell surface receptors, proteases, as well as growth factors and cytokines. Conserved domains such as thrombospondin type 1 repeats (TSRs), kringle domains as well as critical amino acid residues present in these domains are involved in their functions. By exploiting these properties, several small peptides have been designed, synthetically made and being tested for therapeutic efficacy. Peptides derived from type 1 repeat of thrombospondin, alpha 4 and beta 1 chains of laminin, arginine rich N terminus of endostatin, leucine rich repeat 5 of decorin, pigment epithelium derived factor and N terminal of parathyroid hormone are examples of small antiangiogenic peptides derived from endogenous proteins. Such bioactive peptides are further modified physico-chemically to increase their potency and stability. In addition, phage-display library screening and combinatorial approach are also in use to identify novel antiangiogenic peptides targeting tumour and various proteins. This review will provide a comprehensive summary of the current status of the antiangiogenic peptides and their relevance for drug designing and development. Several critical issues that need to be resolved in translating this concept into clinical practice are also discussed.

Eales' disease: oxidant stress and weak antioxidant defence.

Eales' disease (ED) is an idiopathic retinal periphlebitis characterized by capillary non-perfusion and neovascularization. In addition to the existing system, a new staging system has been proposed by Saxena et al. Immunological, molecular biological and biochemical studies have indicated the role of human leucocyte antigen, retinal S antigen autoimmunity, Mycobacterium tuberculosis genome, free radical damage and possibly hyperhomocysteinemia in its etiopathogenesis, which appears multifactorial. Oxidant stress has been shown by increase in the levels of thiobarbituric acid reactive substances (lipid oxidation) in the vitreous, erythrocytes, platelets and monocytes. A decrease in vitamins E and C both in active and healed vasculitis, superoxide dismutase, glutathione and glutathione peroxidase showed a weakened antioxidant defence. Epiretinal membrane from patients of ED who underwent surgery showed, by immunolocalization, presence of carboxy methyl lysine, an advanced glycation end product formed by glycoxidation and is involved in angiogenesis. OH. free radical accumulation in monocytes has been directly shown by electron spin resonance spectrometry. Free radical damage to DNA and of protein was shown by the accumulation of 8 hydroxy 2 deoxyguanosine (in leucocytes) and nitrotyrosine (in monocytes), respectively. Nitrosative stress was shown by increased expression of inducible nitric oxide synthase in monocytes in which levels of iron and copper were increased while those of zinc decreased. A novel 88 kDa protein was found in serum and vitreous in inflammatory condition and had antioxidant function. Platelet fluidity was also affected. Oral, methotrexate in low dosage (12.5 mg/week for 12 weeks) as well as oral vitamin E (400 IU) and C (500 mg) daily for 8 weeks are reported to have beneficial effects.

Biochemistry of homocysteine in health and diseases.

The amino acid homocysteine (Hcy), formed from methionine has profound importance in health and diseases. In normal circumstances, it is converted to cysteine and partly remethylated to methionine with the help of vit B12 and folate. However, when normal metabolism is disturbed, due to deficiency of cystathionine-beta-synthase, which requires vit B6 for activation, Hcy is accumulated in the blood with an increase of methionine, resulting into mental retardation (homocystinuria type I). A decrease of cysteine may cause eye diseases, due to decrease in the synthesis of glutathione (antioxidant). In homocystinurias type II, III and IV, there is accumulation of Hcy, but a decrease of methionine, thus, there is no mental retardation. Homocysteinemia is found in Marfan syndrome, some cases of type I diabetes and is also linked to smoking and has genetic basis too. In hyperhomocysteinemias (HHcys), clinical manifestations are mental retardation and seizures (type I only), ectopia lentis, secondary glaucoma, optic atrophy, retinal detachment, skeletal abnormalities, osteoporosis, vascular changes, neurological dysfunction and psychiatric symptoms. Thrombotic and cardiovascular diseases may also be encountered. The harmful effects of homocysteinemias are due to (i) production of oxidants (reactive oxygen species) generated during oxidation of Hcy to homocystine and disulphides in the blood. These could oxidize membrane lipids and proteins. (ii) Hcy can react with proteins with their thiols and form disulphides (thiolation), (iii) it can also be converted to highly reactive thiolactone which could react with the proteins forming -NH-CO- adducts, thus affecting the body proteins and enzymes. Homocystinuria type I is very rare (1 in 12 lakhs only) and is treated with supplementation of vit B6 and cystine. Others are more common and are treated with folate, vit B12 and in selected cases as in methionine synthase deficiency, methionine, avoiding excess. In this review, the role of elevated Hcy levels in cardiovascular, ocular, neurologial and other diseases and the possible therapeutic measures, in addition to the molecular mechanisms involved in deleterious manifestations of homocysteinemia, have been discussed.

Plasma homocysteine and total thiol content in patients with exudative age-related macular degeneration.

PURPOSE: Exudative age-related macular degeneration (ARMD) is one of the debilitating ocular complications, which results in permanent blindness. Elevated homocysteine (Hcys) levels have been associated in the development of several vascular diseases. Vascular and oxidative stress theories have been implicated for the development of choroidal neovascularization in exudative ARMD. The aim of the present study was to investigate the possible role of plasma Hcys and thiol content (tSH) as a risk factor for the development of exudative ARMD. METHOD: A total of 16 patients with exudative ARMD and 20 age-matched controls were recruited for the study. Plasma Hcys levels were analysed using Reverse Phase High Performance Liquid Chromatography. Plasma glutathione (GSH) content was determined using o-phthalaldehyde (OPA) derivatization and subsequent detection by fluorimeter. Plasma tSH levels were determined by using thiol-specific reagent dithionitrobenzoic acid (DTNB) spectrophotometrically. RESULTS: Plasma Hcys levels in exudative ARMD were elevated three-fold (18+/-5.0 microM) when compared to healthy controls (6.7+/-1.8 microM). There was a two-fold decrease in the GSH and tSH in exudative ARMD when compared with controls. Negative correlation was observed between diminished tSH and Hcys levels (r=-0.4837, P=0.05). Similarly plasma Hcys levels negatively correlated with GSH content (r=-0.6620, P<0.05). CONCLUSION: Results from our present study revealed that there is an elevated Hcys level and diminished thiol pool content in exudative ARMD that are significant.

Primary vitrectomy for combined rhegmatogenous retinal detachment and choroidal detachment with or without oral corticosteroids: a pilot study.

PURPOSE: The occurrence of choroidal detachment (CD) in eyes with primary rhegmatogenous retinal detachment (RRD) is relatively uncommon (2%-4.5%). Recent reports suggest that primary vitrectomy yields better anatomic success than scleral buckling. However, for these inflamed eyes with low intraocular pressure, the influence of preoperative oral steroids on reattachment rates has not been elucidated yet. METHODS: Twenty eyes with combined RRD and CD that underwent primary vitrectomy were randomized to receive oral steroids (for 1 week) or no oral steroids before surgery. RESULTS: Preoperative clinical data such as mean age, lens status, Snellen visual acuity, duration of macular detachment, CD (size and extent), and retinal detachment characteristics (e.g., extent, number of retinal breaks, atrophic or tractional retinal break, size of retinal break, and location of retinal break) were similarly distributed in both groups. Single-operation anatomic success was 81.8% (9/11) among those patients who received preoperative oral steroids and was 66.7% (6/9) among those who did not receive preoperative oral steroids. After reoperation, anatomic success was 100% in both groups. The mean follow-up was 20.1 months. CONCLUSION: The results suggest that administration of oral steroids before primary vitrectomy in eyes with combined RRD and CD improves reattachment rates.

Bilateral corneal fibrosis in homocystinuria: case report and transmission electron microscopic findings.

PURPOSE: To report the unusual occurrence of bilateral, superficial, corneal fibrosis with pannus formation in a young woman with homocystinuria, and to describe the light and transmission electron microscopic findings of these deposits. METHODS: Excision of the corneal lesion and amniotic membrane transplantation was performed in both eyes. The excised material was studied using light and transmission electron microscopy. RESULTS: Whitish, elevated, irregular masses with superficial vascularization in the peripheral cornea were noted in both eyes. Histopathologic evaluation of the excised corneal tissue revealed variable epithelial thickness with melanin pigment in the basal layer. A fibrovascular pannus, fibrosis and disruption of the Bowman's layer, and fibrosis of anterior stroma were evident. Transmission electron microscopy revealed numerous empty intracytoplasmic vacuoles in the corneal epithelial cells and intracytoplasmic inclusions containing fibrillogranular material in the cytoplasm of keratocytes. CONCLUSION: We report the unusual association of corneal fibrosis and scarring in a young woman with homocystinuria. The deposits revealed the presence of membrane-bound inclusions containing fibrillogranular material in the corneal epithelium and keratocytes. Although the etiology of these deposits is not clear, the condition improved following excision of these lesions and amniotic membrane transplantation.

Eales disease--an update.

Eales disease, first described by Henry Eales in 1880, remains an enigma. The disease, observed more commonly in the Indian subcontinent than in the rest of the world, occurs in young healthy adult males, initially presenting as retinal periphlebitis and later as retinal ischemia that may lead to vascular alterations and neovascularization. Recurrent vitreous hemorrhage with or without retinal detachment is the common sequelae. In recent years, immunological, molecular biological, and biochemical studies have indicated the role of human leukocyte antigen, retinal autoimmunity, mycobacterium tuberculosis genome, and free radical mediated damage in the etiopathogenesis of this disease. However, its etiology appears to be multifactorial. The management depends on the stage of the disease and consists of medical treatment with oral corticosteroids in the active inflammatory stage and laser photocoagulation in the advanced retinal ischemia and neovascularization stages. The results of vitreoretinal surgery have been found to be satisfactory in case of vitreous hemorrhage with or without retinal detachment.

Immunolocalization and quantification of advanced glycation end products in retinal neovascular membranes and serum: a possible role in ocular neovascularization.

PURPOSE: Earlier studies have revealed the association of advanced glycation end products (AGE) with the pathogenesis of various micro and macro vascular complications. The purpose of the present study is to localize AGEs, namely carboxy methyl lysine (CML-AGE) and methyl glyoxal-derived AGEs (MG-AGE), in retinal neovascular membranes and to quantify them in serum samples. METHODS: Surgically excised retinal neovascular membranes and serum samples obtained from patients with diabetic retinopathy, Eales' disease and nondiabetics were studied. Immunolocalization of AGEs namely CML-AGE and MG-derived AGEs was done using avidin biotin complex method and quantification was done by enzyme linked immunosorbent assay (ELISA). RESULTS: CML-AGE immunoreactivity was detected in all cases of Eales' disease and 61% cases of diabetic retinopathy and none in idiopathic epiretinal membrane (ERM). MG-AGE immunoreactivity was observed in approximately 15% of diabetic retinopathy and none in Eales' disease and and idiopathic ERM. Quantification of AGEs in serum samples revealed statistically significant increased levels of MG-AGE in diabetes, in relation to nondiabetics with idiopathic ERM and CML-AGE in Eales' disease, in relation to diabetics and nondiabetics with idiopathic ERM. CONCLUSION: Results from this study suggest that AGEs formed through glycation and glycoxidation may play an important role in the development of retinal neovascularization. The immunoreactivity of CML-AGEs in neovascular membrane and its increased levels in serum suggest that inspite of the normoglycemic status, glycoxidation and lipid peroxidation due to oxidative stress may trigger retinal neovascularization in Eales' disease, while MG-AGEs in diabetic membrane and serum suggest the role of glycation. Thus the mechanism of neovascularization in different pathological conditions could be different.

Purification and characterization of a novel 88 kDa protein from serum and vitreous of patients with Eales' disease.

Eales' disease is a perivasculitis that affects the peripheral retina of young adults and results in recurrent vitreous hemorrhage. Although increased oxidative stress and decreased antioxidant defense have been reported to be associated with Eales' disease, the exact cause for the disease and its pathogenesis are not known. Here is reported the identification, purification and characterization of a new protein from the serum and vitreous of patients with Eales' disease. This protein was purified using preparative electrophoresis and HPLC. The purified protein had a retention time of 9.2 min in RP HPLC. Its molecular weight as determined by gel permeation chromatography was 88 kDa hence, it was termed as 88 kDa protein. Alcian blue and Schiffs staining revealed 88 kDa protein to be a glycoprotein. Proteins purified from both serum and vitreous exhibited anti lipid peroxidation effect on erythrocyte when added during in vitro assay of thiobarbuteric acid reactive substances (TBARS). In addition to this property the protein also has Fe(2+)sequestering effect. The anti TBARS activity of 88 kDa protein was completely inhibited by 0.1 m M concentration of parachlromercuric benzoate (PCMB) and 5,5' dithiobis(2-nitrobenzoic acid) DTNB. The total thiol content (cysteine) of the purified 88 kDa protein was found to be 8% by mass. Eighty eight kDa protein from both the sources namely vitreous and serum are immunologically identical when studied using polyclonal antibodies raised in goat against purified serum protein. The N terminal sequence of 88 kDa protein by automated Edman's degradation chemistry is A D D P N S L S P S A F A E A L A L L R D S X L A R F V. The protein and DNA data base search revealed no match to 88 kDa protein and hence this was considered as unique protein. Further knowledge on the in vivo function of 88 kDa protein is very important to understand its role in the pathogenesis of Eales' disease.

Homocystinuria with congenital/developmental cataract.

The aim of the study is to screen patients for homocystinuria with and without cataract and analyse for homocystine and methionine. Fifty-eight samples from 29 patients, i.e., plasma and urine collected after overnight fasting were analysed by the screening test for homocystine, and paper chromatography for homocystine and methionine. Out of 29 homocystinuric patients, 24 had cataract. Only one had appreciable amounts of methionine in his serum. He also had mental retardation as expected and belongs to Type I. The other types did not have methionine but had only homocystine. There was no mental retardation or ectopia lentis. So they belonged to Types II, III or IV. As there is excess methionine in Type I, with low cystine, cataract may be due to deficiency of cysteine and reduced glutathione and might be averted by suitable therapy, i.e., high cystine-low methionine diet with B6. In other types with low methionine, cataract may be due to decreased availability of amino acids for the synthesis of lens proteins; the treatment of choice should be B12, and folate with methionine.