Research opportunities in vision: a report of the U.S.-Indo workshops on collaborative research.
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Animals↗
Biomedical subjects
D Balasubramanian
Publications and source records attributed to D Balasubramanian.
Myocilin gene implicated in primary congenital glaucoma.
Primary congenital glaucoma (PCG) has been associated with CYP1B1 gene (2p21), with a predominantly autosomal recessive mode of inheritance. Our earlier studies attributed CYP1B1 mutations to only 40% of Indian PCG cases. In this study, we included 72 such PCG cases where CYP1B1 mutations were detected in only 12 patients in heterozygous condition, implying involvement of other gene(s). On screening these patients for mutations in myocilin (MYOC), another glaucoma-associated gene, using denaturing high-performance liquid chromatography followed by sequencing, we identified a patient who was double heterozygous at CYP1B1 (c.1103G>A; Arg368His) and MYOC (c.144G>T; Gln48His) loci, suggesting a digenic mode of inheritance of PCG. In addition, we identified the same MYOC mutation, implicated for primary open angle glaucoma, in three additional PCG patients who did not harbor any mutation in CYP1B1. These observations suggest a possible role of MYOC in PCG, which might be mediated via digenic interaction with CYP1B1 and/or an yet unidentified locus associated with the disease.
Aryl Hydrocarbon Hydroxylases↗
Allelic heterogeneity of the carbohydrate sulfotransferase-6 gene in patients with macular corneal dystrophy.
Allelic heterogeneity of the carbohydrate sulfotransferase-6 gene in patients with macular corneal dystrophy. Macular corneal dystrophy (MCD) is an autosomal recessive disorder characterized by grayish white opacities in the cornea. It is caused by mutations in the carbohydrate sulfotransferase-6 (CHST6) gene, which codes for the enzyme corneal N-acetylglucosamine-6-sulfotransferase. This enzyme catalyzes the sulfation of keratan sulfate, an important component of corneal proteoglycans. We screened 31 patients from 26 families with MCD for mutations in the coding region of the CHST6 gene. Twenty-six different mutations were identified, of which 14 mutations are novel. The novel mutations are one nonsense mutation found in one patient (Trp2Ter), one frameshift (insertion plus deletion) mutation in two patients (His335fs), and 12 missense mutations (Leu3Met, Ser54Phe, Val56Arg, Ala73Thr, Ser98Leu, Cys165Trp, Ser167Phe, Phe178Cys, Leu193Pro, Pro204Arg, Arg272Ser, and Arg334Cys) in 11 patients. These data demonstrate a high degree of allelic heterogeneity of the CHST6 gene in patient populations with MCD from Southern India, where this disease may have a relatively higher prevalence than in outbred communities.
Adolescent↗
Desmoplastic infantile ganglioglioma -- a case report.
Desmoplastic infantile ganglioglioma is a very rare supratentorial tumor occurring in the first two years of life. A five-month-old female infant presented with recurrent seizures, large head and loss of acquired milestones. Computerized Tomographic Scan of brain showed a large subarachnoid cyst with a solid intensely contrast enhancing tumor in the right temporoparietal region with severe degree of mass effect. Craniotomy and total excision of the tumor followed subsequently by subduro-peritoneal shunt for the extracerebral fluid collection was done. The child made good recovery. Histopathology revealed features of desmoplastic infantile ganglioglioma, viz., marked desmoplastic component with glial and neuronal elements. Immunohistochemistry showed positive staining for glial fibrillary acidic protein (GFAP) with areas of synaptophysin and chromogranin positivity. Desmoplastic infantile ganglioglioma is a rare tumor of infancy, which has excellent prognosis after total excision. No adjuvant therapy is required. This is the first Indian report of desmoplastic infantile ganglioglioma out of less than fifty cases reported worldwide.
Astrocytes↗
Successful reconstruction of damaged ocular outer surface in humans using limbal and conjuctival stem cell culture methods.
When the ocular outer surface is badly damaged, subsequent corneal transplantation fails due to the absence of basal cells that are needed to support the graft. With the realization that the limbus and the conjunctiva have adult stem cells that can be cultured, it has been possible for us to explant culture these on de-epithelized human amniotic membrane, and to graft the resulting viable and transparent epithelium to 125 needy human patients with success. Ultrastructural, histological, biochemical and immunological assays establish the identity of the cells and the tissue formed.
Amnion↗
Transglutaminase-mediated cross-linking of alpha-crystallin: structural and functional consequences.
Aggregation and covalent cross-linking of the crystallins, the major structural proteins of the eye lens, increase light scattering by the lens leading to opacification and cataract. Disturbance of calcium homeostasis in the tissue is one of the factors implicated in cataractogenesis. Calcium-activated transglutaminase (TG)-catalyzed cross-linking of some lens proteins has been reported earlier. We show here that alpha-crystallin, a major structural protein in the lens and a member of the small heat shock protein family, is also a substrate for TG-mediated cross-linking, indicating the presence of donor Lys and acceptor Gln residues in the protein. Upon TG-catalyzed dimerization, the secondary and tertiary structures of the protein are altered, and its surface hydrophobicity reduced. The chaperone-like property of the protein, suspected to be one of its functions in situ, is substantially reduced upon such cross-linking. These results, taken together with earlier ones on lens beta-crystallins and vimentin, suggest that TG-mediated events might compromise lens function. Also, since alpha-crystallin occurs not only in the lens but in other tissues as well, such TG-catalyzed cross-linking and the associated alterations in its structure and activity would be of general pathological interest.
Animals↗
Enzymatic, clinical and histologic evaluation of corneal tissues in experimental fungal keratitis in rabbits.
Mycotic keratitis, being frequently refractive to most of the currently available antifungal therapy, continues to pose a therapeutic challenge to the clinician. In keratitis of infectious etiology stromal dissolution may be brought about by a combination of agent and host factors. An understanding of the source and nature of corneal tissue damage is essential for evolving more effective therapeutic modalities in the treatment of fungal keratitis. In the present study, we have characterized the extracellular proteases produced in vitro by corneal fungal pathogens namely the Aspergillus flavus and Fusarium solani when collagen was provided as the sole nitrogen source. In addition, fungal infected rabbit corneas were investigated for proteolytic activities and nature of inflammatory reaction. Gelatin zymography detected protease bands with molecular mass ranging from 100 to 200 kDa in the culture extracts of A. flavus, and a single major band of molecular mass approximately 200 kDa in the culture extracts of F. solani. A basal proteolytic activity of mass 65 kDa was visualized in all uninfected and infected rabbit corneal extracts. Infected corneas in addition revealed the presence of additional proteolytic species of mass 92 and 200 kDa. The enzyme inhibitory profile suggested that fungal cultures in vitro contained predominantly serine protease activity and to a lesser extent metalloprotease activity. However, fungal infected corneal homogenates showed the presence of metalloproteinase activity alone, the enzymatic activities entirely being sensitive to ethylene diamine tetra acetate (EDTA), a metalloprotease inhibitor. Interestingly, the serine proteolytic activity detected in fungal cultures in vitro was not present in the fungal infected corneas in vivo. However, the possible role of fungal serine proteases in the activation of corneal matrix metalloproteinases (MMPs) cannot be ruled out. Based on the criteria of molecular mass, proteolytic activity in the presence of calcium at neutral pH, and sensitivity to inhibition by a metalloprotease inhibitor, the 65 and 92 kDa gelatinases were identified as MMP 2 and MMP 9, respectively. The expression of 92 and 200 kDa gelatinases correlated positively with the amount of polymorphonuclear cells present in the infected tissues. Activated resident corneal cells or inflammatory cells may largely contribute to the increased proteolytic activities in fungal infected corneas resulting in tissue matrix degradation in fungal keratitis.
Animals↗
Antioxidant properties of green and black tea, and their potential ability to retard the progression of eye lens cataract.
Aqueous extracts of green and black tea are shown to quench reactive oxygen species such as singlet oxygen, superoxide and hydroxyl radicals, prevent the oxidative cross-linking of test proteins and inhibit single strand breakage of DNA in whole cells. They are also seen to be able to counteract the oxidative insult mounted by cigarette smoke. In rats in which cataract was induced by subcutaneous injection of selenite, administration of green or black tea extracts led to a retardation of the progression of lens opacity, suggesting the potential cataracto-static ability of tea.
Animals↗
Identification of a novel fluorophore, xanthurenic acid 8- O -beta-D-glucoside in human brunescent cataract.
We have identified the chemical structure of a novel protein-unbound fluorescent glucoside (Fl-Glc), found to be far more abundant in the human brunescent cataractous lens nuclei than in non-brunescent ones. Our earlier experiments showed that long-term incubation of the protein-free filtrate of non-brunescent cataractous nuclei generated increasing amounts of a particular yet to be characterized fluorophore (Fl-X). High performance liquid chromatography (HPLC) analyses revealed Fl-X and Fl-Glc to be identical. HPLC-electrospray ionization-mass spectrometry (HPLC-ESI-MS) disclosed the molecular weights (MW) of Fl-X and its beta-glucosidase-digest (Fl-X-aglycon) to be 367 and 205, respectively. Fl-X-aglycon and authentic xanthurenic acid (MW = 205) not only eluted at exactly the same retention time on HPLC but also revealed their protonated ions at the same m/z of 206.1 by positive ion analysis on HPLC-ESI-MS. These results suggest that Fl-X ( = Fl-Glc) is a beta-glucoside of xanthurenic acid. Fl-Glc was finally identified as xanthurenic acid 8- O -beta- D -glucoside because the retention times of both completely agreed with three kinds of HPLC conditions.
Cataract↗
Structural studies on some dityrosine-cross-linked globular proteins: stability is weakened, but activity is not abolished.
We have carried out conformational and stability studies on three proteins that have previously been shown to undergo dityrosine (DT) cross-linking. They include the monomers and dimers of DT-cross-linked calmodulin and the dimers of bovine pancreatic ribonuclease A and bovine eye lens gammaB-Crystallin. In each of these cases, we find the secondary and tertiary structure of the parent protein to be largely maintained. The DT dimer is, however, weaker than the parent. In this sense, the properties of these DT dimers are somewhat similar to those of glutaraldehyde-cross-linked protein crystals. In contrast, the intramolecularly DT-linked monomeric protein that we studied (DT monomer of calmodulin) is seen to have suffered greater changes in its conformation and stability. These results gain significance in light of the growing identification of DT formation as a marker of oxidative stress, aging, and disease.
Calmodulin↗
beta-carbolines that accumulate in human tissues may serve a protective role against oxidative stress.
beta-Carbolines are tricyclic nitrogen heterocycles formed in plants and animals as Maillard reaction products between amino acids and reducing sugars or aldehydes. They are being detected increasingly in human tissues, and their physiological roles need to be understood. Two beta-carboline carboxylates have been reported to accumulate in the human eye lens. We report here on the identification of another beta-carboline, namely 1-methyl-1-vinyl -2, 3,4-trihydro-beta-carboline-3-carboxylic acid, in the lenses of some cataract patients from India. Analysis of these three lenticular beta-carbolines using photodynamic and antioxidant assays shows all of them to be inert as sensitizers and effective as antioxidants; they quench singlet oxygen, superoxide and hydroxyl radicals and inhibit the oxidative formation of higher molecular weight aggregates of the test protein, eye lens gamma-crystallin. Such antioxidative ability of beta-carbolines is of particular relevance to the lens, which faces continual photic and oxidative stress. The beta-carboline diacid IV is also seen to display an unexpected ability of inhibiting the thermal coagulation of gamma-crystallin and the dithiothreitol-induced precipitation of insulin. These results offer experimental support to earlier suggestions that one of the roles that the beta-carbolines have is to offer protection against oxidative stress to the human tissues where they accumulate.
Aged↗
Ultraviolet radiation and cataract.
While solar radiation falling on earth comprises light in the infrared, visible, UVA, UVB, and even UVC ranges, the light incident on, and thus important to the biology of, the eye lens is essentially in the visible and UVA regions. Thus, direct photochemical damage to the lens from UVB radiation is minor, though long-term UVA (and even visible range) irradiation is seen to lead to lens malfunction. Short-term exposure of the lens in vivo to UVA light leads to compromised optical and biochemical properties which are repaired in time, while higher doses affect permanent damage. Such longer wavelength light-mediated changes in the lens occur through photodynamic means, affected by some of the compounds that accumulate in the lens over a period of time, which act as sensitizers. Isolation and chemical identification of over a dozen such compounds has been done, and their photoactive properties have been studied. While several of these are photodynamic and generate reactive oxygen species when UVA light is shone on them, other compounds that accumulate in the lens act as antioxidants.
Cataract↗
Molecular genetics of cataract.
Studies on hereditary congenital cataracts have led to the identification of genes involved in formation of these cataracts. Knowledge of the structure and function of a particular gene and the effect of disease-associated mutations on its function are providing insights into the mechanisms of cataract. Identification of the disease gene requires both the relevant clinical data as well as genetic data on the entire pedigree in which the disease is found to occur. Genes for hereditary cataract have been mapped by genetic linkage analysis, in which one examines the inheritance pattern of DNA markers throughout the genome in all individuals of the pedigree, and compares those with the inheritance of the disease. Cosegregation of a set of markers with disease implies that the disease gene is present at the same chromosomal location as those markers. The genes so far identified for hereditary cataracts in both humans and animal models encode structural lens proteins, gap junction proteins, membrane proteins and regulatory proteins involved in lens development. Understanding of the mechanisms of hereditary cataract may also help us understand the manner in which environmental and nutritional factors act on the lens to promote opacification.
Animals↗
An atypical form of alphaB-crystallin is present in high concentration in some human cataractous lenses. Identification and characterization of aberrant N- and C-terminal processing.
Two unique polypeptides, 22.4 and 16.4 kDa, were prominent in some human cataracts. Both proteins were identified as modified forms of the small heat shock protein, alphaB-crystallin. The concentration of total alphaB-crystallin in most of these cataracts was significantly increased. The 22.4-kDa protein was subsequently designated as alphaB(g). Mass spectrometric analyses of tryptic and Asp-N digests showed alphaB(g) is alphaB-crystallin minus the C-terminal lysine. alphaB(g) constituted 10-90% of the total alphaB-crystallin in these cataracts and was preferentially phosphorylated over the typical form of alphaB-crystallin. Human alphaB(g) and alphaB-crystallin were cloned and expressed in Escherichia coli. The differences in electrophoretic mobility and the large difference in native pI values suggest some structural differences exist. The chaperone-like activity of recombinant human alphaB(g) was comparable to that of recombinant human alphaB-crystallin in preventing the aggregation of lactalbumin induced by dithiothreitol. The mechanism involved in generating alphaB(g) is not known, but a premature termination of the alphaB-crystallin gene was ruled out by sequencing the polymerase chain reaction products of the last exon for the alphaB-crystallin gene from lenses containing alphaB(g). The 16.4-kDa protein was an N-terminally truncated fragment of alphaB(g). The high concentration of alphaB-crystallin in these cataracts is the first observation of this kind in human lenses.
Amino Acid Sequence↗
Artificial chaperoning of insulin, human carbonic anhydrase and hen egg lysozyme using linear dextrin chains--a sweet route to the native state of globular proteins.
Linear dextrins (alpha-1,4-D-glucopyranoside chains) are known to possess amphiphilic surfaces and solubilize lipophilic compounds. We have assessed the ability of this amphiphilic surface of dextrin to inhibit the self-aggregation and assist the refolding of proteins. Addition of decameric dextrin, or dextrin-10, in the renaturation buffer improves the refolding yield of human carbonic anhydrase from its guanidinium chloride-induced denatured state. It is also seen to inhibit the self-aggregation of insulin. The ability of dextrin-10 to interact with cetyltrimethylammonium bromide and postpone its critical micellar concentration allows the use of dextrin-10 as a 'detergent stripping agent' in a novel artificial chaperoning process described earlier. The aggregation of human carbonic anhydrase and lysozyme upon refolding is prevented by cetyltrimethylammonium bromide due to the formation of a protein-detergent complex; dextrin-10 strips off the detergent from the complex and allow the proteins to fold, thus increasing the renaturation yield. Dextran-4 (the alpha-1,6-D-glucopyranoside chain), which does not exhibit amphiphilic properties, does not help in such artificial chaperoning.
Animals↗
Structure and stability of the dityrosine-linked dimer of gammaB-crystallin.
Oxidative damage to proteins leads to a variety of modifications such as racemization, carbonyl compound formation, new fluorophores, aggregation, crosslinking and insolubility, several of which are markers of pathogenesis. A particular modification that has been associated with abnormal and pathological situations is the dityrosine crosslink in proteins, thought to be responsible for the reduced solubility and elasticity of proteins, and plaque formation. Dityrosine crosslinking has been suspected to occur in the crystallins of the eye lens during cataract. We focus attention here on the generation, structure and conformational stability of such a dityrosine-linked protein of the eye lens. We find this crosslink to be readily generated photodynamically in the presence of sensitizers. Among the crystallins, crosslinking occurs most readily in the gamma-crystallins under these conditions. We have isolated, purified and studied the properties of the dityrosine-linked dimer of the eye lens protein gammaB-crystallin. While the dityrosine crosslink does not alter the secondary structure of the protein, it changes the tertiary structure in a subtle manner. This alteration destabilizes the dimer, which denatures more readily than the parent monomer, and also makes it precipitate more readily, a point of relevance to cataractogenesis of the eye lens. Comparison of these results with those reported on other dityrosine-dimerized proteins suggests that while the conformation of these proteins might not be altered in a major manner upon dityrosine linkage, the dimer is structurally less stable and displays reduced solubility, both of which are of pathological importance.
Animals↗
The bacterial pigment xanthomonadin offers protection against photodamage.
Xanthomonas oryzae pv. oryzae is a bacterial pathogen that causes leaf blight, a serious disease of rice. Most members of the genus Xanthomonas produce yellow, membrane bound, brominated aryl polyene pigments called xanthomonadins whose functional role is unclear. We find that pigment-deficient mutants of X. oryzae pv. oryzae exhibit hypersensitivity to photobiological damage. A clone containing the xanthomonadin biosynthetic gene cluster alleviates the hypersensitivity of the pigment-deficient mutant. Extracts containing xanthomonadin provide protection against photodynamic lipid peroxidation in liposomes. These results lead us to suggest a role for the pigment, namely protection against photodamage.
Anisoles↗