Exfoliation syndrome associated with LOXL1 gene polymorphisms in a Black patient from Latin America: a case report / Síndrome de esfoliação associada a polimorfismos do gene LOXL1 em paciente negro da América Latina: relato de caso

ABSTRACT A 89-year-old Black female with a 6-year history of advanced open-angle glaucoma was referred to the Glaucoma Service of the Ophthalmology Department - Federal University of São Paulo (UNIFESP). Best-corrected visual acuity was 20/400 in the right eye and 20/60 in the left eye. Pseudoexfoliation material was observed at the iris border, angle, and the anterior lens surface. Anterior biomicroscopy revealed exfoliation material forming an evident peripheral zone and a central disc separated by a clear intermediate zone on the anterior lens surface OU. Gonioscopy showed an open-angle Sampaolesis's line and whitish material deposits OU. Fundus examination revealed a cup-to-disc ratio of 1.0 OU with peripapillary atrophy. Genetic analysis for single nucleo­tide polymorphisms of the lysyl oxidase-like 1 gene linked to exfoliation syndrome identified two such single nucleotide polymorphisms, rs1048661 and rs216524.

RESUMO Uma mulher negra de 89 anos com um histórico de seis anos de glaucoma avançado de ângulo aberto avançado foi encaminhada ao Serviço de Glaucoma do Departamento de Oftalmologia da Universidade Federal de São Paulo (UNIFESP). A acuidade visual melhor corrigida era 20/400 no olho direito e 20/60 no olho esquerdo. Material pseudo-exfoliativo foi observado na borda iriana, ângulo e superfície anterior do cristalino. A biomicroscopia de segmento anterior demonstrou material exfoliativo formando uma zona periférica evidente e um disco central separado por uma zona intermediária livre na cápsula anterior do cristalino. A gonioscopia mostrou uma linha de Sampaolesi de ângulo aberto e depósitos esbranquiçados. O exame de fundo de olho revelou disco óptico com escavação total em ambos os olhos com atrofia peripapilar. A análise genética para polimorfismos de nucleotídeo único do gene semelhante à lysyl oxidase-like 1 ligado à síndrome de esfoliação identificou dois desses polimorfismos de nucleotídeo único, rs1048661 e rs216524.

Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus

Background: Glycine oxidase (GO), a type of D-amino acid oxidase, is of biotechnological interest for its potential in several fields. In our previous study, we have characterized a new glycine oxidase (BceGO) from Bacillus cereus HYC-7. Here, a variant of N336K with increased the affinity against all the tested substrate was obtained by screening a random mutant library of BceGO. It is observed that the residue N336 is invariable between its homogeneous enzymes. This work was aimed to explore the role of the residue N336 in glycine oxidase by site-directed mutagenesis, kinetic assay, structure modeling and substrate docking. Results: The results showed that the affinity of N336H, N336K and N336R increased gradually toward all the substrates, with increase in positive charge on side chain, while N336A and N336G have not shown a little significant effect on substrate affinity. The structure modeling studies indicated that the residue Asn336 is located in a random coil between -J-18 and a-10. Also, far-UV CD spectra-analysis showed that the mutations at Asn336 do not affect the secondary structure of enzyme. Conclusion: Asn336 site was located in a conserved GHYRNG loop which adjoining to substrate and the isoalloxazine ring of FAD, and involved in the substrate affinity of glycine oxidase. This might provide new insight into the structure-function relationship of GO, and valuable clue to redesign its substrate specificity for some biotechnological application.

Isolation, purification, and characterization of L-glutamate oxidase from Streptomyces sp. 18G

An extracellular L-glutamate oxidase (GLOD) was purified from soil-isolated Streptomyces sp 18G. The enzyme had a molecular weight of approximately 120,000 and consisted of two identical subunits, each with a molecular weight of 61,000. The isoelectric point was pH 8.5 and the enzyme had an optimal pH between 7.0-7.4. GLOD showed the maximum activity at 37ºC. The GLOD activity was stable at pH ranging from 6.5 to 7.0 for 1 hr. Among 21 amino acids tested for substrate specificity, L-glutamate was almost exclusively oxidized. D-glutamate and L-aspartate were oxidized but only to extents of 0.79 percent and 0.53 percent, respectively.

Polymorphisms of octopine dehydrogenase (Odh) in mollusks and implications for the neutralism-selectionism hypothesis

Octopine dehydrogenase (Odh) was examined in several species of bivalves and gastropods and complemented with bibliographic data, to assess the controversy between neutralism and selectionism in explaining the maintenance of genetic variation in natural populations. This debate was the center of the molecular evolution and population genetic research in the 1970s and 1980s, but waned thereafter, without resolution. Although DNA data have been produced, implications are not understood. We examined the polymorphims of Odh in several species of bivalves and gastropods, and the kinetic properties (apparent Km) of the different isozymes in the scallop Euvola ziczac that indicates an apparent case of overdominance of the heterozygous individuals. The question "which of the two hypothesis is correct" has shifted with time to "how much influence did each factor have in the maintenance of genetic variation".

Biochemical characterization of NADPH diaphorase in the chick embryo retina: stimulation by calcium ions and inhibition by arginine analogs

Nitric oxide is an important intercellular messenger in the central nervous system. NADPH-diaphorase, reported to be identical to nitric oxide synthase, is prsent in specific groups of cells in several neural tissues, including the retina. We determined NADPH-diaphorase activity in homogenates of the chick embryo retina. The enzyme activity was measured spectophotometrically at 585 nm after incubating retinal total homogenates (100-150 µg protein) with 1mMNADPH and 0.5 mM nitroblue tetrazolium in 50 mMTris buffer, pH8.1, at 37ºC. NADPH-diaphorse was detected in 14-day old retinas and 53-65 per cent of the enzyme activity was inhibited by 3 mM NG-nitro-L-arginine (NARG), the arginine analog. One mM L-N5-(1-iminoethyl)ornithine (NIO) was the most potent inhibitor (63 per cent inhibition) while 3 mM NG-nitro-L-arginine methyl ester (NAME) (33 per cent inhibition) and I mMNG-monomethyl-L-arginine acetate (NMMA) (14 per cent inhbition) were less effective. Enzyme activity was increased by 48 per cent by 2 mM calcium chloride, and effect reversed by 1 mMEGTA or EDTA. Basal enzyme levels were also partially inhibited by the chelators, indicating the presence of calcium-dependent and -independent isoforms of nitric oxide synthase in the retina. The results show that the NADPH-diaphorase assay is sample and sensitive and that the different isoforms of nitric oxide synthase expressed in chick retinal cells during development can be demonstrated

Negative inotropic effect of carbachol on rat atria mediated by nitric oxide

En este trabajo nosotros mostramos que la aurícula aislada de rata sintetiza óxido nítrico (ON) y este actúa como un mensajero intracelular incrementando la producción de GMPc, que a su vez modula el efecto contráctil inhibitorio ejercido por la activación muscarínica. El carbacol activando al receptor muscrínico M2 estimula el ciclo de los fosfoinosítidos y a la óxido nítrico sintaza con la consiguiente producción de ON. Los inhibidores de fosfolipasa C, proteína quinasa C, calcio/calmodulina, óxido nítrico sintaza y guanilato ciclasa, desvían hacia la derecha la curva dosis-respuesta del carbacol sobre la contractilidad. Más aun, en nitroprusiato de sodio y el 8-bromo GMPc indujeron un efecto inotrópico negativo, similar a las bajas concentraciones de carbacol. Estos reultados sugieren que el carbacol activando a receptores muscarínicos de tipo M2 ejerce un efecto inotrópico negativo asociado a un incremento en la producción de ON. Este mecanismo parece ser secundario a la estimulación del ciclo de los fosfoinosítidos vía la activación de la fosfolipas C; que desencadeando reacciones en cascada llevan a la producción de ON, el cual contribuye al efecto inotrópico negativo ejercido por bajas concentraciones de carbacol

Possible involvement of nitric oxide in estrogen-induced uterine edema in the immature rat

We examined whether nitric oxide mediates estrogen-induced uterine edema in the immature rat. Immature Wistar rats (19-21 days) received estradiol-17 beta (E2) in a single sc dose of 10 micrograms/animal and 6 h later the animals were sacrificed and the changes in uterine wet and dry weights were determined. E2 treatment caused a 93 per cent increase in uterine wet weight (control, N = 6, 39.88 +/- 3.2 mg; E2 treated, N = 6, 76.8 +/- 4.9 mg), but not in dry weight, suggesting that it induces uterine edema. Pretreatment with L-nitroarginine methyl ester (L-NAME), a competitive antagonist of nitric oxide synthetase, at doses of 10 and 20 mg/kg, ip, caused a dose-related reduction (59 and 86 per cent ) in the uterine wet weight increase induced by E2. Furthermore, L-arginine (300-600 mg/kg, sc), the nitric oxide precursor, was able to reverse L-NAME (20 mg/kg)-induced decreases in uterine weight by 47 and 62 per cent , respectively. The results suggest that nitric oxide is the principal mediator involved in estrogen-induced uterine edema in the immature rat

Purification and partial characterization of an l-amino acid oxidase from bushmaster snake (Surucucu pico de jaca) Lachesis muta venom

L-amino-acid oxidase(L-AO) form the venom of Lachesi muta muta was purified 72 times (38%) by gel filtration on Sephadex G-100, followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephacryl S-300. The protein was shown to be homogeneous by polyacrylamide gel electroforesis, immunoelectrophoresis, immunodiffusion and isoelectric focusing. Its specific activity was 44.4 units/mg protein, using 7.5 mM L-leucine as substrate a nd O-dianisidine as electron donor,at pH 7.6 and 25§C. The increase in absorbance at 436 nm was recorded. The enzyme was characterized as a glycoprotein with an S20,w=6.72,MW=138,000 and pI=5.2. It presented maxima at 389 and 460 nm and contained 2 mol of FAD per mole protein

Radiometric studies on the oxidation of (U-14C) L-amino acids by drug-susceptible and drug-resistant mycobacteria

Um sistema radiométrico foi utilizado para estudar os padröes de oxidaçäo dos (U-C)L-aminoácidos por micobactérias sensíveis e resistentes a drogas. Foram usadas duas cepas do M. tuberculosis sensíveis a todas as drogas, H Rv e Erdman. As micobactérias resistentes foram M. tuberculosis H Rv resistente a 5 ug/ml de hidrazida, M. bovis, M. avium, M. intracellulare, M. kansasii e M. chelonei. As micobactérias foram inoculadas em frascos estéreis contendo o meio líquido 7H9 e um dos (U-C)L-aminoácidos. Cada micobactéria apresentou um padräo de oxidaçäo de aminoácidos, mas estes padröes näo foram suficientemente diferentes para identificá-la. Aminoácidos complexos como a prolina, fenilalanina e tirosina näo tiveram utilidade na identificaçäo das micobactérias, pois praticamente todos os microorganismos foram incapazes de oxidá-los. Nenhuma combinaçäo de aminoácidos foi capaz de separar as micobacterias sensíveis das resistentes a drogas

Purificacion y separacion de isoenzimas de L-aminoacido oxidasa del veneno de Bothrops asper. / Purification and isolation of isoenzymes of L-amino acid oxidase from the venom of Bothrops asper

Se purifico y se separo del veneno de serpientes Bothrops asper capturadas en Costa Rica en tres isoenzimas la L-aminoacido oxidasa, empleando solamente dos pasos: calentamiento en presencia de L-leucina y cromatografia en columnas de DEAE-celulosa. Las isoenzimas fueron denominadas l, 2a y 2b, segun su velocidad electroforetica hacia el anodo, siendo 1 la mas rapida y 2b la mas lenta. La pureza de las isoenzimas 1 y 2b se demostro por electroforesis en gel de acrilamida y por estudios de velocidad de sedimentacion en la ultracentrifuga analitica. El peso molecular, calculado por equilibrio de sedimentacion, fue de 125.000 en ambas

Estudio comparativo de las isoenzimas de L-aminoacido oxidasa del veneno de Bothrops asper. / Comparative study of the isoenzymes of L-amino acid oxidase from the venom of Bothrops asper

Se hizo un estudio comparativo entre las isoenzimas 1 y 2b de la L-aminoacido oxidasa (E.C.1.4.3.2) del veneno de serpientes Bothrops asper capturadas en Costa Rica. La composicion de aminoacidos de la L-aminoacido oxidasa-1 (la mas anodica) mostro mayor numero de residuos de los acidos aspartico y glutamico, mientras la L-aminoacido oxidasa-2b resulto mas rica en lisina y alanina. Ambas isoenzimas tienen subunidades con pesos moleculares de 60.000 y 57.000, segun se determino por electroforesis en gel de acrilamida-dodecil-sulfato de sodio. Los mapas peptidicos demostraron un alto grado de homologia entre ambas isoenzimas, y el numero de peptidos que se obtuvo indica que tambien las subunidades de cada isoenzima son homologas