RESUMO
In this study, methanolic extract of Saraca asoca bark was evaluated for its aphrodisiac potential using male and female Wistar albino rats. Male rats were dosed daily for 54 days at a dose of 100 mg/kg p.o. Sexual activity of male rats was assessed after 14, 28, 42 and 54 days of the study. Male rats were placed in a glass chamber lit with a dim red light (10W) followed by the introduction of sexually receptive female rats in a ratio of 1:1. Improvement in sexual behaviour of male rats was characterised by an increase in both mount frequency and intromission frequency and decrease or reduction in mount latency and intromission latency compared to normal control. After completion of the study, the effect of the S. asoca extract on sperm count, sperm motility and sperm morphology was also assessed. The extract of S. asoca bark was found to be safe as it did not affect these sperm parameters. From this study, it was found that methanolic extract of S. asoca bark plays a role in enhancing sexual behaviour and potential without causing reproductive toxicity.
Assuntos
Afrodisíacos/farmacologia , Fabaceae/química , Extratos Vegetais/farmacologia , Comportamento Sexual Animal/efeitos dos fármacos , Animais , Afrodisíacos/química , Afrodisíacos/isolamento & purificação , Masculino , Metanol/química , Modelos Animais , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Ratos , Ratos Wistar , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacosRESUMO
BACKGROUND AND OBJECTIVES: Six patients died and one patient survived following infusion of a specific lot of intravenous immunoglobulin (IVIG) within half an hour in May 2008. This study elucidated the underlying pathogenesis. MATERIALS AND METHODS: A variety of protein fractionation and identification approaches were employed to determine the abnormal components in IVIG products obtained from the hospital where the patients were treated. Animal studies using mice and monkeys were conducted to elucidate the pathophysiological mechanisms. In animal experiments, the effect and distribution of immunoglobulin was investigated using HE staining and immunohistochemistry (IHC) separately, while platelets and fibrinogen depletion were utilized to determine a possible link between thromboembolism formation in animals and the lethal effect of the IVIG. The size and distribution of the protein aggregates were determined with Coulter Counter Multisizer-3 after the dilution of the IVIG with plasma, and the lethal effect of the protein aggregates was simulated with artificial microparticles. RESULTS: The IVIG retrieved from the hospital was found to have striking similarities to the heat-treated IVIG in terms of protein aggregation profiles and lethal effects. Post-mortem examination indicated that immunoglobulin aggregates were mainly found in the lung of the animals, while depletion of platelets and fibrinogen from the IVIG preparations failed to prevent the death of the animals. Similar amount of artificial microparticles caused animal death in similar fashion. CONCLUSIONS: Our findings indicate that the retrieved IVIG exerted its lethal effects by blocking the pulmonary circulation without markedly altering the coagulation cascade or immunological events.
Assuntos
Imunoglobulinas Intravenosas/efeitos adversos , Embolia Pulmonar/etiologia , Tromboembolia/etiologia , Animais , Coagulação Sanguínea , Haplorrinos , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , CoelhosRESUMO
The standardised extract of root of safed musli (Chlorophytum borivilianum) was evaluated for its aphrodisiac potential and safety profile on reproductive system. Wistar albino rats were trained to provide sexual experience under a dim red light (10 W) in a glass tank. Male and female rats were placed periodically in the glass tank in a particular order, that is male followed by introduction of the receptive female. Dosing of extract was carried out for 54 days at 125 and 250 mg kg-1 p.o to male rats. On 14th and 28th days, the animals were observed from the cage side for sexual behaviours. Safed musli at both dose levels enhanced sexual vigour and libido which might be useful for treatment of sexual dysfunction in male till 28th day. Safety profile was assessed after 54 days of drug treatment, where both doses showed an increase in sperm count and increase in sperm motility. Thus, it can be stated that both doses possessed the spermatogenic potential, which would be highly beneficial in treating oligospermia or low sperm count. After 54 days of study, there was increase in sperm abnormality (%) at both doses, but not more than 10%, which indicated that this formulation will not induce infertility.
Assuntos
Afrodisíacos/farmacologia , Asparagaceae , Libido/efeitos dos fármacos , Extratos Vegetais/farmacologia , Comportamento Sexual Animal/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Animais , Feminino , Masculino , Ratos , Ratos Wistar , Contagem de EspermatozoidesRESUMO
Cancer is a complex disease characterized by a loss in the normal cell regulatory mechanisms that govern cell survival, proliferation, and differentiation. Current chemotherapeutics, as anticancer agents, are developing resistance to single drug and also to treatment therapies involving multiple drugs. Cross resistance associated with the specificity and selectivity of existing drugs has restricted the application of chemotherapy. Alternatively, these limitations have given better insight in understanding the underlying molecular mechanisms responsible for the development of various stages in cancer. In the light of this, continuous efforts are being made in order to identify and validate newer anticancer targets. This review presents some of the important targets that have been already reported, such as aromatase, farnesyl transferase, histone deacetylase, tyrosine kinase and cyclin-dependent kinase. A few molecules designed against these targets have successfully reached clinical trials. However, only limited marketed drugs are available from these classes. Besides, the review also highlights some of the other important targets and strategies that have also drawn considerable attention in the area of anticancer drug development such as, cancer stem cells and monoclonal antibodies. Further, the integration of the tools in molecular biology with the results from preclinical and clinical trials would strengthen the effectiveness of treatment regimens in cancer patients. There lies a much scope for designing promising lead compounds and treatment therapies against these established targets.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/genética , Aromatase/genética , Aromatase/uso terapêutico , Farnesil-Difosfato Farnesiltransferase/genética , Farnesil-Difosfato Farnesiltransferase/uso terapêutico , Histona Desacetilases/genética , Histona Desacetilases/uso terapêutico , Humanos , Neoplasias/patologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/uso terapêuticoRESUMO
Recombinant, human, erythropoietin (rhEPO) is a glycoprotein hormone which is prescribed throughout the world to treat anaemia caused by chronic kidney disease or chemotherapy. rhEPO is at the forefront of the recent emergence of biosimilar medicines, with numerous products now available worldwide. Due to its complex glycosylation profile, which has a crucial influence upon biological activity, therapeutic rhEPO preparations must be closely monitored to ensure consistency, safety and efficacy. Here, we have compared twelve rhEPO preparations from eleven manufacturers in China and one in Japan, measuring in vivo biological activity and exploring its relationship with glycosylation through sialic acid content determination, isoform distribution via capillary electrophoresis (CE), O-glycan profiling, and N-glycan mapping using a novel anion-exchange/hydrophilic interaction chromatography-mass spectrometry (AEX/HILIC-MS) approach. We observed differences between glycosylation profiles, including the varying occurrence of sialic acid O-acetylation, extension of N-glycan antennae with N-acetyllactosamine units, and the distribution of sialic acids across multi-antennary structures. The presence of unusually high levels of suspected penta- and hexa-anionic N-glycans in several samples is consistent with elevated rhEPO isoform acidity, which is reflected by slightly elevated in vivo bioactivities. This aside, the observed differences in glycosylation profile do not appear to have a significant influence upon biological activity in mice. Nonetheless, with the continued emergence of biosimilars, the study highlights the importance of monitoring glycosylation profiles in biological medicines, in order to detect and account for divergence between products, as well as the presence of unusual or unexpected glycans.
Assuntos
Eritropoetina/química , Proteínas Recombinantes/química , Animais , China , Eletroforese Capilar/métodos , Glicosilação/efeitos dos fármacos , Humanos , Japão , Espectrometria de Massas/métodos , Camundongos , Camundongos Endogâmicos BALB C , Ácido N-Acetilneuramínico/química , Polissacarídeos/química , Isoformas de Proteínas/químicaRESUMO
Proteins, made up of either single or multiple chains, are designed to carry out specific biological functions. We found an interesting example of a two-chain protein where administration of one of its chains leads to a diametrically opposite outcome than that reported for the full-length protein. Clusterin is a highly glycosylated protein consisting of two chains, α- and ß-clusterin. We have investigated the conformational features, cellular localization, lipid accumulation, in vivo effects and histological changes upon administration of recombinant individual chains of clusterin. We demonstrate that recombinant α- and ß-chains exhibit structural and functional differences and differ in their sub-cellular localization. Full-length clusterin is known to lower lipid levels. In contrast, we find that ß-chain-treated cells accumulate 2-fold more lipid than controls. Interestingly, α-chain-treated cells do not show such increase. Rabbits injected with ß-chain, but not α-chain, show ~40% increase in weight, with adipocyte hypertrophy, liver and kidney steatosis. Many, sometimes contrasting, roles are ascribed to clusterin in obesity, metabolic syndrome and related conditions. Our findings of differential localization and activities of individual chains of clusterin should help in understanding better the roles of clusterin in metabolism.
Assuntos
Clusterina/metabolismo , Metabolismo dos Lipídeos , Animais , Linhagem Celular , Forma Celular , Clusterina/administração & dosagem , Clusterina/química , Humanos , Masculino , Camundongos , Chaperonas Moleculares/metabolismo , Coelhos , Frações Subcelulares/metabolismo , Fatores de Tempo , Aumento de PesoRESUMO
The presence of the 34-kDa hyaluronan binding protein, a new member of the 'hyaladherins' family, was demonstrated in a wide variety of cell lines by immunoblot analysis. This protein was observed to be highly phosphorylated in transformed fibroblasts compared to normal fibroblasts. Phosphorylation was enhanced in the presence of its ligand i.e., hyaluronan, but not in the presence of other glycosaminoglycans. The phosphorylated form of this hyaluronan binding protein was shown to be present on the cell surface and could be detected in serum-free medium. The regulation of the cellular and cell surface phosphorylation of HA-binding protein by HA, PMA and calyculin-A was demonstrated in different cell lines. Hyaluronan enhanced the phosphorylation of PLC-gamma in association with increased formation of inositol 1,4,5-triphosphate, both of which were specifically blocked by pretreatment of the cells with purified anti-hyaluronan binding protein antibodies. The data presented here indicate a role for the 34-kDa hyaluronan binding protein in cellular signal transduction.
Assuntos
Receptores de Hialuronatos/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Animais , Linhagem Celular , Linhagem Celular Transformada , Membrana Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Receptores de Hialuronatos/isolamento & purificação , Ácido Hialurônico/metabolismo , Immunoblotting , Inositol 1,4,5-Trifosfato/metabolismo , Isoenzimas/antagonistas & inibidores , Cinética , Toxinas Marinhas , Oxazóis/farmacologia , Fosfolipase C gama , Fosforilação , Polyomavirus , Inibidores de Proteínas Quinases , Proteínas Quinases/metabolismo , Ratos , Pele , Estaurosporina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
The shift that occurs in the fluorescence emission wavelength upon changing the excitation wavelength towards the red edge of the absorption band is termed red edge excitation shift (REES). We have monitored the REES of intrinsic protein fluorescence of freshly isolated intact lenses, of individual crystallins in their native, denatured and photodamaged states and also of crystallin mixtures. The observed REES values for the lenses from different species are different suggesting that the mobilities and packing of the crystallins may vary with the species. Lens photodamage in all the cases resulted in an increase of REES. Denaturation of crystallins in solution reduces REES and renaturation restores it. Mixtures of alpha- and beta-crystallins prepared either by directly mixing equimolar solutions or mixing them in 4 M urea followed by dialysis (reconstituting) gave similar REES values indicating the absence of any specific interactions in dilute solutions. Possible existence of induced alterations facilitating inter-crystallin interactions at high protein concentration is suggested.
Assuntos
Cristalinas/química , Cristalino/química , Espectrometria de Fluorescência , Animais , Bovinos , Galinhas , Cristalinas/efeitos da radiação , Feminino , Cristalino/efeitos da radiação , Luz , Masculino , Desnaturação Proteica , Ratos , Ratos WistarRESUMO
Alpha-crystallin exhibits chaperone-like properties in preventing aggregation of proteins. We have studied the effect of alpha-crystallin on the refolding of denatured-disulfide intact and denatured-reduced lysozyme and RNase A. Alpha-crystallin does not have any effect on the refolding of both the denatured-disulfide intact enzymes. However, it inhibits the aggregation and oxidative renaturation of denatured-reduced lysozyme. Interestingly, it has no effect on the refolding of denatured-reduced RNase A. In order to probe the molecular basis of this differential behavior of alpha-crystallin towards lysozyme and RNase A, we have carried out circular dichroism and fluorescence studies on the refolding of denatured-reduced RNase A. It exhibits an extended conformation with little difference in the exposed hydrophobicity during the refolding process. We have earlier shown the presence of an aggregation-prone, refolding-competent, molten-globule-like intermediate on the refolding pathway of lysozyme. Alpha-crystallin binds to this intermediate, prevents its aggregation and inhibits its oxidative refolding. It was earlier believed that alpha-crystallin, unlike other chaperones, does not recognize intermediates on the refolding pathway but only recognizes intermediates on the unfolding pathway of proteins. Our present study clearly shows that it recognizes the refolding intermediates as well.
Assuntos
Chaperoninas/metabolismo , Cristalinas/metabolismo , Muramidase/química , Dobramento de Proteína , Ribonuclease Pancreático/química , Dicroísmo Circular , Dissulfetos , Cinética , Muramidase/metabolismo , Oxirredução , Desnaturação Proteica , Ribonuclease Pancreático/metabolismo , Espectrometria de FluorescênciaRESUMO
Alpha-crystallin, a multimeric protein present in the eye lens, is known to have chaperone-like activity in preventing the aggregation of enzymes and other crystallins. We have studied the chaperone-like activity of this protein towards the aggregation of insulin B chain, induced by reducing the interchain disulphide bond with dithiothreitol. At room temperature, there is no detectable protection (at a 1:1 (w/w) ratio of insulin: alpha-crystallin) against the aggregation of insulin B chain by alpha-crystallin, whereas it completely prevents this aggregation at 40 degrees C. We have monitored the temperature dependence of the protection of aggregation by alpha-crystallin; the protection increases sharply above 30 degrees C and reaches almost 100% by 41 degrees C. Probing the hydrophobic surfaces of alpha-crystallin with the hydrophobic fluorphore 8-anilino-1 naphthalene sulfonate suggests that the hydrophobic surfaces of alpha-crystallin are exposed to a greater extent above 30 degrees C. A complete prevention of the aggregation is achieved at 27.6 degrees C by increasing the concentration of alpha-crystallin by more than 8 fold. Similar temperature dependent chaperone-like activity of alpha-crystallin is observed towards the aggregation of zeta-crystallin, an enzyme crystallin from guinea pig. We have earlier shown that alpha-crystallin exposes hydrophobic surface(s) at temperatures above 30 degrees C. These results support our earlier hypothesis [Raman, B. and Rao, Ch.M. (1994) J. Biol. Chem. 269, 27264-27268] that the chaperone-like activity of alpha-crystallin is more pronounced in its structurally perturbed state.
Assuntos
Chaperoninas/química , Chaperoninas/metabolismo , Cristalinas/química , Cristalinas/metabolismo , Animais , Bovinos , Cristalinas/isolamento & purificação , Insulina/química , Cristalino/metabolismo , Substâncias Macromoleculares , Espectrometria de Fluorescência , Temperatura , TermodinâmicaRESUMO
We have investigated the role of recombinant human alphaA- and alphaB-crystallins in the heat-induced inactivation and aggregation of citrate synthase. Homo-multimers of both alphaA- and alphaB-crystallins confer protection against heat-induced inactivation in a concentration-dependent manner and also prevent aggregation. Interaction of crystallins with early unfolding intermediates of citrate synthase reduces their partitioning into aggregation-prone intermediates. This appears to result in enhanced population of early unfolding intermediates that can be reactivated by its substrate, oxaloacetate. Both these homo-multimers do not form a stable complex with the early unfolding intermediates. However, they can form a soluble, stable complex with aggregation-prone late unfolding intermediates. This soluble complex formation prevents aggregation. Thus, it appears that the chaperone activity of alpha-crystallin involves both transient and stable interactions depending on the nature of intermediates on the unfolding pathway; one leads to reactivation of the enzyme activity while the other prevents aggregation.
Assuntos
Citrato (si)-Sintase/química , Cristalinas/química , Dobramento de Proteína , Cromatografia em Gel , Citrato (si)-Sintase/metabolismo , Cristalinas/metabolismo , Cristalinas/farmacologia , Relação Dose-Resposta a Droga , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/fisiologia , Temperatura Alta , Humanos , Substâncias Macromoleculares , Chaperonas Moleculares/metabolismo , Ácido Oxaloacético/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Desnaturação Proteica/efeitos dos fármacos , Desnaturação Proteica/fisiologia , Proteínas Recombinantes/metabolismoRESUMO
Refolding of denatured-reduced lysozyme and the effect of co-refolding it with other proteins such as RNase A, bovine serum albumin, histone, myelin basic protein, alcohol dehydrogenase and DNase I on the renaturation yield and the aggregation of lysozyme have been studied. Basic proteins consistently increase the renaturation yield of the basic protein lysozyme (10-20% more than in their absence) with little or no aggregation. On the other hand, co-refolding of lysozyme with acidic proteins leads to aggregation and a significant decrease in renaturation yields. Our results show that hetero-interchain interactions (non-specific interactions) occur when the basic protein lysozyme is refolded together with acidic proteins such as bovine serum albumin, alcohol dehydrogenase or DNase I. Our results also suggest that the net charge on proteins plays a significant role in such non-specific aggregation. These results should prove useful in understanding the hetero-interchain interactions between folding polypeptide chains.
Assuntos
Muramidase/química , Dobramento de Proteína , Proteínas/metabolismo , Animais , Bovinos , Galinhas , Muramidase/metabolismo , Ligação Proteica , Desnaturação ProteicaRESUMO
To understand the differences in the binding specificities within the aspartic proteinase family of enzymes, we have carried out studies to determine the inhibition constants of a set of related compounds with various members of the human enzyme family. The inhibition constants (Ki values) were determined by competitive inhibition of the hydrolysis of chromogenic octapeptide substrates in the pH range of 3-5. For comparison, inhibition of monkey renin was studied by RIA at pH 6.0. All inhibitors were based on the general structure 4-(morpholinylsulfonyl)-L-Phe-P2-(cyclohexyl)Ala psi[isostere]-P1'-P2'. The isosteric replacements of the scissile peptide bond included difluorohydroxyethylene, 1,2-diols, 1,3-diols, and difluoroketones. Side chain substituents in P2 include hydrogen, allyl, ethylthio, (methoxycarbonyl)methyl, N-methylthiouridobutyl, imidazolylmethyl, and 4-amino-2-thiazolylmethyl. Our measurements have identified potent and selective inhibitors which are useful in evaluating the differences in the specificities among selected enzymes of this family.
Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Dipeptídeos/química , Morfolinas/química , Inibidores de Proteases/metabolismo , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Catepsina D/antagonistas & inibidores , Catepsina D/metabolismo , Catepsina E , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Simulação por Computador , Dipeptídeos/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Morfolinas/metabolismo , Pepsina A/antagonistas & inibidores , Pepsina A/metabolismo , Inibidores de Proteases/química , Renina/antagonistas & inibidores , Renina/metabolismo , Relação Estrutura-AtividadeRESUMO
PURPOSE: zeta-crystallin is a quinone oxido-reductase, recruited in the eye lens of hystricomorphic rodents and camels. A deletion mutation constituting the NADPH-binding domain causes congenital cataract in a strain of guinea pigs. The presence of large quantities of a-crystallin, a molecular chaperone, does not provide any protection against this. In order to investigate whether the underlying reason for the lack of protection is the formation of a folding-incompetent protein, we have expressed the mutant protein in a heterologous system along with other known chaperones. METHODS: We expressed the mutant zeta-crystallin in E. coli along with other chaperones such as GroEL/ES and DnaK/DnaJ/GrpE and then analyzed whether these chaperones could increase the amount of protein partitioning into the soluble fraction of E. coli cells. RESULTS: These chaperones were unable to rescue the mutant protein from partitioning into inclusion bodies, although they could increase the yield of soluble wild-type zeta-crystallin. CONCLUSIONS: The deletion of 34 amino acids, constituting the NADPH-binding domain of zeta-crystallin, makes the protein incompetent to fold correctly and thus form insoluble aggregates. It perhaps suggests why the mutant strain of guinea pigs have cataract at birth even though their lenses contain high amounts of alpha-crystallin. This study also shows that certain mutations can render proteins incompetent to fold into soluble molecules despite abundant assistance.
Assuntos
Catarata/genética , Cristalinas/metabolismo , Cristalino/metabolismo , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Catarata/congênito , Catarata/metabolismo , Clonagem Molecular , Cristalinas/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cobaias , Chaperonas Moleculares/genética , Dados de Sequência Molecular , NADP/metabolismo , Conformação Proteica , Deleção de Sequência , TransfecçãoRESUMO
PURPOSE: alpha-Crystallin belongs to a class of small heat shock proteins and is shown to prevent aggregation of several proteins. We have shown that the temperature-induced structural perturbation leads to several fold enhanced activity. The purpose of this study was to investigate the availability and specificity of the hydrophobic sites that might become available at elevated temperatures. Specifically, we address the following question: Is there an increased exposure of fixed number of hydrophobic sites as a function of temperature or does a new set of sites become available at elevated temperatures? METHODS: alpha-Crystallin target protein complexes were made at two different temperatures and this complex was investigated for its chaperone-like activity towards the same target protein and also other target proteins. DTT-induced aggregation of insulin, alpha-lactalbumin, thermal aggregation of betaL- and gamma-crystallin, and photo-aggregation of gamma-crystallin were used as model systems. Increased light scattering was used to monitor the progress of aggregation. RESULTS: alpha-Crystallin target protein complex prepared at 37 degrees C temperature was effective against thermal aggregation of betaL-crystallin as well as non-thermal aggregation at elevated temperatures. However, the complex prepared at high temperature was ineffective at lower temperatures as well as with other target proteins at both temperatures. CONCLUSIONS: More target protein binding sites become available at elevated temperatures. The sites available at low temperature are a subset of the total sites available at elevated temperatures.
Assuntos
Cristalinas/metabolismo , Insulina/metabolismo , Lactalbumina/metabolismo , Animais , Sítios de Ligação , Bovinos , Cromatografia em Gel , Cristalinas/química , Temperatura Alta , Cristalino/química , Cristalino/metabolismo , Luz , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Ligação Proteica , Desnaturação Proteica , Espalhamento de RadiaçãoRESUMO
We have evaluated tumour characteristics, local recurrence rates and prognostic markers in 40 women with symptomatic palpable breast cancer proven by cytology, but in whom routine two-view mammography failed to detect a radiological abnormality. False negative mammograms were identified by cross-referencing all negative mammograms performed at the Royal Victoria Infirmary during the period 1995-1999, with pathological records at the same institution. The average age was 48 years. The majority of the tumours were invasive ductal carcinomas, 35 with an average size of 24 mm. There were 16 Grade II and 15 Grade III tumours. Lymphovascular invasion was seen in 18 on histology and six patients had distant metastases. Of those patients treated by conservation therapy there has been only one local recurrence, with a median follow-up of 18 months. We conclude that mammographically invisible tumours are of common histological type, are frequently high grade and node positive and occur mainly in the younger age group. However, BCT remains a viable option in the treatment of these tumours.
RESUMO
Since most of the known factors that are associated with cataract formation are oxidative in nature, one would expect that a highly reductive environment might arrest or retard the progress of cataract formation. Reduced nucleotides, both NADH and NADPH, are potent reductants with a large negative redox potential of -320 mV. Lenses of certain species contain high levels of these nucleotides, presumably due to the presence of taxon specific crystallins. We have utilized this situation to investigate whether the levels of reduced pyridine nucleotides modulate photo-oxidative damage to the lens. We have monitored the time dependent loss of tryptophan fluorescence upon photodamage for lenses from guinea pig, rabbit and frog (Rana) that contain high levels of pyridine nucleotides and compared with the lenses from rat, Xenopus and a mutant strain of guinea pig that contain significantly lower amounts of these nucleotides. About 75% and 90% of the initial fluorescence intensity is lost in the case of rat and Xenopus lenses, respectively, after a total of 35 min exposure. Rabbit, guinea pig and frog lenses, under identical conditions, show only about 35-40% loss of the initial fluorescence. It appears that the lenses that contain high levels of reduced nucleotides are less susceptible to photodamage. The observed anti-oxidative role of reduced nucleotides in the lenses indicates the possibility of testing reductants (NADPH, NADH and their functional analogues) as potential candidates to therapeutically intervene in the process of cataractogenesis.
Assuntos
Cristalino/efeitos da radiação , NADP/metabolismo , NAD/metabolismo , Raios Ultravioleta , Animais , Cobaias , Técnicas In Vitro , Cristalino/metabolismo , Oxirredução , Coelhos , Rana catesbeiana , Ratos , Ratos Sprague-Dawley , Espectrometria de Fluorescência , Triptofano/análise , XenopusRESUMO
We have studied the aerobic photolysis of the tryptophan residues of the proteins melittin and beta-lactoglobulin when the proteins are in ordered conformations and when they are in randomly coiled states. The results suggest that the conformational status of the protein is a factor that influences the photolysis of the constituent tryptophan residues. This point appears to be of relevance to the photo-oxidation of the tryptophan residues of the eye lens proteins crystallins.
Assuntos
Venenos de Abelha/efeitos da radiação , Cristalinas/efeitos da radiação , Lactoglobulinas/efeitos da radiação , Meliteno/efeitos da radiação , Conformação Proteica , Triptofano , Raios Ultravioleta , Aerobiose , FotóliseRESUMO
Shift in the wavelength of emission upon shift in the excitation wavelength towards the red edge of the absorption band is termed Red Edge Excitation Shift (REES). This effect is observed only in situation where the fluorophore mobility with respect to the surrounding matrix is considerably reduced. We have observed such red edge excitation effect in the intact eye lens. The REES observed for a normal lens is different from that seen in a photodamaged lens and hence appears to be a potential tool to monitor the changes in the state of the lens. Photodamage experiments with tryptophan in polyethylene glycol (PEG) and intact eye lens indicate that the red edge photon can also cause photodamage.
Assuntos
Cristalino/fisiologia , Animais , Técnicas In Vitro , Masculino , Ratos , Análise EspectralRESUMO
A simple and sensitive post electrophoresis fluorescent staining technique for proteins on polyacrylamide gels using 5-dimethylamino-1-naphthalene sulfonyl chloride (dansyl chloride) has been developed. Dansyl chloride staining increases the sensitivity, 0.125 micrograms protein per band can be visualised by this technique. The staining method appears to be applicable to all types of proteins including proteoglycans.