Low glucose under hypoxic conditions induces unfolded protein response and produces reactive oxygen species in lens epithelial cells

Aging is enhanced by hypoxia and oxidative stress. As the lens is located in the hypoglycemic environment under hypoxia, aging lens with diabetes might aggravate these stresses. This study was designed to examine whether low glucose under hypoxic conditions induces the unfolded protein response (UPR), and also if the UPR then generates the reactive oxygen species (ROS) in lens epithelial cells (LECs). The UPR was activated within 1 h by culturing the human LECs (HLECs) and rat LECs in <1.5 mM glucose under hypoxic conditions. These conditions also induced the Nrf2-dependent antioxidant-protective UPR, production of ROS, and apoptosis. The rat LECs located in the anterior center region were the least susceptible to the UPR, whereas the proliferating LECs in the germinative zone were the most susceptible. Because the cortical lens fiber cells are differentiated from the LECs after the onset of diabetes, we suggest that these newly formed cortical fibers have lower levels of Nrf2, and are then oxidized resulting in cortical cataracts. Thus, low glucose and oxygen conditions induce the UPR, generation of ROS, and expressed the Nrf2 and Nrf2-dependent antioxidant enzymes at normal levels. But these cells eventually lose reduced glutathione (GSH) and induce apoptosis. The results indicate a new link between hypoglycemia under hypoxia and impairment of HLEC functions

Techniques for accurate protein identification in shotgun proteomic studies of human, mouse, bovine, and chicken lenses

Analysis of shotgun proteomics datasets requires techniques to distinguish correct peptide identifications from incorrect identifications, such as linear discriminant functions and target/decoy protein databases. We report an efficient, flexible proteomic analysis workflow pipeline that implements these techniques to control both peptide and protein false discovery rates. We demonstrate its performance by analyzing two-dimensional liquid chromatography separations of lens proteins from human, mouse, bovine, and chicken lenses. We compared the use of International Protein Index databases to UniProt databases and no-enzyme SEQUEST searches to tryptic searches. Sequences present in the International Protein Index databases allowed detection of several novel crystallins. An alternate start codon isoform of βA4 was found in human lens. The minor crystallin γN was detected for the first time in bovine and chicken lenses. Chicken γS was identified and is the first member of the γ-crystallin family observed in avian lenses

αA-Crystallin Peptide 66SDRDKFVIFLDVKHF80 Accumulating in Aging Lens Impairs the Function of α-Crystallin and Induces Lens Protein Aggregation

The eye lens is composed of fiber cells that are filled with α-, β- and γ-crystallins. The primary function of crystallins is to maintain the clarity of the lens through ordered interactions as well as through the chaperone-like function of α-crystallin. With aging, the chaperone function of α-crystallin decreases, with the concomitant accumulation of water-insoluble, light-scattering oligomers and crystallin-derived peptides. The role of crystallin-derived peptides in age-related lens protein aggregation and insolubilization is not understood.We found that αA-crystallin-derived peptide, (66)SDRDKFVIFLDVKHF(80), which accumulates in the aging lens, can inhibit the chaperone activity of α-crystallin and cause aggregation and precipitation of lens crystallins. Age-related change in the concentration of αA-(66-80) peptide was estimated by mass spectrometry. The interaction of the peptide with native crystallin was studied by multi-angle light scattering and fluorescence methods. High molar ratios of peptide-to-crystallin were favourable for aggregation and precipitation. Time-lapse recordings showed that, in the presence of αA-(66-80) peptide, α-crystallin aggregates and functions as a nucleus for protein aggregation, attracting aggregation of additional α-, β- and γ-crystallins. Additionally, the αA-(66-80) peptide shares the principal properties of amyloid peptides, such as β-sheet structure and fibril formation.These results suggest that crystallin-derived peptides such as αA-(66-80), generated in vivo, can induce age-related lens changes by disrupting the structure and organization of crystallins, leading to their insolubilization. The accumulation of such peptides in aging lenses may explain a novel mechanism for age-related crystallin aggregation and cataractogenesis

Comparative enzyme studies of Microsporum canis and Microsporum cookei in relation to their pathogenicity and diversity : a thesis presented in partial fulfillment of the requirement for the degree of Doctor of Philosophy in Microbiology at Massey University

Infections by dermatophytes can be contracted from animals, humans, soil or contaminated fomites. In the genus Microsporum, some species e.g. M. canis are commonly associated with cats and dogs which act as an important reservoir for human infections. Others, e.g. M. cookei are nonpathogenic and found in the soil. The present studies have investigated the incidence of these ecologically contrasting species on cats, dogs and in the soil, their enzyme expression, and enzyme types as identified by proteinase inhibitors, gelatin/SDS-PAGE and multilocus enzyme electrophoresis, and have led to an investigation of their phenotypic variation. The primary aim was to attempt to detect differences in enzyme production which might be related to mechanisms of pathogenicity of M. canis. Isolation procedures employed were the hairbrush technique for small animals and the keratin-baiting technique for soil with samples being cultured on SDA containing antibiotics. Soil samples revealed 19 fungal genera, three being of keratinolytic fungi, representing 50% of total isolations. Trichophyton species were the most common (39% samples) but M. cookei was isolated from 6.8%. Fungi isolated from cats and dogs represented 20 genera, with the predominant isolates being keratinolytic fungi (51.9% of total samples). Cats were the major carriers of keratinolytic fungi (Chrysosporium, Microsporum and Trichophyton). M. canis was frequently isolated (18.5% of cats) and its distribution had a seasonal variation, with a peak appearing in May-June. All isolates of M. canis were of the "-" mating type. M. cookei isolates were of both the "+" and "-" mating types, but "+" types were predominant. Biochemical assays showed that M. canis produced higher proteinase and keratinase activities in shake cultures than in stationary cultures. Elastase activity was greater in stationary cultures. M. cookei's proteinase and keratinase activities were lower but again greater in shake cultures. There was no detectable keratinase activity in stationary cultures of M. cookei, and no significant difference in elastinolytic activity between shake and stationary cultures. Growth in shake culture produces the "pseudo-parasitic" morphology which mimics that found in infection, therefore, the differing enzyme expression of the two Microsporum species may be a reflection of their differing ecological roles. Characterisation of the enzymes with chemical inhibitors revealed that M. canis and M. cookei produced serine proteinases, but only M. canis produced cysteine and probably aspartic and metallo-proteinases. The serine and cysteine proteinases are considered likely to be of particular significance in the pathogenesis of M. canis infections. Using substrate copolymerised gel electrophoresis (gelatin\SDS-PAGE), shake and stationary cultures were again compared for enzyme expression. Among the six different Mr proteinases (122 KDa, 64 KDa, 62 KDa, 44 KDa, 36 KDa, and 28 KDa) expressed by M. canis, three (122 KDa, 62 KDa and 28 KDa) were found to be more highly expressed in shake cultures. In contrast, M. cookei isolates expressed seven different proteinases (67 KDa, 64 KDa, 63 KDa, 62 KDa. 54 KDa, 52 KDa, and 42 KDa), of which two (67 KDa, 64 KDa) were expressed only in stationary cultures and one (52 KDa) although expressed in shake cultures was more highly expressed in stationary cultures. Possibly the high and low Mr proteinases expressed by M. canis are more important in its pathogenicity than the middle range proteinases also detected in M. cookei. Multilocus enzyme electrophoresis using starch gels and examining eight enzymes, showed M. canis to be phenotypically more diverse than M. cookei as measured by the normalised Shannon-Wiener diversity statistic. M. canis showed a substantial within population variability (84.9%) by geographical region, with a moderate level (21.7%) of interpopulation differentiation. Cluster analysis confirmed this diversity and also revealed a possible grouping of isolates from clinical infections, and based on the accumulated data of these studies, EST pnenotype 9 although present in a few carrier isolates was commonly associated with isolates from clinical cases and perhaps deserves further investigation

INSECT AND MYCOFLORA INTERACTIONS IN MAIZE FLOUR

Maize flour treated with or without Tribolium castaneum was investigated for the presence of some fungi. Fusarium moniliforme had the highest occurrence of 36.7%, 28.1% and 33.3% while Aspergillus flavus/parasiticus had a frequency of 3.2%, 3.1% and 3% on primary isolation media of czapek dox agar (CDA), potato dextrose agar (PDA) and sabouraud dextrose agar (SDA) respectively, in maize flour without T. castaneum. The frequency of F. moniliforme reduced in maize flour with T. castaneum to 11.1%, 12.1% and 18.8% on CDA, PDA and SDA while A. flavus/parasiticus increased in occurrence after introducing T. castaneum to 22.2%, 18.2% and 12.3% on the three respective media. Fourteen and 7 fungal genera were isolated from maize flour with and without F. castaneum respectively. Two fungal species isolated from maize flour without T. castaneum were Cladosporium cladosporioides and C. lunata. Ten species isolated from maize flour with T. castaneum were A. pullulans, Auxarthron spp., C. herbarum, Eurotium sp., Phoma glomerata, Neosauorya spp., Scopulariopsis brevicaulis, Rhizopus oryzae, R. stolonifer and Wallemia sebi. These results suggest an association and a synergistic interaction between important spoilage and mycotoxigenic fungi with T. castaneum such as A. flavus/parasiticus and some mildly parasitic fungal colonies but an antagonistic interaction with F. moniliforme. Key words: Tribolium castaneum, storage fungi, synergistic/antagonistic interactions, mycotoxins (Af. J. Food and Nutritional Sciences: 2001 1(1): 3-8

Characterisation of a novel UV filter in the lens of the thirteen-lined ground squirrel (Ictidomys tridecemlineatus)

Structural analysis of a novel UV filter present in the lens of the thirteen-lined ground squirrel has shown that it is related in structure to N-acetyl-3-hydroxykynurenine. This finding is consistent with the fact that the squirrel lenses also contain high levels of this tryptophan metabolite. Analysis of both NMR and mass spectrometric data suggested that the novel UV filter compound forms by condensation of proline with N-acetyl-3-hydroxykynurenine. Its absorption maximum at 340 nm is more than 20 nm lower than that of the kynurenines and it may therefore assist in filtering the more damaging shorter wavelengths of UVA