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Burkholderia pseudomallei causes melioidosis, a common source of pneumonia and sepsis in Southeast Asia and Northern Australia that results in high mortality rates. A caprine melioidosis model of aerosol infection that leads to a systemic infection has the potential to characterize the humoral immune response. This could help identify immunogenic proteins for new diagnostics and vaccine candidates. Outbred goats may more accurately mimic human infection, in contrast to the inbred mouse models used to date. B. pseudomallei infection was delivered as an intratracheal aerosol. Antigenic protein profiling was generated from the infecting strain MSHR511. Humoral immune responses were analyzed by ELISA and western blot, and the antigenic proteins were identified by mass spectrometry. Throughout the course of the infection the assay results demonstrated a much greater humoral response with IgG antibodies, in both breadth and quantity, compared to IgM antibodies. Pre-infection sera showed multiple immunogenic proteins already reactive for IgG (7-20) and IgM (0-12) in most of the goats despite no previous exposure to B. pseudomallei. After infection, the number of IgG reactive proteins showed a marked increase as the disease progressed. Early stage infection (day 7) showed immune reaction to chaperone proteins (GroEL, EF-Tu, and DnaK). These three proteins were detected in all serum samples after infection, with GroEL immunogenically dominant. Seven common reactive antigens were selected for further analysis using ELISA. The heat shock protein GroEL1 elicited the strongest goat antibody immune response compared to the other six antigens. Most of the six antigens showed the peak IgM reactivity at day 14, whereas the IgG reactivity increased further as the disease progressed. An overall MSHR511 proteomic comparison between the goat model and human sera showed that many immune reactive proteins are common between humans and goats with melioidosis., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: To compare levels of S-glutathiolation and S-cysteinylation occurring at more than 60 cysteine residues of 12 different guinea pig lens water-soluble nuclear crystallins following treatment of the animals with hyperbaric oxygen (HBO)., Methods: Guinea pigs (initially 18 months old) were treated 30X (3X per week for 10 weeks) with HBO (2.5 atm 100% O(2) for 2.5 h) as a model to study the formation of nuclear cataract. This treatment produces a moderate increase in lens nuclear light scatter (compared to denser scatter occurring after 80 HBO treatments), with five- to sixfold increases in levels of protein-bound glutathione (PSSG) and protein-bound cysteine (PSSC). Trypsin digests of lens nuclear water-soluble proteins were analyzed with two-dimensional liquid chromatography and mass spectrometry to identify specific cysteine residues binding either glutathione or cysteine. Lens nuclei of age-matched untreated animals were used as controls., Results: All major crystallins, except αB, were modified to some extent by either S-glutathiolation or S-cysteinylation. Overall, 72% of the cysteine residues of guinea pig lens nuclear crystallins were shown to be capable of binding glutathione, cysteine, or both molecules. The crystallin with the highest level of modification was βA1/A3 (six of eight -SH groups), and that with the lowest (two of five -SH groups) was βA2. O(2)-induced increases in PSSG levels were 2.8, 2.4, and 4.1 times control for γA-, γB-, and γC-crystallins, respectively. Comparable increases in PSSC levels for the three γ-crystallins were 2.3, 2.7, and 2.4 times control, respectively. βB2-crystallin showed the highest amount of O(2)-induced PSSG formation of any of the crystallins, as well as a substantial level of control PSSG, and nearly all of this was due to a single residue, C67, a site also present in human βB2-crystallin. Overall, 32 of the 44 modified cysteine residues were homologous with the human., Conclusions: This large-scale study successfully identified lens crystallin cysteine residues that bound glutathione and/or cysteine under normal or oxidative stress conditions. The high percentage of protein -SH groups that are modified by S-thiolation in the guinea pig lens nucleus demonstrates the substantial protein sulfhydryl redox buffer capability present in the center of the lens. The results suggest that PSSG and PSSC formation may act to delay O(2)-induced insolubilization of γA-, γB-, and γC-crystallins, and β-crystallins, but with a greater effect on the γ-crystallins at an early stage of oxidative stress. The study has shown that technological approaches are now available to investigate in considerable detail the role of specific lens -SH groups in nuclear cataractogenesis.

It is known that fluorescence, much of it caused by UVA light excitation, increases in the aging human lens, resulting in loss of sharp vision. This study used an in vivo animal model to investigate UVA-excited fluorescence in the rabbit lens, which contains a high level of the UVA chromophore NADH, existing both free and bound to λ-crystallin. Also, the ability of a Class I (senofilcon A) soft contact lens to protect against UVA-induced effects on the rabbit lens was tested. Rabbit eyes were irradiated with UVA light in vivo (100 mW/cm(2) on the cornea) for 1 h using monochromatic 365 nm light. Irradiation was conducted in the presence of either a senofilcon A contact lens, a minimally UV-absorbing lotrafilcon A contact lens, or no contact lens at all. Eyes irradiated without a contact lens showed blue 365 nm-excited fluorescence initially, but this changed to intense yellow fluorescence after 1 h. Isolated, previously irradiated lenses exhibited yellow fluorescence originating from the lens nucleus when viewed under 365 nm light, but showed normal blue fluorescence arising from the cortex. Previously irradiated lenses also exhibited a faint yellow color when observed under visible light. The senofilcon A contact lens protected completely against the UVA-induced effects on fluorescence and lens yellowing, whereas the lotrafilcon A lens showed no protection. The UVA-exposure also produced a 53% loss of total NADH (free plus bound) in the lens nucleus, with only a 13% drop in the anterior cortex. NADH loss in the nucleus was completely prevented with use of a senofilcon A contact lens, but no significant protection was observed with a lotrafilcon A lens. Overall, the senofilcon A lens provided an average of 67% protection against UVA-induced loss of four pyridine nucleotides in four different regions of the lens. HPLC analysis with fluorescence detection indicated a nearly six-fold increase in 365 nm-excited yellow fluorescence arising from lens nuclear λ-crystallin after the in vivo UVA exposure. It is concluded that UVA-induced loss of free NADH (which fluoresces blue) may have allowed the natural yellow fluorescence of λ-crystallin and other proteins in the lens nucleus to become visible. Increased fluorescence exhibited by UVA-exposed λ-crystallin may have been the result of a UVA-induced change in the conformation of the protein occurring during the initial UVA-exposure in vivo. The results demonstrate the greater susceptibility of the lens nucleus to UVA-induced stress, and may relate to the formation of human nuclear cataract. The senofilcon A contact lens was shown to be beneficial in protecting the rabbit lens against effects of UVA light, including changes in fluorescence, increased yellowing and loss of pyridine nucleotides., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Zeta-crystallin is an NADPH-binding protein consisting of four identical 35kD subunits. The protein possesses quinone oxidoreductase activity, and is present in large amounts in the lenses of camelids, certain hystricomorphic rodents, and the Japanese tree frog, and in lower catalytic amounts in certain tissues of various species. In this study, recombinant methods were used to produce substantial quantities of his-tagged recombinant mouse zeta-crystallin, which was then purified to homogeneity. The yield of pure recombinant mouse zeta-crystallin was five times that obtained previously for purification of recombinant guinea pig zeta-crystallin. The quinone oxidoreductase activity of purified his-tagged recombinant mouse zeta-crystallin was comparable to that of purified native guinea pig lens zeta-crystallin, and to that previously reported for recombinant guinea pig zeta-crystallin. The method permits production of substantial amounts of recombinant zeta-crystallin for conducting studies on the biological role of this interesting protein, which exists in such high concentration in the lenses of certain species.

The role of UVA radiation in the formation of human nuclear cataract is not well understood. We have previously shown that exposing guinea pigs for 5 months to a chronic low level of UVA light produces increased lens nuclear light scattering and elevated levels of protein disulfide. Here we have used the technique of dynamic light scattering (DLS) to investigate lens protein aggregation in vivo in the guinea pig/UVA model. DLS size distribution analysis conducted at the same location in the lens nucleus of control and UVA-irradiated animals showed a 28% reduction in intensity of small diameter proteins in experimental lenses compared with controls (P < 0.05). In addition, large diameter proteins in UVA-exposed lens nuclei increased five-fold in intensity compared to controls (P < 0.05). The UVA-induced increase in apparent size of lens nuclear small diameter proteins was three-fold (P < 0.01), and the size of large diameter aggregates was more than four-fold in experimental lenses compared with controls. The diameter of crystallin aggregates in the UVA-irradiated lens nucleus was estimated to be 350 nm, a size able to scatter light. No significant changes in protein size were detected in the anterior cortex of UVA-irradiated lenses. It is presumed that the presence of a UVA chromophore in the guinea pig lens (NADPH bound to zeta crystallin), as well as traces of oxygen, contributed to UVA-induced crystallin aggregation. The results indicate a potentially harmful role for UVA light in the lens nucleus. A similar process of UVA-irradiated protein aggregation may take place in the older human lens nucleus, accelerating the formation of human nuclear cataract.

Purpose: To characterize gene expression patterns in guinea pig ocular tissues and identify orthologs of human genes from NEIBank expressed sequence tags., Methods: RNA was extracted from dissected eye tissues of 2.5-month-old guinea pigs to make three unamplified and unnormalized cDNA libraries in the pCMVSport-6 vector for the lens, retina, and eye minus lens and retina. Over 4,000 clones were sequenced from each library and were analyzed using GRIST for clustering and gene identification. Lens crystallin EST data were validated using two-dimensional electrophoresis (2-DE), matrix assisted laser desorption (MALDI), and electrospray ionization mass spectrometry (ESIMS)., Results: Combined data from the three libraries generated a total of 6,694 distinctive gene clusters, with each library having between 1,000 and 3,000 clusters. Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank. Complete cDNA sequences were obtained for many guinea pig lens proteins, including alphaA/alphaAinsert-, gammaN-, and gammaS-crystallins, lengsin and GRIFIN. The ratio of alphaA- to alphaB-crystallin on 2-DE gels was 8: 1 in the lens nucleus and 6.5: 1 in the cortex. Analysis of ESTs, genome sequence, and proteins (by MALDI), did not reveal any evidence for the presence of gammaD-, gammaE-, and gammaF-crystallin in the guinea pig. Predicted masses of many guinea pig lens crystallins were confirmed by ESIMS analysis. For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin. The guinea-pig ortholog of NRL, a key rod photoreceptor-specific transcription factor, was also represented in EST data. In the 'rest-of-eye' library, the most abundant transcripts included decorin and keratin 12, representative of the cornea., Conclusions: Genomic analysis of guinea pig eye tissues provides sequence-verified clones for future studies. Guinea pig orthologs of many human eye specific genes were identified. Guinea pig gene structures were similar to their human and rodent gene counterparts. Surprisingly, no orthologs of gammaD-, gammaE-, and gammaF-crystallin were found in EST, proteomic, or the current guinea pig genome data.

Major UV filters have been identified in the lens of the 13 lined ground squirrel (Spermophilus tridecemlineatus). These were found to be N-acetyl-3-hydroxykynurenine and N-acetyl-kynurenine, in addition to a small quantity of 3-hydroxykynurenine. The level of N-acetyl-3-hydroxykynurenine measured in the ground squirrel lens, 8.2mM, is approximately 11 times the concentration of 3-hyroxykynurenine glucoside reported previously for the human lens. Two additional UV filters of related structure were also present; however, their structures are still under investigation. HPLC elution profiles indicated that the ground squirrel lens cortex and nucleus contained comparable amounts of alpha-, beta(H)-, beta(L)-, and gamma-crystallins. Levels of GSH in the cortex and nucleus were 12.4 and 7.4mM, respectively. Such high concentrations of GSH may act to inhibit oxidation of the 3-hydroxykynurenine and N-acetyl-3-hydroxykynurenine. N-Acetylated kynurenines are less labile than those with free alpha-amino groups since N-acetyl-alpha-amino groups do not undergo spontaneous deamination. This modification thus stabilises the squirrel UV filters. In addition, because deamination is prevented, the decomposition products will not be involved in binding to lens proteins. Because of the similarity of the UV filters present in the ground squirrel to those in man, this species may be a suitable animal model for investigating the effects of UV radiation on cataract, and other ocular diseases, thought to involve exposure to light.

Purpose: The role of oxygen in the formation of lens high-molecular-weight (HMW) protein aggregates during the development of human nuclear cataract is not well understood. The purpose of this study was to investigate lens crystallin aggregate formation in hyperbaric oxygen (HBO)-treated guinea pigs by using in vivo and in vitro, Methods: methods. Guinea pigs were treated three times weekly for 7 months with HBO, and lens crystallin aggregation was investigated in vivo with the use of dynamic light-scattering (DLS) and in vitro by HPLC analysis of water-insoluble (WI) proteins. DLS measurements were made every 0.1 mm across the 4.5- to 5.0-mm optical axis of the guinea pig lens., Results: The average apparent diameter of proteins in the nucleus (the central region) of lenses of HBO-treated animals was nearly twice that of the control animals (P < 0.001). Size distribution analysis conducted at one selected point in the nucleus and cortex (the outer periphery of the lens) after dividing the proteins into small-diameter and large-diameter groups, showed in the O2-treated nucleus a threefold increase in intensity (P < 0.001) and a doubling in apparent size (P = 0.03) of large-diameter aggregate proteins, compared with the same control group. No significant changes in apparent protein diameter were detected in the O2-treated cortex, compared with the control. The average diameter of protein aggregates at the single selected location in the O2-treated nucleus was estimated to be 150 nm, a size capable of scattering light and similar to the size of aggregates found in human nuclear cataracts. HPLC analysis indicated that one half of the experimental nuclear WI protein fraction (that had been dissolved in guanidine) consisted of disulfide cross-linked 150- to 1000-kDa aggregates, not present in the control. HPLC-isolated aggregates contained alphaA-, beta-, gamma-, and zeta-crystallins, but not alphaB-crystallin, which is devoid of -SH groups and thus does not participate in disulfide cross-linking. All zeta-crystallin present in the nuclear WI fraction appeared to be there as a result of disulfide cross-linking., Conclusions: The results indicate that molecular oxygen in vivo can induce the cross-linking of guinea pig lens nuclear crystallins into large disulfide-bonded aggregates capable of scattering light. A similar process may be involved in the formation of human nuclear cataract.

The pathogenic fungus Cryptococcus neoformans produces an extracellular PLB1 (phospholipase B1), shown previously to be a virulence factor. A novel phospholipase (LPL1) with only LPL (lysophospholipase) and LPTA (transacylase) activities has now been characterized in C. gattii, and found to be a 66-kDa glycoprotein (by SDS/PAGE), with a native molecular mass of 670 kDa. The pI was 6.3, and it was active at high temperatures (to 70 degrees C), as well as at both acidic and neutral pH values. It was stimulated by calcium and palmitoyl carnitine at pH 7.0, but not at pH 5.0, and palmitoyl lysophosphatidylcholine was the preferred substrate. Sequencing indicated that LPL1 is a novel cryptococcal lysophospholipase, and not the gene product of CnLYSO1 or PLB1. A protein with only LPL and LPTA activities was subsequently isolated from two strains of C. neoformans var. grubii. A PLB1 enzyme was isolated from both C. gattii and a highly virulent strain of C. neoformans var. grubii (H99). In both cases, all three enzyme activities (PLB, LPL and LPTA) were present in one 95-120 kDa glycoprotein (by SDS/PAGE) with pI 3.9-4.3. Characterization of PLB1 from C. gattii showed that it differed from that of C. neoformans in its larger native mass (275 kDa), high PLB activity relative to LPL and LPTA, and preference for saturated lipid substrates. Differences in the properties between the secreted phospholipases of the two cryptococcal species could contribute to phenotypic differences that determine their respective environmental niches and different clinical manifestations.

Extracellular phospholipase (PL) activities comprising phospholipase B, lysophospholipase and lysophospholipase transacylase have been identified in culture supernatants of Cryptococcus neoformans and contribute to virulence. We found that PL production was optimal after fungal growth at 30 degrees C and secretion at 37 degrees C for all six C. neoformans isolates studied (four C. neoformans var. neoformans and two C. neoformans var. gattii). No increase in PL activity was found in one strain, NU-2, in low iron or tissue culture media, conditions where upregulation of other virulence factors has been reported. The most virulent strains in an intravenous mouse model of infection were best able to produce PL at growth and secretion temperatures of 37 degrees C, in tissue culture media and under assay conditions of pH 7.0.

Phane worm (an edible larval stage of the emperor moth Imbrasia belina Westwood) is an important food source, and its harvesting is an economic activity in rural Botswana. When the larva is feeding on leaves and later during processing, phane gets contaminated with fungi from the leaves and soil. We examined 73 jars, each containing approximately 608 g (+/-0.25 g) of processed phane stored under laboratory conditions (temperature range 20 to 24 degrees C and 50 to 80% relative humidity) and combined intestinal contents of five phane squeezed into each of 74 Duran bottles for fungi. Ninety seven percent of 74 samples of intestinal contents and 57.5% of 73 laboratory-stored phane were positive for either molds and/or yeasts. Yeast population in intestinal contents ranged from 2 x 10(1) CFU/g to 5 x 10(3) CFU/g, whereas molds ranged from 1 x 10(1) CFU/g to 2 x 10(2) CFU/g. Laboratory-stored phane had a mold population of 1 x 10(2) CFU/g to 6 x 10(5) CFU/g. Species of Chaetomium 13.8%, Aspergillus 12.4%, Fusarium 5.5%, and Mucor racemosus 4.1% were the most prevalent in intestinal contents of phane, whereas Aspergillus 42.1%, Penicillium 33.9%, and Mucorales 5.7% were predominant in laboratory-stored phane. The important mycotoxigenic fungi A. flavus, A. parasiticus, A. ochraceus, P. aurantiogriseum, P. citrinum, and P. verrucosum were isolated mainly from the laboratory-stored phane. The genera isolated from both intestinal phane contents and laboratory-stored phane were Alternaria, Aspergillus, Chaetomium, Drechslera, Fusarium, Mucor, Phoma, and Penicillium, suggesting recontamination of phane during drying and storage.

Isozyme variation among 54 isolates of Microsporum canis, 18 Microsporum cookei isolates and two Diheterospora isolates were studied using starch gel electrophoresis. Of eight enzymes examined, four were polymorphic (EST, G6P, MDH and PEP), having from two to four electrophoretic forms. Within each species, consistent and reproducible isozyme patterns of the eight enzyme systems were obtained. Phenotypic diversity (H) in M. canis was higher than in M. cookei (H = 0.459 and H = 0.408 respectively), but phenotypic differentiation of M. canis isolates from different geographical regions (Auckland, Wellington and Palmerston North, New Zealand) was low, with a proportion of total diversity (Gst) of 0.151 found among the localities. The results suggest that the isolates of M. canis from different geographical regions are closely related, supporting the theory of a common lineage.

Fifty-four isolates of Microsporum canis (Arthroderma otae) from humans, cats and dogs obtained from Auckland, Palmerston North and Wellington, New Zealand and 18 M. cookei and two Diheterospori spp. from soils were examined for variation using eight isozyme loci. M. canis isolates were from infected and non-infected cases. Isozyme analysis separated the three species which were further subdivided into electrophoretic types (ETs). Clustering analysis using normalized percentage disagreement (PTC) average linkage method revealed two clusters for M. cookei with two subclusters in cluster 2. M. canis had three main divisions (clusters 3, 4 and 5) and Diheterospora formed a separate division. The presence of isolates from different sources in the same clusters and lack of statistical significance as measured by confidence intervals suggests the existence of isolates with common lineage.

Of 236 soil samples baited with wool, some 71.2% (168) produced fungal growth. Gliocladium (25.0%), Paecilomyces (14.8%), Trichophyton (11.9%) species were the most prevalent in soil moistened with sterile distilled water (SDW) containing antibiotics. On the other hand, in soil moistened with SDW only, Trichophyton (32.6%), Paecilomyces (27.5%), Diheterospora (16.5%), Gliocladium (13.6%) and Fusarium (13.1%) species were more common. Of the known potential pathogens, the Paecilomyces and Fusarium species were frequently isolated from soil collected in parks, cleared areas, paddocks, rivers and roadsides. All five sites are areas of human and animal activity. The keratinolytic species were Microsporum cookei, M. gypseum complex, Trichophyton ajelloi and T. terrestre, which are regarded as nonpathogens with the exception of M. cookei and M. gypseum complex.

Characterization by proteinase inhibitors of the enzymes produced by Microsporum spp. revealed that Microsporum canis and Microsporum cookei produce serine proteinase(s), but only M. canis expresses aspartic and cysteine proteinases and probably a metalloproteinase. Both M. canis and M. cookei expressed metalloelastinolytic proteinases. All the proteinase types have been implicated in the pathogenicity of a wide range of microorganisms.

Enzyme expression of 67 isolates of two Microsporum species, M. canis and M. cookei, were compared in both shake and stationary cultures using substrate copolymerized SDS-PAGE. Most M. canis isolates expressed more proteolytic bands in shake culture, while M. cookei isolates expressed more in stationary culture. M. canis isolates expressed up to six proteinases of different relative mobilities (122, 64, 62, 45, 31 and 25 kDa). M. cookei expressed up to seven proteinases in stationary culture (67, 66, 64, 62, 45, 42 and 39 kDa). Those of 67 and 66 kDa were not expressed in shake culture. The proteinases expressed by M. cookei were similar to those expressed by M. canis except for 122 and 25 kDa. With the exception of isolates from non-infected cats, 25 kDa was also commonly expressed by isolates from infected hosts in the shake culture treatment. The differences in enzyme expression obtained may reflect differences in the contrasting ecological roles of the two species.

A total of 178 cats and 59 dogs in Palmerston North, New Zealand were sampled for the presence of keratinophilic fungi on their pelage; 57.8% had fungi. The fungi were classified in 20 genera with the predominant species being members of the genera; Chrysosporium, Microsporum and Trichophyton. Cats were the major carriers of keratinolytic fungi. 18.5% of the cats and 5.1% of the dogs were either carriers or infected with M. canis. Microsporum canis was a frequent isolate and its distribution had a seasonal variation, with a peak appearing in May-June.

The 4-methylumbelliferyl-N-acetyl-beta-D-galactosaminide (UAG) test was evaluated in parallel with chlamydospore (CHL) and germ-tube (GT) tests for the identification of Candida spp. The UAG test gave 86.6% correct identification of C. albicans and C. tropicalis. Non-C. albicans and non-C. tropicalis yeasts gave correct negative results in UAG tests. Only one isolate of Trichosporon beigelii gave a "false" positive reaction. The UAG test, which can be completed within 30 min, is a reliable test for screening non-C. albicans and non-C. tropicalis yeasts.

13 isolates of Trichophyton violaceum and 7 Microsporum langeroni from Zambia and Cameroon were tested in vitro for their sensitivity to griseofulvin. No strain was found resistant. All the 13 T. violaceum were inhibited by 4-8 micrograms/ml while 4 M. langeroni were inhibited by 0.5-1 microgram/ml.

A study of tinea capitis in urban and rural schools of Lusaka revealed two dermatophytes, vis., Microsporum langeroni and Trichophyton violaceum as the causative agents of tinea capitis among school children. Tinea capitis was more common among boys (18.0%) than girls (14.7%), with prevalence of 16.8% and a peak infection at 9-11 years for both sexes. Clinically, most cases due to both M. langeroni and T. violaceum were non-inflammatory. The inflammatory cases were caused by T. violaceum. The higher number of infected children from squatter and low-cost areas was statistically highly significant compared to children from high-cost areas. A higher number of rural children were infected with tinea capitis than children in urban Lusaka. T. violaceum was the only dermatophyte species isolated in rural Lusaka, while in urban Lusaka, both T. violaceum and M. langeroni were isolated.