The mycotoxin 2-Amino-14,16-dimethyloctadecan-3-ol (AOD) has been isolated from cultures of the fungus Fusarium avenaceum, one of the most prevalent Fusarium species. AOD is an analogue of sphinganine and 1-deoxysphinganine, important intermediates in the de novo biosynthesis of cellular sphingolipids. Here we studied cellular effects of AOD using the human liver cell line HepG2 as a model system. AOD (10 μM) induced a transient accumulation of vacuoles in the cells. The effect was observed at non-cytotoxic concentrations and was not linked to cell death processes. Proteomic analyses indicated that protein degradation and/or vesicular transport may be a target for AOD. Further studies revealed that AOD had only minor effects on the initiation rate of macropinocytosis and autophagy. However, the AOD-induced vacuoles were lysosomal-associated membrane protein-1 (LAMP-1) positive, suggesting that they most likely originate from lysosomes or late endosomes. Accordingly, both endosomal and autophagic protein degradation were inhibited. Further studies revealed that treatment with concanamycin A or chloroquine completely blocked the AOD-induced vacuolization, suggesting that the vacuolization is dependent of acidic lysosomes. Overall, the results strongly suggest that the increased vacuolization is due to an accumulation of AOD in lysosomes or late endosomes thereby disturbing the later stages of the endolysosomal process.

Storage of human retinal pigment epithelium (hRPE) can contribute to the advancement of cell-based RPE replacement therapies. The present study aimed to improve the quality of stored hRPE cultures by identifying storage medium additives that, alone or in combination, contribute to enhancing cell viability while preserving morphology and phenotype. hRPE cells were cultured in the presence of the silk protein sericin until pigmentation. Cells were then stored for 10 days in storage medium plus sericin and either one of 46 different additives. Individual effects of each additive on cell viability were assessed using epifluorescence microscopy. Factorial design identified promising additive combinations by extrapolating their individual effects. Supplementing the storage medium with sericin combined with adenosine, L-ascorbic acid and allopurinol resulted in the highest cell viability (98.6 ± 0.5%) after storage for three days, as measured by epifluorescence microscopy. Flow cytometry validated the findings. Proteomics identified 61 upregulated and 65 downregulated proteins in this storage group compared to the unstored control. Transmission electron microscopy demonstrated the presence of melanosomes after storage in the optimized medium. We conclude that the combination of adenosine, L-ascorbic acid, allopurinol and sericin in minimal essential medium preserves RPE pigmentation while maintaining cell viability during storage.

AIM: This was to determine the prevalence of Lactobacilli (LB) species in different stages of caries progression and are considered as secondary invaders of existing carious lesions and specialists for caries progression. METHODS: Carious dentine samples were collected from 70 primary molars (M) during step-wise (S1, S2: n = 35 M) or one-step (O1: n = 35 M) caries treatment and after 11 months of temporary restorations (S3, O2). LB were identified by selected physiological and biochemical characteristics, ratio of lactic acid isomers, electrophoretic mobilities of lactic acid dehydrogenases, and shotgun mass mapping by MALDI mass spectrometry. RESULTS: LB were isolated from 46% of soft dentine samples (S1). The prevalence of LB from hard dentine collected during caries excavation (O1) reached 34%, after 8 weeks of temporary filling (S2) 11%, and 9% each after 11 months of temporary restoration (S3, O2). The mean total bacterial counts (cfu) of soft dentine (S1) were 3.6 × 105. From hard dentine during caries excavation (O1 ) 4.4×104 cfu were calculated, at S2 3.7 × 103 cfu, at S3 0.1 × 103 cfu, and at O2 1.8 × 103 cfu. The percentages of LB in the cfu for LB positive dentine samples were for S1 / S2 / S3 / O1 / O2: 60% (16 M)/34% (4 M)/54% (3 M)/57% (9 M), and 64% (3 M). Five LB species were identified from carious dentine: L. paracasei subsp. paracasei, L. paracasei subsp. tolerans, L. rhamnosus, L. gasseri, and L. alimentarius. CONCLUSIONS: While L. rhamnosus and L. paracasei subsp. paracasei occurred in all caries progression stages, the other species were found only sporadically. L. paracasei subsp. paracasei and L. rhamnosus might be the specialists of the LB in carious progression.

A new shotgun proteomics approach was employed to identify degraded proteins. Jurkat T-cells were induced to undergo apoptosis by Fas (CD95/Apo-1) stimulation. The proteins were separated by large (30 cm) sodium dodecyl sulphate-polyacrylamide gel electrophoresis and identified by liquid chromatography-tandem mass spectrometry after digestion of 100 gel slices with trypsin. The molecular masses of the individual gel slices were calculated through the known theoretical masses of the identified proteins. Proteins were defined as degradation candidates if either the empirical determined molecular mass was at most 80 % of the theoretical value, or if proteins were identified in clearly different gel slices. In this manner, the degradation of 11 already identified apoptosis-modified proteins was confirmed and nine until now unknown degradation candidate proteins identified. Degradation during apoptosis must be verified by additional techniques such as in vitro caspase assays as shown for nucleolin and Rho GDI2. The results presented confirm the suitability of a shotgun approach for the identification of putative protease targets.

The purple ink of the sea hare Aplysia punctata contains a 60 kDa protein with tumoricidal activity. This A. punctata ink toxin (APIT) kills tumor cells within 6--8h in an apoptosis independent manner by the production of high amounts of hydrogen peroxide which induce a necrotic form of oxidative stress. Here, we describe the biochemical features of APIT associated with its anti-tumor activity. APIT is a weakly glycosylated FAD-binding L-amino acid oxidase that catalyzes the oxidative deamination of L-lysine and L-arginine and thereby produces hydrogen peroxide (H(2)O(2)), ammonia (NH(4)(+)) and the corresponding alpha-keto acids. The tumoricidal effect is completely abrogated in the absence of the amino acids L-lysine and L-arginine. The enzyme is stable at temperatures from 0 to 50 degrees C. Similar to other FAD-binding enzymes, it is resistant against tryptic digest. Even digest with proteinase K fails to degrade the enzyme. Cloning of the APIT gene and subsequent sequencing revealed a FAD-binding domain followed by a so-called GG-motif, which is typical for L-amino acid oxidases. Strongest homology exists to escapin, aplysianin A precursor, the cyplasins L and S and achacin.

Lactobacillus salivarius subsp. salivarius UCC118 is a probiotic bacterium that was originally isolated from human intestinal tissues and was subsequently shown in a pilot study to alleviate symptoms associated with mild-moderate Crohn's disease. Strain UCC118 can adhere to animal and human intestinal tissue, and to both healthy and inflamed ulcerative colitis mucosa, irrespective of location in the gut. In this study, an enzymatic technique has been combined with proteomic analysis to correlate bacterial growth phase with the presence of factors present in the cell wall of the bacterium. Using PAGE electrophoresis, it was determined that progression from lag to log to stationary growth phases in vitro correlated with increasing prominence of an 84kD protein associated with in vitro adherence ability. Isolated proteins from the 84kD band region were further separated by two-dimensional electrophoresis, resolving this band into 20 individual protein spots at differing isoelectric points. The protein moieties were excised, trypsin digested and subjected to tandem mass spectrometry. The observed proteins are analogous to those reported to be associated with the Listeria monocytogenes cell-wall proteome, and include DnaK, Ef-Ts and pyruvate kinase. These data suggest that at least some of the beneficial attributes of probiotic lactobacilli, and in particular this strain, may be due to nonpathogenic mimicry of pathogens and potentially be mediated through a form of attenuated virulence.

Peptide mass fingerprinting by MALDI-MS and sequencing by tandem mass spectrometry have evolved into the major methods for identification of proteins following separation by two-dimensional gel electrophoresis, SDS-PAGE or liquid chromatography. One main technological goal of proteome analyses beside high sensitivity and automation was the comprehensive analysis of proteins. Therefore, the protein species level with the essential information on co- and post-translational modifications must be achieved. The power of peptide mass fingerprinting for protein identification was described here, as exemplified by the identification of protein species with high molecular masses (spectrin alpha and beta), low molecular masses (elongation factor EF-TU fragments), splice variants (alpha A crystallin), aggregates with disulfide bridges (alkylhydroperoxide reductase), and phosphorylated proteins (heat shock protein 27). Helpful tools for these analyses were the use of the minimal protein identifier concept and the software program MS-Screener to remove mass peaks assignable to contaminants and neighbor spots.

Proteome analysis of Jurkat T cells induced to undergo apoptosis by CD95 (Fas/Apo-1) treatment was performed to identify modified proteins. We used stable isotope labeling with amino acids in cell culture (SILAC) using leucine to identify proteins of apoptotic and control Jurkat T cells by 2-DE and MALDI-MS. Out of 224 spots analyzed, we quantified 213 spots with 3.5 leucine-containing peptide pairs on average; 28 proteins with a relative abundance of higher than 1.5 were found. Five new modified proteins including calcyclin binding protein, cytosolic acyl coenzyme A thioester hydrolase, heterogeneous ribonucleoprotein M, replication factor C 37-kDa subunit, and tropomyosin 4 chain were identified as being modified in response to apoptosis. In comparison to differential proteome analysis via silver-stained 2-D gels and PMF of total Jurkat T cell lysates, 15 additional apoptosis-modified proteins were identified though 8 proteins were not found. The described approach using SILAC instead of silver staining for relative quantification was simpler to perform regarding the number of required 2-D gels, that cumbersome gel comparisons were avoided, and more apoptosis-modified proteins were identified, but with a higher demand on data interpretation of the mass spectra obtained.

Marine snails of the genus Aplysia possess numerous bioactive substances. We have purified a 60 kDa protein, APIT (Aplysia punctata ink toxin), from the defensive ink of A. punctata that triggers cell death with profound tumor specificity. Tumor cell death induced by APIT is independent of apoptosis but is characterized by the rapid loss of metabolic activity, membrane permeabilization, and shrinkage of nuclei. Proteome analysis of APIT-treated tumor cells indicated a modification of peroxiredoxin I, a cytoplasmic peroxidase involved in the detoxification of peroxides. Interestingly, knockdown of peroxiredoxin I expression by RNA interference sensitized cells for APIT-induced cell death. APIT induced the death of tumor cells via the enzymatic production of H2O2 and catalase completely blocked APITs' activity. Our data suggest that H2O2 induced stress and the modulation of peroxiredoxins might be a promising approach for tumor therapy.

Global approaches like proteome or transcriptome analyses have been performed extensively to identify candidate genes or proteins involved in biological and pathological processes. Here we describe the identification of proteins implicated in the regulation of apoptosis using proteome analysis and the functional validation of targets by RNA interference. A high-throughput platform for the validation of synthetic small interfering RNAs ( siRNAs) by quantitative real-time PCR was established. Genes of the identified factors were silenced by automated siRNA transfection, and their role in apoptotic signaling was investigated. Using this strategy, nine new modulators of apoptosis were identified. A subsequent detailed study demonstrated that hepatoma-derived growth factor ( HDGF) is required for TNFalpha-induced release of pro- apoptotic factors from mitochondria. The strategy described here may be used for hypothesis-free, global gene function analysis.