Proteomic scan for tyrosinase peptide antigenic pattern in vitiligo and melanoma: role of sequence similarity and HLA-DR1 affinity

Immune responses contribute to the pathogenesis of vitiligo and target melanoma sometimes associated with vitiligo-like depigmentation in some melanoma patients. We analyzed the sera from patients with vitiligo and cutaneous melanoma for reactivity toward tyrosinase peptide sequences 1) endowed with low level of similarity to human proteome, and 2) potentially able to bind HLA-DR1 Ags. We report that the tyrosinase autoantigen was immunorecognized with the same molecular pattern by sera from vitiligo and melanoma patients. Five autoantigen peptides composed the immunodominant anti-tyrosinase response: aa95-104FMGFNCGNCK; aa175-182 LFVWMHYY; aa176-190FVWMHYYVSMDALLG; aa222-236IQKLTGDENFTIPYW, and aa233-247 IPYWDWRDAEKCDIC. All of the five antigenic peptides were characterized by being (or containing) a sequence with low similarity level to the self proteome. Sera from healthy subjects were responsive to aa95-104FMGFNCGNCK, aa222-236IQKLTGDENFTIPYW, and aa233-247 IPYWDWRDAEKCDIC, but did not react with the aa175-182LFVWMHYY and aa176-190FVWMHYYVSMDALLG peptide sequences containing the copper-binding His180 and the oculocutaneous albinism I-A variant position F176. Our results indicate a clear-cut link between peptide immunogenicity and low similarity level of the corresponding amino acid sequence, and are an example of a comparative analysis that might allow to comprehensively distinguish the epitopic peptide sequences within a disease from those associated to natural autoantibodies. In particular, these data, for the first time, delineate the linear B epitope pattern on tyrosinase autoantigen and provide definitive evidence of humoral immune responses against tyrosinase.     

F-Like Plasmid Sequences in Enteric Bacteria of Diverse Origin, with Implication of Horizontal Transfer and Plasmid Host Range

Seventy-eight bacterial isolates from human, animal, and plant hosts, representing eight species of the family Enterobacteriaceae, were screened for F-like plasmid sequences. Of the examined human Escherichia coli strains, 28% harbored one or two of the three F-like, RepFI replication regions, while 35% of the examined animal and all phytopathogenic strains harbored RepFIA-specific sequences. Comparative analysis of Salmonella, Shigella, Erwinia, and E. coli plasmid RepFI sequences showed 100% or very high homology, indicating frequent and recent interspecies gene transfer. The high incidence of RepFIA sequences in enteric bacterial species, including Klebsiella and Erwinia, showed that F-like plasmids are successful in avoiding natural barriers to establishment of horizontally transferred DNA and that in the natural environment conjugal transfer is efficient in diverse ecological niches. Received: 26 March 2001 / Accepted 12 July 2001     

Identification of the origin of replications and partial characterization of plasmid pRK100

In search for the evolutionary origin of the conjugative F-like plasmid pRK100, the plasmid's functional replication regions were identified. Additionally targeted genetic analysis was used to investigate origins of other regions of the plasmid. Construction of minireplicons via ligation of Tn1725 with plasmid fragments and targeted cloning of putative replication regions, followed by sequence analysis indicated two functional replication regions, a F plasmid related RepFIB and a R1 plasmid related RepFIIA replication region. Partial nucleotide sequencing of regions of the plasmid revealed genes that encode a putative enterochelin iron uptake system previously associated with an Escherichia coli pathogenicity island, PAI III536, and the pColV-like aerobactin genes. In addition, a homologue of the R100 plasmid related rmoA gene was found that exhibits strong similarity to hha/ymoA encoding the Hha/YmoA class of modulators of gene expression. PCR and hybridization experiments further demonstrated that pRK100 harbors multiple IS2 and IS3 insertion sequences that may have facilitated in the acquisition of elements from other DNA molecules. These data together with the previous identification of a F-like tra region and a pColIa-like colicin Ia, indicate that pRK100 has a highly mosaic structure with elements derived from many different known large natural plasmids.     

Computational peptide dissection of Melan-a/MART-1 oncoprotein antigenicity

We have mapped the linear antigenic determinant of a commercial MAb raised in the mouse against the melanoma-associated-antigen Melan-A/MART-1. The B cell epitope on the Melan-A/MART-1 oncoprotein is located in the 15-mer amino acid sequence 101-115 PPAYEKLSAEQSPPP, within residues 102-106. The definition of the antigenic sequence on Melan-A/MART-1 oncoprotein was reached following analyses of MHC II binding potential and similarity level to the mouse proteome, that put into evidence the 15-mer amino acid sequence 101-115 PPAYEKLSAEQSPPP as the top scoring peptide in binding H2-A(d) molecules and the epitopic sequence residues 102-106 (i.e., the peptide sequence PAYEK) as having low-similarity level to the mouse proteome. Dot-blot epitope mapping immunoassay identified proline residue 102 as critical, based on its effect on antibody recognition. The present study adds to previous companion reports in validating the hypothesis that low-similarity to the host's proteome and binding potential to MHC II molecules are essential concurring factors in the modulation of the pool of epitopic sequences.     

Effect of arsenic trioxide on metallothionein and its conversion to different arsenic metabolites in hen liver

The metabolism of arsenic, its affinity to metallothionein (MT), its influence on selenium levels, and its biotransformation to different metabolites in the liver tissue of laying hens exposed to arsenic trioxide (As2O3) was investigated. The experiment was performed with two groups of hens fed for 19 d with either a standard diet or with the same diet enriched in arsenic (30 microg/g). The major findings were as follows: 1. After 19 d exposure, about 65% of the total liver As was found in the water-soluble phase (100,000g centrifuged supernatant). In liver supernatant, As binding was found mostly in the range of very low-molecular-weight proteins (Mr < 10,000). Although after exposure the amount of MT-like proteins increased, the As bound to it was only in trace amounts. The protein was identified by convential procedures as Zn,Cu-thionein with traces of selenium and arsenic. 2. Arsenic exposure resulted in almost unchanged Se levels regarding its tissue concentrations and distribution between supernatant and pellet, where about 10% of total Se was found in the supernatant. On the contrary, As exposure did affect Cd levels. Tissue Cd concentration was slightly diminished, but the percentage of tissue Cd found in the water-soluble phase was increased from 20% to 40%. 3. In methanol extracts of tissue and supernatant of the As-exposed group, only two arsenic compounds were detected, As(III) and dimethylarsinic acid (DMA), the latter prevailing.     

Genotyping of Chlamydia trachomatis directly from urogenital and conjunctiva samples using an ompA gene pyrosequencing-based assay

The aim of the present study was to develop and validate a multitarget pyrosequencing-based protocol for basic Chlamydia trachomatis genotyping directly from clinical samples and to characterize the distribution of genotypes among Slovenian sexually active population. The newly developed combination of assays that targets the variable domains VD-I and VD-IV of the C. trachomatis ompA gene, was optimized and validated with 11 reference C. trachomatis strains and by comparison to complete ompA conventional sequencing. In addition, 183 clinical specimens which were previously diagnosed as C. trachomatis positive were evaluated by pyrosequencing. The pyrosequencing products showed a 100% match to corresponding sections of the respective conventional ompA sequences. Based on our results the most frequent genotype in urogenital samples was E (51.1%) followed by F (21.4%), G and K (6.9%), D (6.1%), H (3.8%), J (2.3%) and Ia and Ja (0.8%). In conjunctiva samples the genotype distribution was E (63.3%), D and F (13.3%), K (6.7%) and G (3.3%). Pyrosequencing thus proved itself to be a rapid method for C. trachomatis typing, which is important for better understanding the pathogenesis and epidemiology of this pathogen.     

Prevalence of ColE1-Like Plasmids and Colicin K Production among Uropathogenic Escherichia coli Strains and Quantification of Inhibitory Activity of Colicin K

Colicin K exhibited pronounced inhibitory activity against uropathogenic Escherichia coli (UPEC) strains. Low prevalence of colicin K production and a relatively high prevalence of ColE1-like plasmids were determined among 215 UPEC strains from Slovenia. Sequencing of the colicin K-encoding pColK-K235 revealed a mosaic structure and the presence of the insertion sequence IS2.     

Respiratory protein interactions in Dehalobacter sp. strain 8M revealed through genomic and native proteomic analyses

Dehalobacter (Firmicutes) encompass obligate organohalide-respiring bacteria used for bioremediation of groundwater contaminated with halogenated organics. Various aspects of their biochemistry remain unknown, including the identities and interactions of respiratory proteins. Here, we sequenced the genome of Dehalobacter sp. strain 8M and analysed its protein expression. Strain 8M encodes 22 reductive dehalogenase homologous (RdhA) proteins. RdhA D8M_v2_40029 (TmrA) was among the two most abundant proteins during growth with trichloromethane and 1,1,2-trichloroethane. To examine interactions of respiratory proteins, we used blue native gel electrophoresis together with dehalogenation activity tests and mass spectrometry. The highest activities were found in gel slices with the highest abundance of TmrA. Protein distributions across gel lanes provided biochemical evidence that the large and small subunits of the membrane-bound [NiFe] uptake hydrogenase (HupL and HupS) interacted strongly and that HupL/S interacted weakly with RdhA. Moreover, the interaction of RdhB and membrane-bound b-type cytochrome HupC was detected. RdhC proteins, often encoded in rdh operons but without described function, migrated in a protein complex not associated with HupL/S or RdhA. This study provides the first biochemical evidence of respiratory protein interactions in Dehalobacter, discusses implications for the respiratory architecture and advances the molecular comprehension of this unique respiratory chain.