Carbocyclic 3'-deoxyadenosine-based highly potent bisubstrate-analog inhibitor of basophilic protein kinases

Carbocyclic analogs of 3'-deoxyadenosine were synthesized as racemates and the resulting stereoisomers were separated by chromatography on a chiral column. The conjugation of obtained compounds with hexa-(D-arginine) via 6-aminohexanoic acid linker led to a highly potent inhibitor of several basophilic protein kinases with some selectivity towards cAMP-dependent protein kinase.     

Bacillus licheniformis bacitracin-resistance ABC transporter: relationship to mammalian multidrug resistance

The nucleotide sequence of the Bacillus licheniformis bacitracin-resistance locus was determined. The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed. The BcrA protein shares a high degree of homology with the hydrophilic ATP-binding components of the ABC family of transport proteins. The bcrB and bcrC genes were found to encode hydro-phobic proteins, which may function as membrane components of the permease. Apart from Bacillus subtilis, these genes also confer resistance upon the Gram-negative Escherichia coli. The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target. In addition to the homology of the nucleotide-binding sites, BcrA protein and mammalian multidrug transporter or P-glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane. The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide-binding protein to determine the Importance of various regions in transport.     

Cross-talk of vitamin D and glucocorticoids in hippocampal cells

There is growing evidence for a role of vitamin D3 signalling in the brain. In this study, we investigated the influence of vitamin D3, in combination with glucocorticoids, on differentiation of the hippocampal progenitor line HIB5, as well as survival of rat primary hippocampal cells. In HIB5, pre-treatment with dexamethasone (Dex) alone inhibited neurite outgrowth and abolished activation of the mitogen-activated protein kinase (MAPK) pathway during platelet-derived growth factor (PDGF)-induced differentiation, consistent with previous findings. Interestingly, pre-treating HIB5 with vitamin D3 significantly reduced these effects of Dex and, in addition, lowered the transactivational function of the glucocorticoid receptor (GR) in transient reporter gene assays. A further impact of vitamin D3 on glucocorticoid effects was observed in a rat primary hippocampal culture known to be particularly sensitive to prolonged GR activation. In this model, Dex induced considerable cell death after 72 h of exposure in vitro. However, 24 h of pre-treatment with low doses of vitamin D3 substantially reduced the degree of Dex-induced apoptosis in primary hippocampal cells. Taken together, our experiments demonstrate a cross-talk between vitamin D3 and glucocorticoids in two hippocampal models, a feature that may have important implications in disorders with dysregulated glucocorticoid signalling, including major depression.     

Molecular characterization of human pathogen Babesia EU1 in Ixodes ricinus ticks from Slovenia

New cases of human babesiosis were recently reported in Europe. The etiological agent was identified as Babesia EU1, a zoonotic pathogen with previously unreported molecular characteristics. On the basis of a comparison of the complete babesial 18S rRNA gene, we have generated strong molecular evidence that Ixodes ricinus ticks from Slovenia are infected with EU1.     

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.