No abstract

The transfer regions of different conjugative plasmids show significant similarities in the genetic organization and in the amino acid sequence of some gene products, especially of proteins from the traG or trbB family. These similarities are also evident on the level of the nucleotide sequences. On the basis of conserved DNA regions we designed degenerate PCR primer pairs to detect specifically tra regions within a collection of bacterial clones isolated from an agricultural soil. Most of the potential transfer-proficient indigenous bacterial isolates were able to mobilize a derivative of the nonconjugative IncQ plasmid RSF1010 into recipient strains. With the help of the primers it should be possible to evaluate the genetic potential for horizontal gene transfer carried out by conjugative plasmids.

Matching of human leukocyte antigen (HLA) alleles between donors and recipients plays a major role in hematopoietic stem cell transplantation (HSCT). Null or questionably expressed HLA allelic variants are a major issue in HLA matching, because the aberrant expression of such alleles can have a major impact on the outcome of HSCT and/or its complications such as graft-versus-host disease. The goal of this study was to investigate the potential of a recently developed cytokine-induced secretion assay to differentiate the expression levels of HLA-A*32:11Q (questionable) into a null (N) or low (L) expression variant. An amino acid mutation at position 164 of HLA-A*32:11Q disrupts the disulfide bridge in the α2 domain. HLA-A*32:11Q is not detectable by standard microlymphocytotoxicity assay. To this end, we cloned soluble HLA-A*32:11Q and a reference allele (HLA-A*32:01) into expression vectors and transfected/transduced HEK293 and K562 cells. Allele-expressing K562 cells were simultaneously transfected/transduced with a β2-microglobulin (B2M)-encoding vector to ensure the intact HLA structure with B2M. After treatment with proinflammatory cytokines, secreted soluble HLA molecules were determined by enzyme-linked immunosorbent assay in the supernatant and intracellular accumulation of the recombinant proteins by flow cytometry. HLA-A*32:11Q was nearly undetectable in untreated transfectants. Cytokine treatment increased the secretion of HLA-A*32:11Q to detectable levels and resulted in intracellular accumulation of the allele. There was no difference in mRNA transcription between the A*32 alleles. On the basis of these results, we recommend reclassification of HLA-A*32:11Q as a low expression (L) variant.

Plasmid analysis of isolates from a small Paracoccus population revealed that all 15 representatives carried at least one endogenous plasmid of 23 or 15 kb in size, in addition to further plasmids of different sizes. It was shown by restriction analysis and hybridization that the 23 and 15 kb plasmids from the different isolates were identical or very similar to each other. By partial sequencing of pOL18/23, one of the 23 kb plasmids, a complete rrn operon with the structural genes for 16S, 23S and 5S rRNA, two genes for tRNA Ile and tRNA Ala within the spacer between the 16S and 23S rRNA genes, and a final tRNA fMet at the end of the operon were discovered. Expression of a green fluorescent protein gene (gfp) after insertion of a DNA fragment from the region upstream of the rRNA genes into a promoter-probe vector demonstrated that the rrn promoter region is functional. The rrn operon encoded by plasmid pOL18/23 is the first complete rrn operon sequenced from a strain of the genus Paracoccus, and only the second example of an rrn operon on a small plasmid.

Integrins are a large family of heterodimeric proteins that are involved in cell adhesion, migration, and proliferation. Integrin diversity and function is regulated by alternative splicing. Membrane-bound and truncated β3-integrins were shown to be key players in cancer metastasis. However, the immunomodulatory functions of the soluble (s) β3-integrin have not been investigated yet. In this study, we described a novel form of sβ3-integrin in acute myeloid leukaemia (AML) patients. Furthermore, we assessed the role of the sβ3-integrin in the modulation of natural killer (NK)-cell activity. Levels of sβ3-integrin were analysed in plasma samples of 23 AML patients and 26 healthy donors by ELISA. The capacity of sβ3-integrin to regulate NK cell activity was investigated using proliferation, cytokine secretion, and cytotoxicity assays. Circulating sβ3-integrin was detected in the plasma of 8 AML patients. NK cells showed significantly higher proliferation rates after stimulation with sβ3-integrin and IL-2, IL-15 (73%). Significant increases in the NK cells’ secreted levels of TNF-α, IFN-γ were measured in presence of sβ3-integrin. In addition, sβ3-integrin caused the upregulation of Granzyme B transcripts levels as well as FasL expression levels in NK cells. Most importantly, significantly higher K562 or AML blast target cell lysis rates were observed when NK cells were exposed to sβ3-integrin. This study reports the identification of a novel sβ3-integrin in AML patients and provides novel insights into its role in the immunomodulation of NK cell activity.