Regulation of Src family kinases involved in T cell receptor signaling by protein-tyrosine phosphatase CD148

CD148 is a receptor-like protein-tyrosine phosphatase known to inhibit transduction of mitogenic signals in non-hematopoietic cells. Similarly, in the hematopoietic lineage, CD148 inhibited signal transduction downstream of T cell receptor. However, it also augmented immunoreceptor signaling in B cells and macrophages via dephosphorylating C-terminal tyrosine of Src family kinases (SFK). Accordingly, endogenous CD148 compensated for the loss of the main SFK activator CD45 in murine B cells and macrophages but not in T cells. Hypothetical explanations for the difference between T cells and other leukocyte lineages include the inability of CD148 to dephosphorylate a specific set of SFKs involved in T cell activation or the lack of CD148 expression during critical stages of T cell development. Here we describe striking differences in CD148 expression between human and murine thymocyte subsets, the only unifying feature being the absence of CD148 during the positive selection when the major developmental block occurs under CD45 deficiency. Moreover, we demonstrate that similar to CD45, CD148 has both activating and inhibitory effects on the SFKs involved in TCR signaling. However, in the absence of CD45, activating effects prevail, resulting in functional complementation of CD45 deficiency in human T cell lines. Importantly, this is independent of the tyrosines in the CD148 C-terminal tail, contradicting the recently proposed phosphotyrosine displacement model as a mechanism of SFK activation by CD148. Collectively, our data suggest that differential effects of CD148 in T cells and other leukocyte subsets cannot be explained by the CD148 inability to activate T cell SFKs but rather by its dual inhibitory/activatory function and specific expression pattern.     

KPC-2-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> isolated from a Czech patient previously hospitalized in Greece and in vivo selection of colistin resistance

Carbapenemase-producing Gram-negative bacteria peak clinical interest due to their ability to hydrolyze most β-lactams, including carbapenems; moreover, their genes spread through bacterial populations by horizontal transfer. Bacteria with acquired carbapenemase have sporadically been reported in the Czech Republic, so far only in Enterobacteriaceae and Pseudomonas aeruginosa. In this study, we described the first finding of a KPC-2-producing strain of Klebsiella pneumoniae, which was isolated from a surgical wound swab, decubitus ulcer, and urine of a patient previously hospitalized in Greece. The patient underwent various antibiotic therapies including a colistin treatment. However, after approximately 20 days of the colistin therapy, the strain developed a high-level resistance to this drug. All the isolates were indistinguishable by pulsed field gel electrophoretic analysis and belonged to the international clone ST258, which is typical of KPC-producing K. pneumoniae isolates. The bla _KPC-2 gene was located on a Tn4401a transposon variant. The OmpK35 and OmpK36 genes analysis performed due to the high resistance level of the strains to β-lactams exhibited no changes in their sequence or in their expression when compared with carbapenem-susceptible isolates.     

Phosphoramidate pronucleotides of cytostatic 6-aryl-7-deazapurine ribonucleosides

A series of O-phenyl methyl-, ethyl- and benzylalanyl phosphoramidate pronucleotides derived from cytostatic 6-aryl-7-deazapurine ribonucleosides were prepared by the cross-coupling reactions of the 2′,3′-isopropylidene protected 6-chloro-7-deazapurine ribonucleoside phosphoramidates with (het)arylboronic acids or -stannanes followed by deprotection. Most of the prepared prodrugs exerted in vitro cytostatic effects against both solid tumor and lymphoid cancer cells within low micromolar range of concentrations. These activities were in general weaker or comparable to the activities of the parent nucleosides. Additional testing of selected prodrugs suggests that the lack of activity improvement over parent nucleosides is not due to the lack of permeability or inefficient catabolism of alanyl-ester by intracellular hydrolases. More likely, active efflux of prodrugs may play a role in their weak cytotoxic activity.     

Kinetic Constant Variability in Bacterial Oxidation of Elemental Sulfur

Wide ranges of growth yields on sulfur (from 2.4 × 10¹⁰ to 8.1 × 10¹¹ cells g⁻¹) and maximum sulfur oxidation rates (from 0.068 to 1.30 mmol liter⁻¹ h⁻¹) of an Acidithiobacillus ferrooxidans strain (CCM 4253) were observed in 73 batch cultures. No significant correlation between the constants was observed. Changes of the Michaelis constant for sulfur (from 0.46 to 15.5 mM) in resting cells were also noted.     

Osteotropic Peptide that differentiates functional domains of the skeleton

HPMA copolymer-d-aspartic acid octapeptide (D-Asp8) conjugates have been found to target the entire skeleton after systemic administration. In a recent study using the ovariectomized rat model of osteoporosis, we surprisingly discovered that D-Asp8 would favorably recognize resorption sites in skeletal tissues, while another bone-targeting moiety, alendronate (ALN), directs the delivery system to both formation and resorption sites. Atomic force microscopy (AFM) analyses reveal that ALN has a stronger binding force to hydroxyapatite (HA) than D-Asp8. In vitro HA binding studies indicate that D-Asp8 is more sensitive to change of HA crystallinity than ALN. Because the bone apatite in the newly formed bone (formation sites) usually has lower crystallinity than the resorption sites (mainly mature bone), we believe that the favorable recognition of D-Asp8 to the bone resorption sites could be attributed to its relatively weak binding to apatite, when compared to bisphosphonates, and the different levels of crystallinity of bone apatite at different functional domains of the skeleton.     

Unequal distribution of genes and chromosomes refers to nuclear diversification in the binucleated Giardia intestinalis

The single-celled parasite Giardia intestinalis (Diplomonadida) has two equally sized nuclei in one cell. The nuclei have been considered identical. We have previously shown that they contain different chromosomal sets and proceed through the cell cycle with some asynchrony. Here, we demonstrate by fluorescence in situ hybridization that several genes from chromosome 5 are lost in one of the two nuclei of the WBc6 Giardia line. The missing segment stretches over at least 50 kb near the 5′ chromosome end. In both WB and WBc6 Giardia cell lines, chromosome 5 is trisomic in one nucleus and monosomic in the other nucleus. The described chromosomal deletion has always been observed at the monosomic chromosome in WBc6; however, the deletion was not detected in the parent line WB. The chromosomal segment was thus initially lost after biological cloning of WB, which gave rise to clone WBc6. We show that Giardia is capable of carrying out gene expression from only one nucleus. The two nuclei display a certain level of diversity, making each of them irreplaceable. The doubled karyomastigonts of diplomonads likely have separate functions both in the mastigont/flagellar organization and in chromosomal and gene content. To our knowledge, our results offer the first methodical approach to differentiating the two, so far indistinguishable nuclei.     

Hidden diversity and evolutionary trends in malacosporean parasites (Cnidaria: Myxozoa) identified using molecular phylogenetics

Malacosporeans represent a small fraction of myxozoan biodiversity with only two genera and three species described. They cycle between bryozoans and freshwater fish. In this study, we (i) microscopically examine and screen different freshwater/marine fish species from various geographic locations and habitats for the presence of malacosporeans using PCR; (ii) study the morphology, prevalence, host species/habitat preference and distribution of malacosporeans; (iii) perform small subunit/large subunit rDNA and Elongation factor 2 based phylogenetic analyses of newly gathered data, together with all available malacosporean data in GenBank; and (iv) investigate the evolutionary trends of malacosporeans by mapping the morphology of bryozoan-related stages, host species, habitat and geographic data on the small subunit rDNA-based phylogenetic tree. We reveal a high prevalence and diversity of malacosporeans in several fish hosts in European freshwater habitats by adding five new species of Buddenbrockia and Tetracapsuloides from cyprinid and perciform fishes. Comprehensive phylogenetic analyses revealed that, apart from Buddenbrockia and Tetracapsuloides clades, a novel malacosporean lineage (likely a new genus) exists. The fish host species spectrum was extended for Buddenbrockia plumatellae and Buddenbrockia sp. 2. Co-infections of up to three malacosporean species were found in individual fish. The significant increase in malacosporean species richness revealed in the present study points to a hidden biodiversity in this parasite group. This is most probably due to the cryptic nature of malacosporean sporogonic and presporogonic stages and mostly asymptomatic infections in the fish hosts. The potential existence of malacosporean life cycles in the marine environment as well as the evolution of worm- and sac-like morphology is discussed. This study improves the understanding of the biodiversity, prevalence, distribution, habitat and host preference of malacosporeans and unveils their evolutionary trends.     

Nickel tolerance of serpentine and non-serpentine Knautia arvensis plants as affected by arbuscular mycorrhizal symbiosis

Serpentine soils have naturally elevated concentrations of certain heavy metals, including nickel. This study addressed the role of plant origin (serpentine vs. non-serpentine) and symbiosis with arbuscular mycorrhizal fungi (AMF) in plant Ni tolerance. A semi-hydroponic experiment involving three levels of Ni and serpentine and non-serpentine AMF isolates and populations of a model plant species (Knautia arvensis) revealed considerable negative effects of elevated Ni availability on both plant and fungal performance. Plant growth response to Ni was independent of edaphic origin; however, higher Ni tolerance of serpentine plants was indicated by a smaller decline in the concentrations of photosynthetic pigments and restricted root-to-shoot Ni translocation. Serpentine plants also retained relatively more Mg in their roots, resulting in a higher shoot Ca/Mg ratio. AMF inoculation, especially with the non-serpentine isolate, further aggravated Ni toxicity to host plants. Therefore, AMF do not appear to be involved in Ni tolerance of serpentine K. arvensis plants.     

Real-Time Analysis of Imatinib- and Dasatinib-Induced Effects on Chronic Myelogenous Leukemia Cell Interaction with Fibronectin

Attachment of stem leukemic cells to the bone marrow extracellular matrix increases their resistance to chemotherapy and contributes to the disease persistence. In chronic myelogenous leukemia (CML), the activity of the fusion BCR-ABL kinase affects adhesion signaling. Using real-time monitoring of microimpedance, we studied in detail the kinetics of interaction of human CML cells (JURL-MK1, MOLM-7) and of control BCR-ABL-negative leukemia cells (HEL, JURKAT) with fibronectin-coated surface. The effect of two clinically used kinase inhibitors, imatinib (a relatively specific c-ABL inhibitor) and dasatinib (dual ABL/SRC family kinase inhibitor), on cell binding to fibronectin is described. Both imatinib and low-dose (several nM) dasatinib reinforced CML cell interaction with fibronectin while no significant change was induced in BCR-ABL-negative cells. On the other hand, clinically relevant doses of dasatinib (100 nM) had almost no effect in CML cells. The efficiency of the inhibitors in blocking the activity of BCR-ABL and SRC-family kinases was assessed from the extent of phosphorylation at autophosphorylation sites. In both CML cell lines, SRC kinases were found to be transactivated by BCR-ABL. In the intracellular context, EC50 for BCR-ABL inhibition was in subnanomolar range for dasatinib and in submicromolar one for imatinib. EC50 for direct inhibition of LYN kinase was found to be about 20 nM for dasatinib and more than 10 µM for imatinib. Cells pretreated with 100 nM dasatinib were still able to bind to fibronectin and SRC kinases are thus not necessary for the formation of cell-matrix contacts. However, a minimal activity of SRC kinases might be required to mediate the increase in cell adhesivity induced by BCR-ABL inhibition. Indeed, active (autophosphorylated) LYN was found to localize in cell adhesive structures which were visualized using interference reflection microscopy.