Serological profiling with Chlamycheck, a commercial multiplex recombinant antigen Western blot assay of chlamydial infections

A new chlamydial test system, the Chlamycheck assay, which uses 4 purified recombinant antigens of Chlamydia trachomatis and Chlamydophila pneumoniae and one antigen of Chlamydophila psittaci, has been developed and commercialized. We investigated the reactivities of the recombinant antigens with sera from a group of 30 patients with acute Chlamydia trachomatis infection, 88 patients consulting for sexually transmitted infections, and 46 patients with serological evidence of Chlamydophila pneumoniae infection. The results obtained from human and infected mouse sera suggest that Chlamycheck serology against multiple proteins may provide additional useful information that is not available by conventional whole elementary body microimmunofluorescence or single-antigen enzyme-linked immunosorbent assay serology. Specific serological profiles were associated with acute versus past Chlamydia trachomatis infection or with Chlamydia trachomatis primo-infection versus infection in a Chlamydophila pneumoniae history context.     

Growth and inorganic composition of ‘Nova’ mandarin plants grafted on two commercial rootstocks in response to salinity and silicon

The effects of salinity and its combination with silicon (Si) were studied in ‘Nova’ mandarin plants grafted on Citrus aurantium L. or Swingle Citrumelo to determine: (1) which combination is more tolerant to salt stress and (2) the impact of Si in limiting the harmful effects of salinity. Six groups of plants were grown in a greenhouse for 120 days and irrigated with: (1) 50 % Hoagland’s solution (Control), (2) 50 % Hoagland’s solution plus 80 mM NaCl (NaCl), and (3) 50 % Hoagland’s solution plus 80 mM NaCl plus 0.5 mM Si (NaCl + Si). Grafted plants exhibited accumulation of Na and Cl in their tissues following exposure to salinity. The ability of S. Citrumelo to retain the toxic ions in the roots in corroboration with the observation that the dry weights (DWs) of S. Citrumelo tissues were not influenced by NaCl treatment indicates that this rootstock is more tolerant to salinity. Silicon supplementation into the saline medium promoted the accumulation of toxic ions, whereas, when compared to NaCl treatment, it increased the DW of S. Citrumelo roots. Mineral concentrations were significantly affected by rootstock, treatment, and their interaction with S. Citrumelo, which presented better nutrient status than Sour Orange; and Si which differed depending on citrus tissue. It appears that S. Citrumelo rootstock is the most tolerant for ‘Nova’ mandarin plants under salinity, whereas salt tolerance in grafted citrus plants is not improved by Si application, indicating that the beneficial role of Si depends on the cultivar or rootstock–scion combinations.     

The frequency of NPM1 mutations in childhood acute myeloid leukemia

Leser-Trélat syndrome is characterized by the eruptive appearance of multiple seborrheic keratoses in association with underlying malignant disease. Usually, the sign of Leser-Trélat is associated with adenocarcinoma, most frequently of the colon, breast, or stomach, but also of the lung, kidney, liver, and pancreas. Herein, we present a case that we believe is the first report of the sign of Leser-Trélat in association with occult gastric adenocarcinoma and the anamnestic oncologic history of five other multiple primitive cancers. Epidermal growth factor receptor (EGFR) immunohistochemical expression analysis of multiple seborrheic keratoses revealed an intense membranous staining in the basal keratinocytes and in all the upper epidermal layers. Patients with the sign of Leser-Trélat should undergo a diagnostic screening programme for malignant disease along with patients with known Leser-Trélat syndrome who present with a recent acute and florid eruption of their seborrheic keratoses. We propose the importance of combining the molecular features of multiple seborrheic keratoses with EGFR immunohistochemistry analyses to determine the likelihood of Leser-Trélat syndrome and the consequent high risk of underlying multiple visceral malignancies.     

The stability of the archaeal HU histone-like DNA-binding protein from Thermoplasma volcanium

The complete genome analysis of the archaeon Thermoplasma volcanium has revealed a gene assigned to encode the histone-like DNA-binding protein HU. Thermoplasma volcanium is a moderate thermophile growing around 60°C and it is adaptable to aerobic and anaerobic environment and therefore it is unique as a candidate for the origin of eukaryotic nuclei in the endosymbiosis hypothesis. The HU protein is the major component of the bacterial nuclei and therefore it is an important protein to be studied. The gene for HUTvo protein (huptvo) was cloned from the genomic DNA of T. volcanium and overexpressed in Escherichia coli. A fast and efficient purification scheme was established to produce an adequate amount of bioactive protein for biochemical and biophysical studies. Highly purified HUTvo was studied for its DNA-binding activity and thermostability. As studied by circular dichroism and high-precision differential scanning microcalorimetry, the thermal unfolding of HUTvo protein is reversible and can be well described by a two-state model with dissociation of the native dimeric state into denatured monomers. The ∆G versus T profile for HUTvo compared to the hyperthermophilic marine eubacterial counterpart from Thermotoga maritima, HUTmar, clearly shows that the archaeal protein has adopted a less efficient molecular mechanism to cope with high temperature. The molecular basis of this phenomenon is discussed. F. Orfaniotou and P. Tzamalis contributed equally to this work.     

ICPBC and C12-ICPBC: two new red emitting, fluorescent Ca2+ indicators excited with visible light

Two new, visible-excited and red-emitting fluorescent Ca(2+) indicators were synthesized and the spectral profiles of their free and Ca(2+) bound forms were studied. The fluorescent properties of these probes are due to the extended conjugation of the chromeno[3',2':3,4]pyrido[1,2a][1,3]benzimidazole chromophore incorporated in their BAPTA-type, Ca(2+) chelating structure. The compounds, namely ICPBC and its N-dodecyl analog C12-ICPBC exhibit Ca(2+) dissociation constants of 7.7 and 18.0 microM, respectively. The fluorescence spectra of the probes showed a clear shift in excitation wavelength maxima upon Ca(2+) binding along with a large Stokes shift and changes in fluorescence intensity, indicating their potential use as Ca(2+) indicators. The ability of ICPBC to trace high calcium spikes was tested in the human HepG2 cell line with positive results.     

New phenotypic aspects of the decidual spiral artery wall during early post-implantation mouse pregnancy

During pregnancy the walls of decidual spiral arteries (SAs) undergo clinically important structural modifications crucial for embryo survival/growth and maternal health. However, the mechanisms of SA remodeling (SAR) are poorly understood. Although an important prerequisite to this understanding is knowledge about the phenotype of SA muscular wall prior to and during the beginning of mouse SAR, this remains largely unexplored and was the main aim of this work. Using histological and immunohistochemical techniques, this study shows for the first time that during early mouse gestation, from embryonic day 7.5 (E7.5) to E10.5, the decidual SA muscular coat is not a homogeneous structure, but consists of two concentric layers. The first is a largely one cell-thick sub-endothelial layer of contractile mural cells (positive for α-smooth muscle actin, calponin and SM22α) with pericyte characteristics (NG2 positive). The second layer is thicker, and evidence is presented that it may be of the synthetic/proliferative smooth muscle phenotype, based on absence (α-smooth muscle actin and calponin) or weak (SM22α) expression of contractile mural cell markers, and presence of synthetic smooth muscle characteristics (expression of non-muscle Myosin heavy chain-IIA and of the cell proliferation marker PCNA). Importantly, immunohistochemistry and morphometrics showed that the contractile mural cell layer although prominent at E7.5-E8.5, becomes drastically reduced by E10.5 and is undetectable by E12.5. In conclusion, this study reveals novel aspects of the decidual SA muscular coat phenotype prior to and during early SAR that may have important implications for understanding the mechanisms of SAR.     

Ribosomes containing mutants of L4 ribosomal protein from Thermus thermophilus display multiple defects in ribosomal functions and sensitivity against erythromycin

Protein L4 from Thermus thermophilus (TthL4) was heterologously overproduced in Escherichia coli cells. To study the implication of the extended loop of TthL4 in the exit-tunnel and peptidyltransferase functions, the highly conserved E56 was replaced by D or Q, while the semiconserved G55 was changed to E or S. Moreover, the sequence -G55E56- was inverted to -E55G56-. When we incorporated these mutants into E. coli ribosomes and investigated their impact on poly(Phe) synthesis, high variations in the synthetic activity and response to erythromycin of the resulting ribosomes were observed. In the absence of erythromycin, ribosomes harboring mutations G55E and E56D in TthL4 protein were characterized by low activity in synthesizing poly(Phe) and decreased capability in binding tRNA at the A site. On the other hand, ribosomes possessing mutations G55E, G55S, G55E-E56G, or E56Q in TthL4 protein were unexpectedly more sensitive to erythromycin. Evidence in support of these findings was drawn by in vivo experiments, assessing the erythromycin sensitivity of E. coli cells expressing wild-type or mutant TthL4 proteins. Our results emphasize the role of the extended loop of L4 ribosomal protein in the exit-tunnel and peptidyltransferase center functions.     

Beta-catenin inhibits T cell activation by selective interference with linker for activation of T cells-phospholipase C-γ1 phosphorylation

Despite the defined function of the β-catenin pathway in thymocytes, its functional role in peripheral T cells is poorly understood. We report that in a mouse model, β-catenin protein is constitutively degraded in peripheral T cells. Introduction of stabilized β-catenin into primary T cells inhibited proliferation and cytokine secretion after TCR stimulation and blunted effector cell differentiation. Functional and biochemical studies revealed that β-catenin selectively inhibited linker for activation of T cells phosphorylation on tyrosine 136, which was associated with defective phospholipase C-γ1 phosphorylation and calcium signaling but normal ERK activation. Our findings indicate that β-catenin negatively regulates T cell activation by a previously undescribed mechanism and suggest that conditions under which β-catenin might be inducibly stabilized in vivo would be inhibitory for T cell-based immunity.