Alterations of Red Cell Membrane Properties in Nneuroacanthocytosis

Neuroacanthocytosis (NA) refers to a group of heterogenous, rare genetic disorders, namely chorea acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington’s disease-like 2 (HDL2) and pantothenate kinase associated neurodegeneration (PKAN), that mainly affect the basal ganglia and are associated with similar neurological symptoms. PKAN is also assigned to a group of rare neurodegenerative diseases, known as NBIA (neurodegeneration with brain iron accumulation), associated with iron accumulation in the basal ganglia and progressive movement disorder. Acanthocytosis, the occurrence of misshaped erythrocytes with thorny protrusions, is frequently observed in ChAc and MLS patients but less prevalent in PKAN (about 10%) and HDL2 patients. The pathological factors that lead to the formation of the acanthocytic red blood cell shape are currently unknown. The aim of this study was to determine whether NA/NBIA acanthocytes differ in their functionality from normal erythrocytes. Several flow-cytometry-based assays were applied to test the physiological responses of the plasma membrane, namely drug-induced endocytosis, phosphatidylserine exposure and calcium uptake upon treatment with lysophosphatidic acid. ChAc red cell samples clearly showed a reduced response in drug-induced endovesiculation, lysophosphatidic acid-induced phosphatidylserine exposure, and calcium uptake. Impaired responses were also observed in acanthocyte-positive NBIA (PKAN) red cells but not in patient cells without shape abnormalities. These data suggest an “acanthocytic state” of the red cell where alterations in functional and interdependent membrane properties arise together with an acanthocytic cell shape. Further elucidation of the aberrant molecular mechanisms that cause this acanthocytic state may possibly help to evaluate the pathological pathways leading to neurodegeneration.     

Detection of Serotype-Specific Antibodies to the Four Dengue Viruses Using an Immune Complex Binding (ICB) ELISA

Background

Dengue virus (DENV) infections are preferentially diagnosed by detection of specific IgM antibodies, DENV NS1 antigen assays or by amplification of viral RNA in serum samples of the patients. The type-specific immunity to the four worldwide circulating DENV serotypes can be determined by neutralization assays. An alternative to the complicated neutralization assays would be helpful to study the serotype-specific immune response in people in DENV hyperendemic areas but also in subjects upon DENV vaccination.

Methods

In consecutive samples of patients with DENV-1- 4 infection type-specific antibodies were detected using an immune complex binding (ICB) ELISA. During incubation of serum samples and enzyme- labeled recombinant envelope domain III (EDIII) antigens immune complexes (ICs) are formed, which are simultaneously bound to a solid phase coated with an Fc–receptor (CD32). After a single washing procedure the bound labeled ICs can be determined. To further improve type-specific reactions high concentrations of competing heterologous unlabeled ED III proteins were added to the labeled antigens.

Results

Follow-up serum samples of 64 patients with RT-PCR confirmed primary DENV-1, -2, -3 or -4 infections were tested against four enzyme-labeled recombinant DENV EDIII antigens. Antibodies to the EDIII antigens were found in 55 patients (sensitivity 86%). A complete agreement between the serotype detected by PCR in early samples and the serotype-specific antibody in later samples was found. Type-specific anti-EDIII antibodies were first detected 9–20 days after onset of the disease. In 21% of the samples collected from people in Vietnam secondary infections with antibodies to two serotypes could be identified.

Conclusions

The data obtained with the ICB-ELISA show that after primary DENV infection the corresponding type-specific antibodies are detected in almost all samples collected at least two weeks after onset of the disease. The method will be of value to determine the distribution of the various type-specific anti–DENV antibodies in DENV endemic areas.     

Constitutive activity of Erk1/2 and NF-κB protects human endometrial stromal cells from death receptor-mediated apoptosis

Apoptosis in the human endometrium plays an essential role for endometrial receptivity and early implantation. A dysbalance of pro- and anti-apoptotic events in the secretory endometrium seems to be involved in implantation disorders and consecutive pregnancy complications. However, little is known about the mechanisms regulating apoptosis-sensitivity in the human endometrium. Therefore this study was performed to identify molecular mechanisms underlying the resistance toward apoptosis in human endometrial stromal cells (ESCs). Human ESCs were isolated from hysterectomy specimens and used as undifferentiated cells or after decidualization in vitro. Cells were incubated with an activating anti-Fas antibody, tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), TNF-α and inhibitors of protein- and RNA-syntheses, a caspase-inhibitor and inhibitors of extracellular signal regulated kinase (Erk)1/2, nuclear factor (NF)-κB and Akt. Apoptosis was measured by flow cytometric detection of hypodiploid nuclei. Caspase-activity was detected by luminescencent assays. Several pro- and anti-apoptotic molecules and the activation of Erk1/2, NF-κB and Akt were analyzed by in-cell Western assays or flow cytometry. Inhibition of protein- and RNA-syntheses differentially sensitized human ESCs for death receptor-mediated apoptosis in a caspase-dependent manner, based on the up-regulation of the death receptors Fas and TRAIL-R2. The constitutive activity of Erk1/2 and NF-κB could be identified as a reason for the apoptosis-resistance of human ESCs. These results suggest the pro-survival signaling pathways Erk1/2 and NF-κB as key regulators of the sensitivity of human ESCs for death receptor-mediated apoptosis. The modulation of these pathways might play an important role in the physiology of implantation.     

Interactions between peptidyl tRNA hydrolase homologs and the ribosomal release factor Mrf1 in S. pombe mitochondria

Mitochondrial translation synthesizes key subunits of the respiratory complexes. In Schizosaccharomyces pombe, strains lacking Mrf1, the mitochondrial stop codon recognition factor, are viable, suggesting that other factors can play a role in translation termination. S. pombe contains four predicted peptidyl tRNA hydrolases, two of which (Pth3 and Pth4), have a GGQ motif that is conserved in class I release factors. We show that high dosage of Pth4 can compensate for the absence of Mrf1 and loss of Pth4 exacerbates the lack of Mrf1. Also Pth4 is a component of the mitochondrial ribosome, suggesting that it could help recycling stalled ribosomes.     

Pharmacological Induction of Transforming Growth Factor-Beta1 in Rat Models Enhances Radiation Injury in the Intestine and the Heart

Radiation therapy in the treatment of cancer is dose limited by radiation injury in normal tissues such as the intestine and the heart. To identify the mechanistic involvement of transforming growth factor-beta 1 (TGF-β1) in intestinal and cardiac radiation injury, we studied the influence of pharmacological induction of TGF-β1 with xaliproden (SR 57746A) in rat models of radiation enteropathy and radiation-induced heart disease (RIHD). Because it was uncertain to what extent TGF-β induction may enhance radiation injury in heart and intestine, animals were exposed to irradiation schedules that cause mild to moderate (acute) radiation injury. In the radiation enteropathy model, male Sprague-Dawley rats received local irradiation of a 4-cm loop of rat ileum with 7 once-daily fractions of 5.6 Gy, and intestinal injury was assessed at 2 weeks and 12 weeks after irradiation. In the RIHD model, male Sprague-Dawley rats received local heart irradiation with a single dose of 18 Gy and were followed for 6 months after irradiation. Rats were treated orally with xaliproden starting 3 days before irradiation until the end of the experiments. Treatment with xaliproden increased circulating TGF-β1 levels by 300% and significantly induced expression of TGF-β1 and TGF-β1 target genes in the irradiated intestine and heart. Various radiation-induced structural changes in the intestine at 2 and 12 weeks were significantly enhanced with TGF-β1 induction. Similarly, in the RIHD model induction of TGF-β1 augmented radiation-induced changes in cardiac function and myocardial fibrosis. These results lend further support for the direct involvement of TGF-β1 in biological mechanisms of radiation-induced adverse remodeling in the intestine and the heart.     

K+ Efflux and Retention in Response to NaCl Stress Do Not Predict Salt Tolerance in Contrasting Genotypes of Rice (Oryza sativa L.)

Sudden elevations in external sodium chloride (NaCl) accelerate potassium (K⁺) efflux across the plasma membrane of plant root cells. It has been proposed that the extent of this acceleration can predict salt tolerance among contrasting cultivars. However, this proposal has not been considered in the context of plant nutritional history, nor has it been explored in rice (Oryza sativa L.), which stands among the world’s most important and salt-sensitive crop species. Using efflux analysis with ⁴²K, coupled with growth and tissue K⁺ analyses, we examined the short- and long-term effects of NaCl exposure to plant performance within a nutritional matrix that significantly altered tissue-K⁺ set points in three rice cultivars that differ in salt tolerance: IR29 (sensitive), IR72 (moderate), and Pokkali (tolerant). We show that total short-term K⁺ release from roots in response to NaCl stress is small (no more than 26% over 45 min) in rice. Despite strong varietal differences, the extent of efflux is shown to be a poor predictor of plant performance on long-term NaCl stress. In fact, no measure of K⁺ status was found to correlate with plant performance among cultivars either in the presence or absence of NaCl stress. By contrast, shoot Na⁺ accumulation showed the strongest correlation (a negative one) with biomass, under long-term salinity. Pharmacological evidence suggests that NaCl-induced K⁺ efflux is a result of membrane disintegrity, possibly as result of osmotic shock, and not due to ion-channel mediation. Taken together, we conclude that, in rice, K⁺ status (including efflux) is a poor predictor of salt tolerance and overall plant performance and, instead, shoot Na⁺ accumulation is the key factor in performance decline on NaCl stress.     

Characterization of the protein fraction of the temporary adhesive secreted by the tube feet of the sea star Asterias rubens

Sea stars are able to make firm but temporary attachments to various substrata by secretions released by their tube feet. After tube foot detachment, the adhesive secretions remain on the substratum as a footprint. Proteins presumably play a key role in sea star adhesion, as evidenced by the removal of footprints from surfaces after a treatment with trypsin. However, until now, characterisation was hampered by their high insolubility. In this study, a non-hydrolytic method was used to render most of the proteins constituting the adhesive footprints soluble. After analysis by SDS-PAGE, the proteins separated into about 25 bands, which ranged from 25 to 450 kDa in apparent molecular weight. Using mass spectrometry and a homology-database search, it was shown that several of the proteins are known intracellular proteins, presumably resulting from contamination of footprint material with tube foot epidermal cells. However, 11 protein bands, comprising the most abundant proteins, were not identified and might correspond to novel adhesive proteins. They were named ‘Sea star footprint proteins’ (Sfps). Tandem mass spectrometry analysis of the protein bands yielded 43 de novo-generated peptide sequences. Most of them were shared by several, if not all, Sfps. Polyclonal antibodies were raised against one of the peptides (HEASGEYYR from Sfp-115) and were used in immunoblotting. They specifically labelled Sfp-115 and other bands with lower apparent molecular weights. The different results suggest that all Sfps might belong to a single family of related proteins sharing similar motifs or, alternatively, they are the products of polymerization and/or degradation processes.     

Expression of Betapapillomavirus Oncogenes Increases the Number of Keratinocytes with Stem Cell-Like Properties

Human papillomaviruses (HPV) of genus Betapapillomavirus (betaPV) are associated with nonmelanoma skin cancer development in epidermodysplasia verruciformis (EV) and immunosuppressed patients. Epidemiological and molecular studies suggest a carcinogenic activity of betaPV during early stages of cancer development. Since viral oncoproteins delay and perturb keratinocyte differentiation, they may have the capacity to either retain or confer a “stem cell-like” state on oncogene-expressing cells. The aim of this study was to determine (i) whether betaPV alters the expression of cell surface markers, such as CD44 and epithelial cell adhesion molecule (EpCAM), that have been associated with epithelial stemness, and (ii) whether this confers functional stem cell-like properties to human cutaneous keratinocytes. Fluorescence-activated cell sorter (FACS) analysis revealed an increase in the number of cells with high CD44 and EpCAM expression in keratinocyte cultures expressing HPV type 8 (HPV8) oncogenes E2, E6, and E7. Particularly through E7 expression, a distinct increase in clonogenicity and in the formation and size of tumor spheres was observed, accompanied by reduction of the epithelial differentiation marker Calgranulin B. These stem cell-like properties could be attributed to the pool of CD44^high EpCAM^high cells, which was increased within the E7 cultures of HPV5, -8, and -20. Enhanced EpCAM levels were present in organotypic skin cultures of primary keratinocytes expressing E7 of the oncogenic HPV types HPV5, -8, and -16 and in clinical samples from EV patients. In conclusion, our data show that betaPV may increase the number of stem cell-like cells present during early carcinogenesis and thus enable the persistence and accumulation of DNA damage necessary to generate malignant stem cells.