Expression of cyclin B1 after induction of senescence and cell death in non-small cell lung carcinoma A549 cells

The purpose of this study was to evaluate the level of mitotic cyclin B1 in the context of senescence and cell death in A549 non-small cell lung carcinoma cells. This was performed through analysis of the cell cycle, the percentage of SA-β-galactosidase-positive, as well as TUNEL-positive cells. Morphological alterations were studied using a transmission electron microscope. Changes in the intracellular level and the presence of cyclin B1 in the nucleus and cytoplasm areas were detected by flow cytometry and confocal fluorescence microscopy, respectively. In the cells exposed to various concentrations of doxorubicin, different kinds of cell death and senescent phenotype were observed. Alterations in the cell cycle and increased polyploidy may be indicative of mitotic catastrophe execution. Changes in cyclin B1 may also be strictly related to its different regulation at mitotic catastrophe and senescence programs.     

A new quill mite species (Acari: Syringophilidae) parasitising tinamous (Aves: Tinamiformes)

Tinamiphilopsis ariconte n. sp., a syringophilid mite, is described from three tinamou species (Tinamidae), Rhynchotus rufescens (Temminck) and Nothura boraquira (Spix) in Paraguay and N. minor (Spix) in Brazil. These data indirectly support the hypothesis that the cheyletoid-like predatory ancestor of the Syringophilidae switched to parasitism before the divergence of the avian hosts into the two major clades Palaeognathae and Neognathae.     

Growth hormone (GH) peak after falling asleep reflects spontaneous nocturnal GH secretion, however is not corresponding to the results of GH stimulating tests in children with short stature

OBJECTIVE: Growth hormone (GH) secretion is characterized by a pulsatile, circadian rhythm, with the highest concentrations at night hours. Evaluation of nocturnal GH secretion may be truncated to 6 hours. Growth hormone stimulating tests are the standard method of assessment of GH secretion. In Poland, the assessment of GH peak during 2 hours after falling asleep was introduced as a screening procedure in children, suspected for GH deficiency. The aim of current study was to compare the results of a screening test with GH secretion during 6-hour nocturnal profile and with the results of GH stimulating tests, as well as with IGF-I secretion in children with short stature. Methods: In 72 short children, GH concentrations were measured every 30 minutes during first 6 hours after falling asleep and in two GH stimulating tests (the cut-off level of GH peak for all the tests was 10.0 ng/ml). Also, IGF-I concentrations were measured and expressed as IGF-I SDS for age and sex. Results: The screening test results correlated significantly with both GH peak in 6-hour profile and mean GH concentration, and the area under the curve (AUC) in 6 hour profile (r= 0.94, r=0.90 and r=0.89, respectively, p<0.05) but not with GH peak in stimulating tests (r=0.07, NS). There was no correlation between IGF-I secretion and any of the analyzed parameters of spontaneous and stimulated GH secretion. Conclusions: The results of screening test seem to reflect overnight GH secretion in short children, remaining, however, discordant with the results of GH stimulating tests and with IGF-I secretion.     

A novel cross-talk between endothelin and ErbB receptors controlling glutamate transporter expression in astrocytes

The endothelin and epidermal growth factor (EGF) systems are central to the control of reactive brain processes and are thought to partly exert these tasks by endothelin-induced transactivation of the epidermal growth factor receptor (EGFR) Here we show that beyond EGFR transactivation, endothelins prevent the ligand-induced internalization of the EGFR. We unravel that endothelins abrogate internalization of the EGFR by either promoting the formation of "internalization-deficient" EGFR/ErB2-heterodimers or by activating c-Abl kinase, a negative regulator of EGFR internalization. We further provide evidence that this cross-talk is operational in the control of astrocytic glutamate transport. Specifically, we establish that the inhibitory effects exerted by endothelins on basal as well as EGF-induced expression of the major astroglial glutamate transporter subtype, glutamate transporter 1, are a direct consequence of the endothelin-dependent retention of the EGFR at the cell surface. Together our findings unravel a previously unknown cross-talk between endothelin and epidermal growth factor receptors, which may have implications for a variety of pathological conditions.     

On measuring the response of mesophyll conductance to carbon dioxide with the variable J method

The response of mesophyll conductance to CO(2) (g(m)) to environmental variation is a challenging parameter to measure with current methods. The 'variable J' technique, used in the majority of studies of g(m), assumes a one-to-one relationship between photosystem II (PSII) fluorescence and photosynthesis under non-photorespiratory conditions. When calibrating this relationship for Populus trichocarpa, it was found that calibration relationships produced using variation in light and CO(2) were not equivalent, and in all cases the relationships were non-linear-something not accounted for in previous studies. Detailed analyses were performed of whether different calibration procedures affect the observed g(m) response to CO(2). Past linear and assumed calibration methods resulted in systematic biases in the fluorescence estimates of electron transport. A sensitivity analysis on modelled data (where g(m) was held constant) demonstrated that biases in the estimation of electron transport as small as 2% (～0.5 μmol m(-2) s(-1)) resulted in apparent changes in the relationship of g(m) to CO(2) of similar shape and magnitude to those observed with past calibration techniques. This sensitivity to biases introduced during calibrations leads to results where g(m) artefactually decreases with CO(2), assuming that g(m) is constant; if g(m) responds to CO(2), then biases associated with past calibration methods would lead to overestimates of the slope of the relationship. Non-linear calibrations were evaluated; these removed the bias present in past calibrations, but the method remained sensitive to measurement errors. Thus measurement errors, calibration non-linearities leading to bias, and the sensitivity of variable J g(m) hinders its use under conditions of varying CO(2) or light.     

Enhanced immunoreactivity of TIMP-2 in the stromal compartment of tumor as a marker of favorable prognosis in ovarian cancer patients

Degradation of the extracellular matrix and basement membrane is a critical step in tumor progression. Matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP 2) act in a coordinated manner to form an integrated system involved in ovarian cancer (OC) progression. In this study, the authors describe the expression of TIMP-2 detected by immunohistochemistry in 6 OC cell lines and in 43 malignant epithelial ovarian tumors (in tumor and stromal compartments) in sections originating from primary laparotomies. No significant correlations between overall and progression-free survival and TIMP-2 expression in tumor compartment were observed. The analysis demonstrated a significant association between enhanced stromal expression of TIMP-2 and better clinical response to cisplatin- and paclitaxel-based chemotherapy. Increased expression of TIMP-2 in the stromal compartment and simultaneous overexpression in both stromal and tumor compartments strongly correlated with increased survival. No significant correlations were found in vitro between resistance to cisplatin, paclitaxel, or topotecan and the expression of TIMP-2 in the OC cell lines, suggesting stromal influences on tumor chemoresistance in the physiological environment. This study supports the concept of TIMP-2 expression in the stromal compartment of OC as a promising marker of prognosis and response to cisplatin- and paclitaxel-based chemotherapy in OC patients.     

A novel mutation (Cys308Phe) of the LDL receptor gene in families from the South-Eastern part of Poland

The purpose of this investigation was to characterize a new mutation in the LDL-receptor (LDLR) gene in three families with clinically diagnosed familial hypercholesterolemia (FH) from the South-Eastern part of Poland. Mutational screening with exon by exon sequencing analysis was performed in all probands. The novel mutation c986G>T (Cys308Phe) in the exon 7 of LDLR gene was found in three apparently unrelated probands with FH. Analysis of the receptor activity of peripheral blood lymphocytes by binding and uptake of DiL-LDL showed a significant reduction (by 24% versus healthy control) of the fluorescent label in the lymphocytes of patients heterozygous for this mutation. Concentrations of serum LDL-C in probands before treatment were between 9.5 and 10.5 mmol/l. All patients had corneal arcus and tendon xanthoma. Clinically, families were characterized by premature coronary artery disease. This mutation occurred relatively frequently in our group of patients with FH, but this could be explained by a founder effect since we demonstrated their common ancestors.     

A practical synthesis of N α -Fmoc protected l-threo-β-hydroxyaspartic acid derivatives for coupling via α- or β-carboxylic group

Abstract  

A simple and practical general synthetic protocol towards orthogonally protected tHyAsp derivatives fully compatible with Fmoc solid-phase peptide synthetic methodology is reported. Our approach includes enantioresolution of commercially available d,l-tHyAsp racemic mixture by co-crystallization with l-Lys, followed by ion exchange chromatography yielding enantiomerically pure l-tHyAsp and d-tHyAsp, and their selective orthogonal protection. In this way N ^α -Fmoc protected tHyAsp derivatives were prepared ready for couplings via either α- or β-carboxylic group onto the resins or the growing peptide chain. In addition, coupling of tHyAsp via β-carboxylic group onto amino resins allows preparation of peptides containing tHyAsn sequences, further increasing the synthetic utility of prepared tHyAsp derivatives.     

New functional ligands for ficolin-3 among lipopolysaccharides of Hafnia alvei

Ficolin-1 (M), ficolin-2 (L), ficolin-3 (H) and mannan-binding lectin (MBL) activate the complement system and have opsonic activity. The specificity of ficolin-3 is poorly characterized and currently limited to a few ligands only. We present new specific targets for human ficolin-3, identified among lipopolysaccharides (LPSs, endotoxin) of Hafnia alvei. The interaction was restricted to LPSs of four strains: 23, Polish Collection of Microorganisms (PCM) 1200, PCM 1203 and PCM 1205 and limited to their O-specific polysaccharides (O-specific PSs) composed of different numbers of oligosaccharide (OS) repeating units (RUs). Moreover, these LPS/ficolin-3 complexes activated the lectin pathway of complement in a C4b-deposition assay in a calcium- and magnesium-dependent way. A neoglycoconjugate of the O-specific PS fraction of H. alvei 1200 LPS with bovine serum albumin (BSA) was prepared and used as a tool for the determination of ficolin-3 concentration and activity in serum. To confirm a structure of the O-specific PS 1200 selected for the conjugate preparation, structural analysis was performed on a series of O-specific PSs released by the mild acid hydrolysis of the LPS. The isolated O-specific PSs, showing the different length distributions, were devoid of a major part of the core OS region and had Hep-Kdo disaccharide at a reducing end. The neoglycoconjugate was a highly selective tool for the determination of ficolin-3 concentration and activity in serum (lectin pathway activation in the C4b deposition assay) and was not affected by MBL, ficolin-1 and ficolin-2 or natural antibodies.     

Thermo-sensitive transient receptor potential vanilloid channel-1 regulates intracellular calcium and triggers chromogranin A secretion in pancreatic neuroendocrine BON-1 tumor cells

Transient receptor potential channels (TRPs) regulate tumor growth via calcium-dependent mechanisms. The (thermosensitive) capsaicin receptor TRPV1 is overexpressed in numerous highly aggressive cancers. TRPV1 has potent antiproliferative activity and is therefore potentially applicable in targeted therapy of malignancies. Recently, we characterized TRPM8 functions in pancreatic neuroendocrine tumors (NETs), however, the role of TRPV1 is unknown. Here, we studied the expression and the role of TRPV1 in regulating intracellular Ca²⁺ and chromogranin A (CgA) secretion in pancreatic NET BON-1 cell line and in primary NET cells (prNET). TRPV1 expression was detected by RT-PCR, Western blot and immunofluorescence. Intracellular free Ca²⁺ ([Ca²⁺]_i) was measured by fura-2; TRPV1 channel currents by the planar patch-clamp technique. Nonselective cation currents were analyzed by a color-coded plot method and CgA secretion by ELISA. Pancreatic BON-1 cells and NETs express TRPV1. Pharmacological blockade of TRPs by La³⁺ (100 μM) or by ruthenium-red (RuR) or by capsazepine (CPZ) (both at 10 μM) suppressed the capsaicin (CAP)- or heat-stimulated increase of [Ca²⁺]_i in NET cells. CAP (20 μM) also increased nonselective cation channel currents in BON-1 cells. Furthermore, CAP (10 μM) stimulated CgA secretion, which was inhibited by CPZ or by RuR (both 10 μM). La³⁺ potently reduced both stimulated and the basal CgA secretion. Our study shows for the first time that TRPV1 is expressed in pancreatic NETs. Activation of TRPV1 translates into changes of intracellular Ca²⁺, a known mechanism triggering the secretion of CgA. The clinical relevance of TRPV1 activation in NETs requires further investigations.