Genetic disregulation of TNF alpha and TNF alpha type II receptors in colon cancer at the II and III stage of disease

The expression of the genes coding TNFalpha and TNF RII receptors (TNF RII: TNFR2 membrane and soluble domain, TNFR2/R7 soluble domain) was analysed in colon cancer at the II and III stage of disease, by estimation of mRNA expression. The study included 80 patients with histopathologically confirmed adenocarcinoma. The number of TNFalpha mRNA, TNFR2 mRNA and TNFR2/R7 mRNA copies were estimated in tumour and healthy tissue. The highest number of mRNA TNF-alpha copies were investigated in all samples of tissue and independently of the stage of disease. Simultaneously, we noticed the largest number of mRNA copies for TNFalpha and TNF R2/R7 in healthy cells at stage III of the disease. It is possible to draw a hypothetical line separating the anti-cancer activity of TNFalpha and its influence on cancer progression.     

Optimization of 131I ablation in patients with differentiated thyroid carcinoma: comparison of early outcomes of treatment with 100 mCi versus 60 mCi

INTRODUCTION: The aim of this study was to compare the early outcomes between two groups of patients with differentiated thyroid carcinoma (DTC) who received 60 or 100 mCi of (131)I for remnant ablation. MATERIAL AND METHODS: 224 DTC patients with primary tumor > 1 cm of diameter or multifocal were randomised into prospective clinical trial. Patients with extrathyroideal extension of primary tumor and nodal metastases or M1 were not enrolled. 99 patients received 60 mCi, and 125--100 mCi of radioiodine as the first ablative dose. RESULTS: The effectiveness of thyroid ablation was evaluated after one year, during endogenous TSH (thyroid stimulating hormone) stimulation, and after two years during Lthyroxine therapy. Whole body scintigraphy (WBS) was performed under thyroxine withdrawal and thyroglobulin serum level was assessed. Distant micrometastases were detected in 9.8% of patients by post-therapy WBS, 11 patients in group A treated with 60 mCi and 11 in group B treated with 100 mCi. In other patients no symptoms of persistent disease were detected. At one year follow up full remission was diagnosed in 176 patients: 76 in group A and 100 in group B. The remaining ones, 13.3% and 11.2% respectively, received the second course of (131)I for remnant ablation. There were no statistically significant differences in Tg (thyroglobulin) serum level either 12 or 24 months after 131I treatment. CONCLUSIONS: Our evaluation of early efficacy of adjuvant radioiodine treatment in low risk DTC patients shows no differences between two radioiodine activities - 60 and 100 mCi in relation to thyroid ablation. Thus, the activity of 60 mCi is recommended.     

Visualisation of microtubules and actin filaments in fixed BY-2 suspension cells using an optimised whole mount immunolabelling protocol

Excellent visualisation of microtubules and actin filaments was obtained in fixed tobacco BY-2 suspension cells after optimising a protocol for whole mount immunolabelling. The procedure is based on modification of fixation, cell wall digestion, dimethyl sulfoxide (DMSO) treatment, post fixation, and blocking. The most critical aspects of successful preservation and visualization of cytoskeletal elements appeared to be: a two-step fixation with paraformaldehyde and glutaraldehyde before enzymatic cell wall digestion and a post fixation with aldehydes thereafter. The method allows the improved visualization of the organisation of the microtubular and actin filament arrays during the successive stages of cell division and at interphase. Although we present the application of our protocols for cytoskeleton labelling, the excellent results show the potential of using this method for the analysis of various proteins and molecules in plant cells.Electronic Supplementary Material Supplementary material is available for this article at     

Relative importance of cellular uptake and reactive oxygen species for the toxicity of alloxan and dialuric acid to insulin-producing cells

The diabetogenic agent alloxan is selectively accumulated in insulin-producing cells through uptake via the GLUT2 glucose transporter in the plasma membrane. In the presence of intracellular thiols, especially glutathione, alloxan generates "reactive oxygen species" (ROS) in a cyclic reaction between this substance and its reduction product, dialuric acid. The cytotoxic action of alloxan is initiated by free radicals formed in this redox reaction. Autoxidation of dialuric acid generates superoxide radicals (O(2)(*-)) and hydrogen peroxide (H(2)O(2)), and finally hydroxyl radicals ((*)OH). Thus, while superoxide dismutase (SOD) only reduced the toxicity, catalase, in particular in the presence of SOD, provided complete protection of insulin-producing cells against the cytotoxic action of alloxan and dialuric acid due to H(2)O(2) destruction and the prevention of hydroxyl radical ((*)OH) formation, indicating that it is the hydroxyl radical ((*)OH) which is the ROS ultimately responsible for cell death. After selective accumulation in pancreatic beta cells, which are weakly protected against oxidative stress, the cytotoxic glucose analogue alloxan destroys these insulin-producing cells and causes a state of insulin-dependent diabetes mellitus through ROS-mediated toxicity in rodents and in other animal species, which express this glucose transporter isoform in their beta cells.     

Bacteriophage translocation

The occurrence of phages in the human body, especially in the gastrointestinal tract, raises the question of their potential role in the physiology and pathology of this system. Especially important is the issue of whether phages can pass the intestinal wall and migrate to lymph, peripheral blood, and internal organs and, if so, the effects such a phenomenon could have (such passage by bacteria, known as bacterial translocation, has been shown to cause various disturbances in humans, from immune defects to sepsis). Available data from the literature support the assumption that phage translocation can take place and may have some immunomodulatory effects. In addition, phages of the gut may play a protective role by inhibiting local immune reactions to antigens derived from gut flora.     

Surfactin isoforms from Bacillus coagulans

Bacillus coagulans has been found to produce several surfactins that are powerful lipopeptide surfactants. Four main components with molecular weights 1007, 1021 and 1035 Da were separated. Their structures have been confirmed by spectrometric and spectroscopic studies and by acid hydrolysis. The compounds were found to represent two pairs of surfactin isoforms in which beta-hydroxy-iso-C14 or anteiso-C15 fatty acids are linked to the [Leu7] or [Val7] heptapeptide moiety by both an amide group and a lactone bond.     

Immunohistochemical detection of the inducible heat shock protein hsp70: a methodological study.

Stress-inducible Hsp70i and constitutively expressed Hsc70 are highly related heat shock proteins. Aberrant expression levels and intracellular localization of these proteins has been suggested as a potential marker in certain tumors. The aim of our study was to work out a reliable, immunohistochemical detection of the stress-inducible Hsp70i protein and enabling discrimination between Hsp70i and Hsc70 proteins in paraffin-embedded human tissues. We tested the effect of several fixative procedures and antigen retrieval on the effectiveness of the Hsp70i detection in murine cells cultured in vitro and in liver of rats subjected to heat shock. For cells grown in vitro, specific Hsp70i immunoreactivity was obtained with all fixatives used. However, samples fixed in 10% formalin and 4% paraformaldehyde required antigen retrieval. In liver tissue embedded in paraffin, regardless the fixative used, positive Hsp70i staining could be visible only if antigen retrieval was applied. We applied this procedure for detection of Hsp70i in routine sections of breast and lung cancers fixed with 10% formalin and found that the application of thermal antigen retrieval significantly enhanced the SPA810 immunoreactivity and reduced background staining. This procedure enabled also the differential detection of Hsp70i and Hsc70 in routine histopathological preparations.     

Differential induction of connexins 26 and 30 in skin tumors and their adjacent epidermis.

Gap junctions (GJs) have been shown to play a role in tumor progression including a variety of keratinocyte-derived and non-keratinocyte-derived skin tumors. Here we show that the synthesis of the GJ proteins connexin 26 and connexin 30 (Cx26 and Cx30) is induced in keratinocyte-derived epithelial skin tumors whereas there is either no change or a downregulation of Cx43. Cx26, Cx30, and Cx43 are absent in non-epithelial skin tumors. Further, Cx26 and Cx30 are induced in the epidermis adjacent to malignant melanoma but absent in the epidermis adjacent to benign non-epithelial skin lesions (melanocytic nevi and angioma). The keratinocyte-derived skin tumors are very heterogeneous regarding the Cx26/Cx30 pattern in the epidermis at the periphery of the tumors. We did not observe any difference in the localization of the very similar proteins Cx26 and Cx30 but a variation in intensity of immunoreactivity. As the staining patterns of Cx26 and Cx30 antibodies are not identical to those of CK6, a marker for hyperproliferation, and CK17, a marker for trauma, we discuss that the induction of these gap junctional proteins exceeds a reflection of reactive hyperproliferative or traumatized epidermis. We further discuss the putative roles of these gap junctional proteins in tumor progression.     

Binding of the low-density lipoprotein by streptococcal collagen-like protein Scl1 of Streptococcus pyogenes

Several bacterial genera express proteins that contain collagen-like regions, which are associated with variable (V) non-collagenous regions. The streptococcal collagen-like proteins, Scl1 and Scl2, of group A Streptococcus (GAS) are members of this 'prokaryotic collagen' family, and they too contain an amino-terminal non-collagenous V region of unknown function. Here, we use recombinant rScl constructs, derived from several Scl1 and Scl2 variants, and affinity chromatography to identify Scl ligands present in human plasma. First, we show that Scl1, but not Scl2, proteins from different GAS serotypes bind the same ligand identified as apolipoprotein B (ApoB100), which is a major component of the low-density lipoprotein (LDL). Scl1 binding to purified ApoB100 and LDL is specific and concentration-dependent. Furthermore, the non-collagenous V region of the Scl1 protein is responsible for LDL/ApoB100 binding because only those rScls, constructed by domain swapping, which contain the V region from Scl1 proteins, were able to bind to ApoB100 and LDL ligands, and this binding was inhibited by antibodies directed against the Scl1-V region. Electron microscopy images of Scl1-LDL complexes showed that the globular V domain of Scl1 interacted with spherical particles of LDL. Importantly, live M28-type GAS cells absorbed plasma LDL on the cell surface and this binding depended on the surface expression of the Scl1.28, but not Scl2.28, protein. Phylogenetic analysis showed that the non-collagenous globular domains of Scl1 and Scl2 evolved independently to form separate lineages, which differ in amino acid sequence, and these differences may account for the variations in binding patterns of Scl1 and Scl2 proteins. Present studies provide insight into the structure-function relationship of the Scl proteins and also underline the importance of lipoprotein binding by GAS.     

Loop formation by the transgeneWAP:6xHishGH in transgenic rabbit fibroblasts, revealed by fluorescencein situ hybridization to nuclear halos

Using fluorescence in situ hybridization (FISH) to somatic nuclear halos from transgenic rabbits WAP:6xHishGH, we present evidence for stability of transgenesis at the chromatin level. FISH performed on fibroblasts from a homozygous individual showed 2 independent loops from both chromosomes of pair 7. On a heterozygous individual, FISH detected a single loop. According to the concept of chromatin loops and their influence on gene expression, this shows that the human growth hormone transgene, which was actively expressed in mammary gland under the influence of the tissue-specific promoter, was inactive in examined skin fibroblasts.