Expression of the E-cadherin repressors Snail, Slug and Zeb1 in urothelial carcinoma of the urinary bladder: relation to stromal fibroblast activation and invasive behaviour of carcinoma cells

Epithelial–mesenchymal transition (EMT) is regulated by interaction of carcinoma and stromal cells and crucial for progression of urinary bladder carcinoma (UBC). Therefore, the influence of activated fibroblasts on the expression of E-cadherin repressors as well as EMT and invasion in UBC was investigated. A correlative analysis of the immunohistochemical expression of fibroblast (ASMA, S100A4, FAP, SDF1, PDGFRβ) and EMT (Snail, Slug, Zeb1, E-cadherin) markers was performed on 49 UBC cases of different stages. The impact of distinguishable growth factor stimulated fibroblasts on invasion, EMT, and E-cadherin repressor expression was investigated in an invasion model. In situ, invasiveness was significantly correlated to the loss of membranous E-cadherin (E-cad_m) and increased Snail, Slug, Zeb1 in tumour cells, as well as to increased ASMA, S100A4, and PDGFRβ in stromal cells. A significant correlation to nodal metastasis could be evidenced for the loss of E-Cad_m, and for an increase in S100A4 and PDGFRβ. Comparison of stromal and EMT markers revealed significant correlations of ASMA to Snail and Slug; of S100A4 to the loss of E-cad_m and Zeb1; and of PDGFRβ to the loss of E-Cad_m, Slug and Zeb1. In vitro, TGFβ1 induced myofibroblasts were the strongest attractants, while aFGF or TGFβ1/aFGF stimulated fibroblasts were the most potent EMT inductors. As shown here for the first time, distinct sub-populations of fibroblasts are to various extents associated with EMT and tumour progression in UBC. These relevant findings might be the basis for the identification of new diagnostic markers and therapeutic targets selectively affecting tumour supporting CAF effects.     

Subthreshold nitric oxide synthase inhibition improves synergistic effects of subthreshold MMP‐2/MLCK‐mediated cardiomyocyte protection from hypoxic injury

Injury of myocardium during ischaemia/reperfusion (I/R) is a complex and multifactorial process involving uncontrolled protein phosphorylation, nitration/nitrosylation by increased production of nitric oxide and accelerated contractile protein degradation by matrix metalloproteinase‐2 (MMP‐2). It has been shown that simultaneous inhibition of MMP‐2 with doxycycline (Doxy) and myosin light chain kinase (MLCK) with ML‐7 at subthreshold concentrations protects the heart from contractile dysfunction triggered by I/R in a synergistic manner. In this study, we showed that additional co‐administration of nitric oxide synthase (NOS) inhibitor (1400W or L‐NAME) in subthreshold concentrations improves this synergistic protection in the model of hypoxia–reoxygenation (H‐R)‐induced contractile dysfunction of cardiomyocytes. Isolated cardiomyocytes were subjected to 3 min. of hypoxia and 20 min. of reoxygenation in the presence or absence of the inhibitor cocktails. Contractility of cardiomyocytes was expressed as myocyte peak shortening. Inhibition of MMP‐2 by Doxy (25–100 μM), MLCK by ML‐7 (0.5–5 μM) and NOS by L‐NAME (25–100 μM) or 1400W (25–100 μM) protected myocyte contractility after H‐R in a concentration‐dependent manner. Inhibition of these activities resulted in full recovery of cardiomyocyte contractility after H‐R at the level of highest single‐drug concentration. The combination of subthreshold concentrations of NOS, MMP‐2 and MLCK inhibitors fully protected cardiomyocyte contractility and MLC1 from degradation by MMP‐2. The observed protection with addition of L‐NAME or 1400W was better than previously reported combination of ML‐7 and Doxy. The results of this study suggest that addition of NOS inhibitor to the mixture of inhibitors is better strategy for protecting cardiomyocyte contractility.     

Are the spectral Doppler indices of ovarian arteries indicative of antral follicular development and predictive of ovulatory responses and embryo yields in superovulated ewes?

Nineteen ewes received 200 mg of pFSH administered in eight decreasing doses from Days 1 to 4, starting three days before CIDR® device removal. Ten ewes received an injection of 350 μg of estradiol benzoate at CIDR® device insertion (Group E) and nine animals served as controls (Group C). B-mode and spectral Doppler ultrasonographic examinations were performed daily throughout superovulatory treatment to enumerate ovarian antral follicles and to determine ovarian blood flow indices, respectively. There were no differences (P > 0.05) in superovulatory responses between left and right ovaries/uterine horns or the two groups of animals. End-diastolic velocity (EDV) and mean velocity (Vm) values were greater (P < 0.05) on Days 1 and 2, and peak systolic velocity (SVp) was greater (P < 0.05) on Day 3 in Group C than in Group E. In Group E 15 correlations was recorded among indices (SVp, Vm, EDV, flow velocity integral-FVI, and pulsatility index-PI) and follicles numbers in different size classes on Days 1, 2 and 4, and seven correlations among indices (SVp, EDV, Vm, and vascular resistance index-RI) and superovulatory/embryo results (numbers of regressing corpora lutea, numbers/percentages of degenerated embryos and viability rates) on Days 1, 2 and 3. In Group C, there were three correlations among EDV and RI and medium-sized/large follicle numbers on Days 1 and 3, and five correlations among indices (EDV, RI and PI) and superovulatory/embryo results (numbers of luteinized unovulated follicles, degenerated embryos and unfertilized eggs) on Days 2 or 4. There was a lack of consistency in the velocimetric correlates of antral follicle numbers and superovulatory responses between the left and right side. Therefore, the usefulness of ovarian arterial indices to predict ovine superovulatory outcomes remains equivocal and requires further confirmatory studies.     

Region-specific effects on brain metabolites of hypoxia and hyperoxia overlaid on cerebral ischemia in young and old rats: a quantitative proton magnetic resonance spectroscopy study

Background  

Both hypoxia and hyperoxia, deregulating the oxidative balance, may play a role in the pathology of neurodegenerative disorders underlain by cerebral ischemia. In the present study, quantitative proton magnetic resonance spectroscopy was used to evaluate regional metabolic alterations, following a 24-hour hypoxic or hyperoxic exposure on the background of ischemic brain insult, in two contrasting age-groups of rats: young - 3 months old and aged - 24 months old.     

Lack of effect of vasoactive intestinal peptide antagonists on blood flow in the rat thyroid

We used three putative vasoactive intestinal peptide (VIP) antagonists: 1) [4Cl-D-Phe⁶,Leu¹⁷]VIP, 2) [N-Ac-Tyr¹,D-Phe²]GRF(1–29)-NH₂, and 3) VIP(10–28) to assess the involvement of endogenous VIP in the regulation of thyroid hormone secretion and thyroid blood flow (BF). We measured thyroid BF in ketamine-pentobarbital-anesthetized rats using the microsphere technique. Increases in thyroid BF induced by VIP administration (30 pmol-1.5 nmol/100 g b.wt.) were not affected by any of the three compounds tested at doses 10–100 times higher than that of VIP. These compounds (3–15 nmol/100 g b.wt.) also failed to affect basal thyroid BF or hormone secretion. Increases in pancreatic and salivary gland BFs induced by VIP (30 pmol/100 g b.wt.) were also not affected by [4Cl-D-Phe⁶,Leu¹⁷]VIP or [N-Ac-Tyr¹,D-Phe²]GRF(1–29)-NH₂ (3 nmol/100 g b.wt.). These results indicate that the three compounds tested are not effective inhibitors of VIP receptors in the thyroid vasculature and, therefore, they cannot be used in the investigation of the functional significance of endogenous VIP in the regulation of thyroid BF.     

1H NMR conformational study of a variety of alpha-anomers of C5-substituted 2'-deoxyuridines: comparison to their antiherpetic beta counterparts

Although alpha-nucleosides are not found in nucleic acid, they do occur as constituents of smaller molecules in living cells, e.g., in vitamin B(12). There are now several examples of alpha-nucleosides exerting a biological activity in some instances equal to, or even exceeding, that of the corresponding beta-anomer. Examples include growth inhibitory properties against mouse leukemia cells and antitumor activity. From stereochemical point of view, alpha-anomers serve as references for studying of interaction of the base with the sugar moiety in beta-anomers and may help in better understanding of structure-activity relationships. One important problem preventing conformational analysis of alpha nucleosides is uncertainty in the determination of vicinal coupling constants from simulation of overlapping sugar proton resonances of strongly coupled spin systems. A successful resolution of near-isochronous H3' and H4' resonances made possible a full conformational analysis for a series of alpha-anomers C5-substituted 2'-deoxyuridines, including methyl, ethyl, isopropyl, fluor, vinyl, and bromovinyl, in comparison to their beta counterparts. Conformation of the sugar ring is determined from proton-proton coupling constants and described in terms of pseudorotation between two main puckering domains C2'endo (S) and C3'endo (N). A thorough analysis of chemical shifts as well as conformation of the sugar ring and C4'-C5' rotamers made possible determination of conformational preferences in equilibrium about the glycosidic bond between two regions, anti and syn. This work provides insights into the role of anomeric configuration of the base in conformational behavior of the sugar moiety, a link in the backbone of nucleic acids.     

The role of resveratrol and melatonin in the nitric oxide and its oxidation products mediated functional and structural modifications of two glycolytic enzymes: GAPDH and LDH

Background

Nitric oxide is a well-known gaseous signaling molecule and protein modifying agent. However, at higher concentrations or during oxidative stress nitric oxide may exert some deleterious effects on protein structure and function. Here we investigated the influence of nitric oxide and products of its oxidation on two glycolytic enzymes: GAPDH and LDH under in vitro nitrosative stress conditions. Secondly, we applied natural antioxidants: melatonin and resveratrol to examine their effects on the enzymes under studied conditions.

Exacerbation of antigen-induced arthritis in IFN-gamma-deficient mice as a result of unrestricted IL-17 response

Proinflammatory Th1 responses are believed to be involved in the induction and perpetuation of rheumatoid arthritis. However, the role of IFN-gamma, the major cytokine produced by Th1 cells, is still incompletely defined. In the present study, we investigated the effects of IFN-gamma deficiency (IFN-gamma(-/-)) on the course of experimental murine Ag-induced arthritis (AIA). In the acute stage of disease, IFN-gamma(-/-) AIA mice showed significantly increased inflammatory responses compared with wild-type C57BL/6 AIA mice, i.e., exacerbated joint swelling, increased delayed-type hypersensitivity reaction, and increased histopathological scores of arthritis. Intraarticular administration of exogenous IFN-gamma at induction of AIA significantly suppressed these acute aggravation effects. Stimulated cells isolated from lymph nodes and spleen of IFN-gamma(-/-) AIA mice showed increased production of IL-2, IL-4, IL-5, IL-6, but most prominently of IL-17. These elevations were paralleled by decreased humoral immune responses, with low serum levels of total and Ag-specific IgG (IgG1, IgG2a(b), IgG2b, IgG3). At immunohistology, the knee joints of IFN-gamma(-/-) AIA mice showed massive neutrophil granulocyte infiltration. Treatment with mAbs neutralizing IL-17 diminished the acute inflammation. In vitro, Th cell expansion and production of IL-17 upon restimulation were effectively and dose dependently inhibited by IFN-gamma. These results clearly demonstrate that IFN-gamma has anti-inflammatory properties during the initial phase of AIA, and indicate that IFN-gamma deficiency exerts disease-promoting effects, preferentially via IL-17-modulated pathways.