Limitations of the scrape-loading/dye transfer technique to quantify inhibition of gap junctional intercellular communication

Gap junctional intercellular communication (GJIC) is recognized as playing an important role in normal cell proliferation and development. Chemically induced alteration of GJIC has been proposed to be associated with abnormal cellular growth and/or tumor promotion. Several in vitro assays are currently used to determine the effects of chemicals on GJIC between cultured mammalian cells. One of these assays, the scrape-loading dye transfer (SLIDT) technique, is based on monitoring the transfer of the fluorescent dye Lucifer yellow from one cell into adjacent cells via functional gap junctions. The objective of our study was to evaluate and compare various approaches for quantifying results obtained with the SL/DT technique. Confluent cultures of either WB rat liver epithelial cells or LC-540 rat leydig cells were exposed to the animal tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), solvent (0.1% ethanol), or culture medium for one hour at 37° C prior to analysis of GJIC. Inhibition of dye transfer was clearly evident following TPA exposure. Quantification of this dye transfer was assessed via four approaches: manually counting the number of labeled cells; measuring the distance of dye travel from the scrape line; quantifying the amount of cellular dye uptake; and determining the distribution of dye away from the scrape line. Our results suggest that while the SL/DT technique can be effectively used as a tool to determine the qualitative presence or absence of GJIC, its use in quantifying changes in GJIC following chemical exposure is limited. Since concentration-dependent responses are critical in chemical testing, application of the SLIDT method should be restricted to a screening assay for qualitatively assessing the presence or absence of GJIC. Another assay (e.g., electrical coupling, microinjection, metabolic cooperation, radioactive metabolite transfer, or fluorescence redistribution after photobleaching) should be considered to quantify changes in GJIC and construct chemical concentration-response curves.Abbreviations FBS, fetal bovine serum - GJIC, gap junctional intercellular communication - HBSS, Hank's balanced saline solution - SL/DT, scrape-loading/dye transfer - TPA, 12-O-tetradecanoylphorbol-13-acetate.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.     

Long-term treadmill exercise improves spatial memory of male APPswe/PS1dE9 mice by regulation of BDNF expression and microglia activation

Increasing evidence suggests that physical activity could delay or attenuate the symptoms of Alzheimer''s disease (AD). But the underlying mechanisms are still not fully understood. To investigate the effect of long-term treadmill exercise on the spatial memory of AD mice and the possible role of β-amyloid, brain-derived neurotrophic factor (BDNF) and microglia in the effect, male APPswe/PS1dE9 AD mice aged 4 months were subjected to treadmill exercise for 5 months with 6 sessions per week and gradually increased load. A Morris water maze was used to evaluate the spatial memory. Expression levels of β-amyloid, BDNF and Iba-1 (a microglia marker) in brain tissue were detected by immunohistochemistry. Sedentary AD mice and wildtype C57BL/6J mice served as controls. The results showed that 5-month treadmill exercise significantly decreased the escape latencies (P < 0.01 on the 4th day) and improved the spatial memory of the AD mice in the water maze test. Meanwhile, treadmill exercise significantly increased the number of BDNF-positive cells and decreased the ratios of activated microglia in both the cerebral cortex and the hippocampus. However, treadmill exercise did not significantly alleviate the accumulation of β-amyloid in either the cerebral cortex or the hippocampus of the AD mice (P > 0.05). The study suggested that long-term treadmill exercise could improve the spatial memory of the male APPswe/PS1dE9 AD mice. The increase in BDNF-positive cells and decrease in activated microglia might underpin the beneficial effect.     

Distinct effects of TGF-beta 1 on CD4+ and CD8+ T cell survival, division, and IL-2 production: a role for T cell intrinsic Smad3

TGF-beta1 is critical for maintaining T cell homeostasis. Smad3 has been implicated in this regulatory process, yet the cellular targets and molecular details remain poorly understood. In this study, we report that TGF-beta1 impairs the entry of CD4+ and CD8+ T cells into the cell cycle as well as their progression through subsequent rounds of division, and show that Smad3 is essential for TGF-beta1 to inhibit TCR-induced division of only CD4+ and not CD8+ T cells. Both CD8+ and CD4+ T cells from Smad3-/- mice were refractory to TGF-beta1-induced inhibition of IL-2 production, thus demonstrating that not all CD8+ T cell responses to TGF-beta1 are Smad3 independent. These TGF-beta1 effects were all T cell intrinsic, as they were reproduced in purified CD4+ and CD8+ T cells. Finally, we found that Smad3 was critical for the survival of CD8+, but not CD4+ T cells following activation ex vivo. The TCR-induced death of Smad3-/- CD8+ T cells was not dependent upon TNF-alpha production. Exogenous TGF-beta1 partially rescued the CD8+ T cells by signaling through a Smad3-independent pathway. TGF-beta1 also enhanced survival of TCR-stimulated CD4+CD44high T cells in a Smad3-independent manner. Collectively, these findings firmly establish for the first time that TGF-beta1 discriminately regulates CD4+ and CD8+ T cell expansion by signaling through distinct intracellular pathways.     

Acute toxicity effects of ibuprofen on behaviour and haematological parameters of African catfish Clarias gariepinus (Burchell, 1822)

Indiscriminate discharge of pharmaceutical waste into the aquatic ecosystem may pose serious health challenges to aquatic biota. The effect of acute exposure to ibuprofen was evaluated using changes in behaviour and haematological parameters under static bio-assay method in Clarias gariepinus. Test specimens were exposed to acute concentrations of ibuprofen (0.28, 0.33, 0.38, 0.43 and 0.48 mg l^?1) for 24, 48, 72 and 96 h durations respectively. Behavioural and phenotypic changes were observed in surviving fish. There were significant (p < 0.05) concentration and duration-dependent increases in erythrocyte (RBC), haemoglobin (Hb), pack cell volume (PCV) and leukocytes (WBC) in treated fish compared to the control. Insignificant decreases (p > 0.05) in mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were observed in treated fish compared to the control. Ibuprofen elicited dose and duration- dependent decrease in neutrophil counts with the decreases being significant (p < 0.05) in the higher doses of 0.43 and 0.48 mg l^?1. Ibuprofen did not elicit any significant changes in monocytes, basophils and eosinophils. Changes observed in this study showed that ibuprofen negatively affected the health of the fish and we recommend that discharge of ibuprofen into the aquatic environment should be monitored and controlled.     

Context-dependent memory: colour versus odour

An olfactory stimulus and a visual stimulus were employed in a context- dependent memory study using a prose passage as the to-be-remembered item. Ninety-five university students (aged 17-35 years) learned the passage of prose in the presence of one of the stimuli and were then asked to recall the passage with the original context either reinstated or not reinstated. The results revealed a significant context-dependent memory effect for the olfactory cue but not for the visual cue. They demonstrate support for the effectiveness of odours as context cues and it is suggested that context-dependent memory processes may underlie the formation and retrieval of odour-evoked autobiographical memories.