Low Levels of GSTA1 Expression Are Required for Caco-2 Cell Proliferation

The colonic epithelium continuously regenerates with transitions through various cellular phases including proliferation, differentiation and cell death via apoptosis. Human colonic adenocarcinoma (Caco-2) cells in culture undergo spontaneous differentiation into mature enterocytes in association with progressive increases in expression of glutathione S-transferase alpha-1 (GSTA1). We hypothesize that GSTA1 plays a functional role in controlling proliferation, differentiation and apoptosis in Caco-2 cells. We demonstrate increased GSTA1 levels associated with decreased proliferation and increased expression of differentiation markers alkaline phosphatase, villin, dipeptidyl peptidase-4 and E-cadherin in postconfluent Caco-2 cells. Results of MTS assays, BrdU incorporation and flow cytometry indicate that forced expression of GSTA1 significantly reduces cellular proliferation and siRNA-mediated down-regulation of GSTA1 significantly increases cells in S-phase and associated cell proliferation. Sodium butyrate (NaB) at a concentration of 1 mM reduces Caco-2 cell proliferation, increases differentiation and increases GSTA1 activity 4-fold by 72 hours. In contrast, 10 mM NaB causes significant toxicity in preconfluent cells via apoptosis through caspase-3 activation with reduced GSTA1 activity. However, GSTA1 down-regulation by siRNA does not alter NaB-induced differentiation or apoptosis in Caco-2 cells. While 10 mM NaB causes GSTA1-JNK complex dissociation, phosphorylation of JNK is not altered. These findings suggest that GSTA1 levels may play a role in modulating enterocyte proliferation but do not influence differentiation or apoptosis.     

Longitudinal Exposure to Selected Pesticides in Drinking Water

The presence and temporal fluctuations of concentrations of insecticides and herbicides in natural waters has been well documented. Little, however, is known about exposure to pesticides through drinking water for the general population. Concentrations often pesticides, including 4,4′DDE and atrazine, were measured up to six times at equally spaced intervals over a 1-year period in drinking water of 80 randomly selected residences in Maryland. Atrazine was detected in 228 (57.9%) of the drinking water samples with a mean of 0.15?µg/L, with standard deviation 0.12?µg/L, median 0.17?µg/L, and range <0.037 to 0.46?µg/L. 4,4′DDE was found in 22 (5.6%) water samples; no other target analytes were detected. Concentrations of atrazine in drinking water were found to vary over a 12-month period with the greatest concentrations in the late summer and fall and the lowest in the early spring. Atrazine concentrations in drinking water were influenced more by differences in levels among residences than by time of year. Seven-day average exposures and exposures per unit body weight to atrazine in drinking water exhibited a similar temporal pattern. Among individuals, drinking water consumption rate was a more important determinant of atrazine exposure (µg/d) and exposure per unit body weight (µg/kg/d) than atrazine concentration in drinking water or body weight.     

Force-frequency relationship and potentiation in mammalian skeletal muscle.

Repetitive activation of a skeletal muscle results in potentiation of the twitch contractile response. Incompletely fused tetanic contractions similar to those evoked by voluntary activation may also be potentiated by prior activity. We aimed to investigate the role of stimulation frequency on the enhancement of unfused isometric contractions in rat medial gastrocnemius muscles in situ. Muscles set at optimal length were stimulated via the sciatic nerve with 50-micros duration supramaximal pulses. Trials consisted of 8 s of repetitive trains [5 pulses (quintuplets) 2 times per second or 2 pulses (doublets) 5 times per second] at 20, 40, 50, 60, 70, and 80 Hz. These stimulation frequencies represent a range over which voluntary activation would be expected to occur. When the frequency of stimulation was 20, 50, or 70 Hz, the peak active force (highest tension during a contraction - rest tension) of doublet contractions increased from 2.2 +/- 0.2, 4.1 +/- 0.4, and 4.3 +/- 0.5 to 3.1 +/- 0.3, 5.6 +/- 0.4, and 6.1 +/- 0.7 N, respectively. Corresponding measurements for quintuplet contractions increased from 2.2 +/- 0.2, 6.1 +/- 0.5, and 8.7 +/- 0.7 to 3.2 +/- 0.3, 7.3 +/- 0.6, and 9.0 +/- 0.7 N, respectively. Initial peak active force values were 27 +/- 1 and 61.5 +/- 5% of the maximal (tetanic) force for doublet and quintuplet contractions, respectively, at 80 Hz. With doublets, peak active force increased at all stimulation frequencies. With quintuplets, peak active force increased significantly for frequencies up to 60 Hz. Twitch enhancement at the end of the 8 s of repetitive stimulation was the same regardless of the pattern of stimulation during the 8 s, and twitch peak active force returned to prestimulation values by 5 min. These experiments confirm that activity-dependent potentiation is evident during repeated, incompletely fused tetanic contractions over a broad range of frequencies. This observation suggests that, during voluntary motor unit recruitment, derecruitment or decreased firing frequency would be necessary to achieve a fixed (submaximal) target force during repeated isometric contractions over this time period.     

Multimodal Advertisement of Pregnancy in Free-Ranging Female Japanese Macaques (Macaca fuscata)

The role of multiple sexual signals in indicating the timing of female ovulation, and discrimination of this timing by males, has been particularly well studied among primates. However the exhibition of pregnancy signals, and how such signals might modulate male post-conception mating decisions, is still poorly understood. Here we aimed to determine if Japanese macaque males use changes in female sexual signals (behavioral, visual and auditory) to discriminate pregnancy and adjust their socio-sexual behaviors. We combined behavioral observations, digital photography and endocrinological (progestogen and estrogen) data, collected systematically during three one-month periods: the pre-conceptive period, the 1^st month of pregnancy and the 2^nd month of pregnancy. We analyzed variation in the probability of detecting male and female socio-sexual behaviors and estrus calls, as well as changes in female face color parameters, in relation to female reproductive state. Based on our focal observations, we found that males did not copulate during the pregnancy period, and that female socio-sexual behaviors generally decreased from the pre-conceptive to post-conceptive periods. Female face luminance decreased from the pre-conceptive month to the pregnancy period whereas face color only varied between the 1^st and 2^nd month of gestation. Our results suggest that Japanese macaque females display sexual cues of pregnancy that males might use to reduce energy wasted on non-reproductive copulations with pregnant females. We hypothesize that females advertize their pregnancy through changes in behavioral, visual and potential auditory signals that males can use to adjust their mating behaviors. We finish by discussing implications for male and female post-conception strategies.     

Rat atrial muscle responses with caffeine: dose-response, force frequency, and postrest contractions.

Caffeine has been reported to have a positive and (or) a negative inotropic effect on cardiac muscle. In this study, the force-frequency and postrest characteristics of rat atrium were studied in the presence of caffeine (1.0-10 mM) to see if the interval between beats affected the response of cardiac muscle to caffeine. When stimulation frequency was 0.5 or 2.0 Hz, there was a positive followed by a negative inotropic response with 1, 5, or 10 mM caffeine. Incomplete relaxation occurred under these circumstances, giving rise to contracture. At low frequency of stimulation (0.1 Hz) caffeine had only a negative inotropic effect, and this effect was greater with 1 mM caffeine than with 5 mM caffeine. In the absence of caffeine, when stimulation at 0.5 or 3 Hz was interrupted, a pause of 2-20 s resulted in potentiation. When caffeine was present (2.0 mM), postrest potentiation was severely attenuated, but the steady-state contraction amplitude within the range 0.5-3.0 Hz was not different. These results are consistent with the hypothesis that caffeine induces a leak of Ca2+ from the sarcoplasmic reticulum, and this Ca2+ is extruded from the cell, possibly by Na+/Ca2+ exchange. Sarcoplasmic reticular uptake of Ca2+ and the translocation to release sites appear not to be affected by caffeine within 1-5 mM concentrations.