Cognitive Deficits Found in a Pro-inflammatory State are Independent of ERK1/2 Signaling in the Murine Brain Hippocampus Treated with Shiga Toxin 2 from Enterohemorrhagic Escherichia coli.

Shiga toxin 2 (Stx2) from enterohemorrhagic Escherichia coli (EHEC) produces hemorrhagic colitis, hemolytic uremic syndrome (HUS), and acute encephalopathy. The mortality rate in HUS increases significantly when the central nervous system (CNS) is involved. Besides, EHEC also releases lipopolysaccharide (LPS). Many reports have described cognitive dysfunctions in HUS patients, the hippocampus being one of the brain areas targeted by EHEC infection. In this context, a translational murine model of encephalopathy was employed to establish the deleterious effects of Stx2 and the contribution of LPS in the hippocampus. The purpose of this work is to elucidate the signaling pathways that may activate the inflammatory processes triggered by Stx2, which produces cognitive alterations at the level of the hippocampus. Results demonstrate that Stx2 produced depression-like behavior, pro-inflammatory cytokine release, and NF-kB activation independent of the ERK1/2 signaling pathway, while co-administration of Stx2 and LPS reduced memory index. On the other hand, LPS activated NF-kB dependent on ERK1/2 signaling pathway. Cotreatment of Stx2 with LPS aggravated the pathologic state, while dexamethasone treatment succeeded in preventing behavioral alterations. Our present work suggests that the use of drugs such as corticosteroids or NF-kB signaling inhibitors may serve as neuroprotectors from EHEC infection.

Tyrosine hydroxylase is short-term regulated by the ubiquitin-proteasome system in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats: possible implications in hypertension.

Aberrations in the ubiquitin-proteasome system (UPS) are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR) and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86 ± 15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2 ± 8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92 ± 22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7 ± 0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasome activity. Proteasome activity was significantly reduced by 67 ± 4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension.

The long-term effects of prenatal nicotine exposure on verbal working memory: an fMRI study of young adults.

BACKGROUND: Using functional magnetic resonance imaging (fMRI), the long-term effects of prenatal nicotine exposure on verbal working memory were investigated in young adults. Participants were members of the Ottawa Prenatal Prospective Study, a longitudinal study that collected a unique body of information on participants from infancy to young adulthood. This allowed for the measurement of an unprecedented number of potentially confounding drug exposure variables including: prenatal marijuana and alcohol exposure and current marijuana, nicotine and alcohol use. METHODS: Twelve young adults with prenatal nicotine exposure and 13 non-exposed controls performed a 2-Back working memory task while fMRI blood oxygen level-dependent responses were examined. Despite similar task performance, participants with more prenatal nicotine exposure demonstrated significantly greater activity in several regions of the brain that typically subserve verbal working memory including the middle frontal gyrus, precentral gyrus, the inferior parietal lobe and the cingulate gyrus. RESULTS: These results suggest that prenatal nicotine exposure contributes to altered neural functioning during verbal working memory that continues into adulthood. Working memory is critical for a wide range of cognitive skills such as language comprehension, learning and reasoning. CONCLUSION: Thus, these findings highlight the need for continued educational programs and public awareness campaigns to reduce tobacco use among pregnant women.

Generating mammalian stable cell lines by electroporation.

Expression of functional, recombinant mammalian proteins often requires expression in mammalian cells (see Single Cell Cloning of a Stable Mammalian Cell Line). If the expressed protein needs to be made frequently, it can be best to generate a stable cell line instead of performing repeated transient transfections into mammalian cells. Here, we describe a method to generate stable cell lines via electroporation followed by selection steps. This protocol will be limited to the CHO dhfr-Urlaub et al. (1983) and LEC1 cell lines, which in our experience perform the best with this method.

Effects of drospirenone/estradiol on steroid receptors and Bcl-2 in the postmenopausal endometrium.

OBJECTIVE: To evaluate the effect of drospirenone with 17ß-estradiol on the histology and expression of estrogen and progesterone receptors and of Bcl-2 protein, in endometrium of postmenopausal women. METHOD: Forty postmenopausal women, including controls, participated in this study evaluating oral hormone replacement treatment combining 2 mg/day of drospirenone with 1 mg/day of 17ß-estradiol administered for a 24-week period. The effect on the endometrium was assessed by histology and the apoptosis marker Bcl-2. The immunoexpression of estrogen (ER) and progesterone (PR) receptors in the endometrium was also measured. RESULTS: No increase in endometrial thickness was evident after either treatment, although endometrial histology was atrophic in most biopsies. The drospirenone/estradiol group showed higher expression of ER and PR in glandular epithelium compared to stroma, but the Bcl-2 protein was more immunoreactive in stroma than in glandular epithelium. Compared to controls, drospirenone/estradiol users showed higher immunoexpression of ER, PR and Bcl-2 in both glandular epithelium and endometrial stroma. CONCLUSION: A 24-week course of drospirenone with 17ß-estradiol resulted in low proliferation and was shown to lead to atrophic endometrium. The novel progestogen drospirenone seems to have favorable effects on the endometrium of postmenopausal women due to its pro-apoptotic action in glandular epithelium.

Assay of carnitine in plasma and urine of healthy adults / 中国医学科学院学报

<p><b>OBJECTIVE</b>To determine the levels of carnitine in plasma and daily excretion of carnitine in urine of healthy adults so as to provide the reference standard for studying the changes of carnitine in patients.</p><p><b>METHODS</b>Carnitine in plasma and urine was assayed with high performance liquid chromatography (HPLC). The levels of total carnitine (TC), free carnitine (FC) and acetyl-carnitine (AC) in fasting plasma and the daily excretion of TC, FC and AC in urine were assayed in 40 healthy adults (20 men and 20 women) with standard diet.</p><p><b>RESULTS</b>Good linearity (r 2 > or = 0.999) was observed in assaying TC, FC and AC. The relative standard deviation (RSD) was lower than 9.1% and bias lower than 5.6%. It was showed that the plasmatic levels of TC, FC and AC in healthy men [(53.1 +/- 8.5), (41.2 +/- 6.1), (6.2 +/- 0.6) mumol/L] were significantly higher than those in healthy women [(45.4 +/- 5.6), (35.2 +/- 4.9), (5.7 +/- 0.7) mumol/L] (P = 0.002, 0.002, 0.035). The daily urinary excretion of TC, FC and AC in healthy men [(386.1 +/- 22.9), (180.5 +/- 31.8), (33.8 +/- 3.3) mumol] were also significantly higher than those in healthy women [(240.1 +/- 35.6), (112.7 +/- 22.6), (29.3 +/- 4.3) mumol] (P < 0.0005, < 0.0005, < 0.0005) when the adults were given standard diet. Both the plasmatic levels and the daily urinary excretion of TC, FC and AC were of significantly positive correlation with lean body mass (LBM) (r = 0.501-0.856). The (TC-FC)/FC ratios in plasma were 0.29 +/- 0.05 for male and 0.29 +/- 0.04 for female.</p><p><b>CONCLUSION</b>Good precision and accuracy were observed in assaying carnitine with HPLC. After standard diet, both the level of carnitine in fasting plasma and the daily urinary carnitine excretion of healthy adults were positively correlated with LBM.</p>