Induction of cortical endoplasmic reticulum by dimerization of a coatomer-binding peptide anchored to endoplasmic reticulum membranes

Cortical endoplasmic reticulum (cER) is a permanent feature of yeast cells but occurs transiently in most animal cell types. Ist2p is a transmembrane protein that permanently localizes to the cER in yeast. When Ist2 is expressed in mammalian cells, it induces abundant cER containing Ist2. Ist2 cytoplasmic C-terminal peptide is necessary and sufficient to induce cER. This peptide sequence resembles classic coat protein complex I (COPI) coatomer protein-binding KKXX signals, and indeed the dimerized peptide binds COPI in vitro. Controlled dimerization of this peptide induces cER in cells. RNA interference experiments confirm that coatomer is required for cER induction in vivo, as are microtubules and the microtubule plus-end binding protein EB1. We suggest that Ist2 dimerization triggers coatomer binding and clustering of this protein into domains that traffic at the microtubule growing plus-end to generate the cER beneath the plasma membrane. Sequences similar to the Ist2 lysine-rich tail are found in mammalian STIM proteins that reversibly induce the formation of cER under calcium control.The current view of the yeast endoplasmic reticulum (ER) discriminates perinuclear ER from cortical ER (cER), which forms a circular structure apposed to the plasma membrane (PM) (1). Both structures are connected by tubulated membranes (2, 3), at least transiently, because ER membranes undergo continuous fission and fusion events (4, 5). cER is a much less prominent feature of most mammalian cells (6). The best-characterized function of cER is its role in the store-operated calcium entry, an ubiquitous Ca²⁺ influx pathway activated in response to depletion of intracellular calcium stores (7).Ist2 is a “yeast peripheral” protein involved in osmotic stress tolerance. It was initially believed to be located at the plasma membrane (8–11), and its cytosolic tail (Ist2ct) has been shown to carry the peripheral targeting signal (8). Ist2ct includes a dimerization domain (amino acids 878–928) and a lysine-rich carboxy terminal tail containing a KKXX-like motif that has been proposed to interact with the PM (11, 12). The nature of the peripheral Ist2 resident sites remains a matter of debate, however. It was once thought that Ist2 reached the PM in a new Golgi-independent manner (10), but more recently it has been concluded that the major residence site is in fact the cER (11).To gain insight into the biogenesis of cER in mammalian cells, we investigated whether Ist2, when expressed in a heterologous system, can serve as a useful marker for this compartment. Interestingly, enrichment of Ist2 chimeric protein at the cER appears to directly modulate the formation and/or maintenance of this ER subdomain. These dynamic changes in peripheral ER structure are absolutely dependent on both microtubules and coat protein complex I (COPI) and suggest a different role of COPI than its classical one.     

A longitudinal study for investigating the exposure level of anesthetics that impairs neurobehavioral performance

There is conflicting evidence on the level of anesthetics that impairs neurobehavioral performance, leading to differences in exposure standards (25 or 50 ppm for N(2)O). Thirty-eight operating room nurses and 23 unexposed nurses were asked to provide information on confounding variables: age, gender, years of schooling, alcohol and coffee consumption, smoking, length of work, symptoms (Euroquest) and results of Block Design test. Afterward, all workers were repeatedly examined (on Monday and Friday of a working week, before and after workshift) for stress and arousal (Mood Scale) and complex reaction times (Color Word Vigilance, CWV), the latter being the outcome. Individual exposure was assessed through urinary end-shift concentrations of nitrous oxide (N(2)O) and isoflurane. According to the highest value of urinary excretion of N(2)O in the week, exposed workers were subdivided in three groups (<13; > or =13 and <27; and > or = 27 microg/l). The values of 13 and 27 microg/l correspond to environmental concentrations of 25 and 50 ppm, respectively. In order to take into account the pre-existing abilities of exposed and reference workers, and investigate the neurobehavioral changes over time, longitudinal data were analyzed by a two-stage regression model and analysis of variance for repeated measures (MANOVA). The former method, controlling for confounding factors and Monday morning CWV (which conveyed the pre-existing ability of the subjects), showed that, with respect to unexposed nurses, reaction times were significantly (p<0.020) higher only in workers with urinary N(2)O> or = 27 microg/l. Therefore, at MANOVA, all subjects were categorized in two classes (N(2)O urinary concentrations or = 27 microg/l), and CWV results were adjusted for the confounding variables and effects of stress and arousal, taken concurrently with CWV. CWV significantly (p<0.039) decreased over a working week (indicating a learning effect) in workers with urinary N(2)O<27 microg/l, while remained steady (indicating impairment of neurobehavioral performance) in those with urinary N(2)O> 27 microg/l.     

Reducing effect of the positive allosteric modulator of the GABAB receptor, GS39783, on alcohol self-administration in alcohol-preferring rats

Rationale and objectives The positive allosteric modulator of the GABA_B receptor, GS39783, has recently been found to suppress acquisition and maintenance of alcohol drinking behavior in selectively bred Sardinian alcohol-preferring (sP) rats exposed to the standard, homecage two-bottle “alcohol vs water” choice regimen. The present study was designed to extend the characterization of the “anti-alcohol” effects of GS39783 to oral self-administration of alcohol under an operant procedure. Materials and methods Two separate groups of male sP rats were trained to lever-press (on an FR4 schedule) to orally self-administer alcohol (15%, v/v) or sucrose (0.3%, w/v) in daily 30-min sessions. Once lever-pressing behavior reached stable levels, the effect of GS39783 (0, 25, 50, and 100 mg/kg, i.g.) on responding for alcohol and sucrose was determined. Results Pretreatment with GS39783 resulted in a significant, dose-dependent reduction in responding for alcohol; at the dose of 100 mg/kg GS39783, the number of lever responses for alcohol was reduced by approximately 50% in comparison to vehicle-treated rats. The effect of GS39783 on alcohol self-administration was specific, as responding for sucrose was completely unaffected by pretreatment with GS39783. Conclusions These data demonstrate the capability of GS39783 to attenuate the reinforcing properties of alcohol in alcohol-preferring rats. These data constitute a further piece of experimental evidence in support of the hypothesized role for the GABA_B receptor in the control of alcohol drinking and reinforcement.     

Fetal blood-brain barrier P-glycoprotein contributes to brain protection during human development

During brain development and blood-brain barrier (BBB) differentiation the expression of P-glycoprotein (P-gp) may complement the protective function of the placental barrier against xenobiotic substances. To establish an immunohistochemical procedure for P-gp detection, different anti-P-gp monoclonal antibodies were first tested on a fibrosarcoma cell line and colonic carcinoma tissue. The protocol was then tested on adult human brains as a BBB-P-gp tissue-specific control and for double labeling with anti-P-gp and the astroglia marker glial fibrillary acidic protein (GFAP). The protocol was then used to analyze the expression and localization of P-gp in human fetuses during cerebral cortex formation. At the earliest examined stage, 12 weeks of gestation (wg), P-gp was detectable as diffuse cytoplasmic labeling of the endothelial cells lining the primary cortex microvessels. At 18 wg, a punctate P-gp staining pattern was detected on cortex and subcortical vessels and on their side branches. At 22 wg, P-gp staining was linear and concentrated on endothelial cell membranes. In all examined ages, GFAP-positive radial glial cells and astrocytes did not stain for P-gp, even at their perivascular processes, whereas faint P-gp labeling was seen on vimentin-reactive radial glia at the earliest examined fetal age. At midgestation, P-gp colocalized with caveolin-pY14 on the abluminal endothelial cell membrane. These results demonstrate that P-gp is expressed early during human cerebral cortical microvessel development, and suggest that at midgestation there may be efflux activity that is regulated by interactions with the caveolar endothelial cell compartment.     

High level of messenger RNA for BRMS1 in primary breast carcinomas is associated with poor prognosis

BRMS1 is regarded as a metastasis suppressor gene for its ability to reduce metastatic potential of human and murine breast cancer cells as well as human melanoma cells. However, BRMS1 association to human tumor progression is not clearly understood. In the present study we analyzed BRMS1 mRNA expression in tumor progression and its potential prognostic value for breast carcinoma. BRMS1 mRNA expression level was quantified by real-time PCR in 47 tumoral, in 14 peritumoral and in 15 metastatic microdissected cellular populations from 47 breast cancer patients with 10-year follow up. We found BRMS1 expression to be higher in carcinoma cells than in matching normal epithelial cell populations in 10 out of 14 cases (p = 0.0005), while lymph-nodal carcinoma cells showed lower BRMS1 expression in 9 out of 15 cases (p = 0.001). Using both in vivo (human mammary breast carcinomas) and in vitro systems (breast cancer cell lines) we were able to demonstrate that BRMS1 overexpression was not a bias effect induced by cell proliferation rate. BRMS1 expression levels did not correlate with standard breast cancer prognostic factors but BRMS1 higher expression was associated with patient shorter disease-free and overall survival. Our findings are apparently inconsistent with the concept of BRMS1 as a metastasis suppressor gene. One possible explanation is that epithelial cells increase their BRMS1 expression as a compensatory response to tumor formation or metastasis progression, which is elevated in proportion to tumor aggressiveness, whereas those cells of the primary tumor that cannot upregulate BRMS1 escape to form metastasis.     

Patterns of hepatocellular carcinoma development in hepatitis B virus and hepatitis C virus related cirrhosis.

To compare incidence, risk factors and morphologic pattern of hepatocellular carcinoma (HCC) development in hepatitis B virus (HBV) and hepatitis C virus (HCV) related cirrhosis, 401 patients were followed prospectively by periodic ultrasound examination for 14-189 months (mean: 84.8+/-36.7). During follow-up, 77 (19.2%) patients developed HCC, with 5 and 10 year cumulative incidence of 10 and 27.5%, respectively. The risk of HCC was significantly higher in HBV and HCV co-infected patients (P=0.014) compared to those with single HBsAg or anti-HCV (antibodies to hepatitis C virus) positivity. In anti-HCV positive cases the annual risk of HCC increased from 2% in the first 5 year period to 4% in the third 5 year period, while it decreased from 2 to 0% in the same time periods in the HBsAg positive group. By Cox's regression, age above 59 years (P=0.001), male sex (P=0.09), longer duration (P=0.04) and more advanced stage (P=0.01) of cirrhosis, lower platelets count (P=0.001) and higher ALT levels were significant risk factors for HCC in anti-HCV positive patients, while only high alpha-fetoprotein (AFP) levels during follow-up (P=0.04) was a significant risk factor for HCC in HBsAg positive cases. The pattern of HCC was nodular in 63 (81.8%) patients and infiltrating in 14 (18.2%), and the former type was associated with older age (P=0.0001), longer duration (P=0.002) and more advanced stage (P=0.0001) of cirrhosis but not with the viral etiology of disease. In contrast, development of infiltrating HCC was unrelated to age and disease duration and stage, and was associated with male sex (P=0.01), HBV infection (P=0.06) and HBV and HCV co-infection (P=0.0001). Our results indicate different incidence profile, risk factors and patterns of morphogenesis of HCC development in HBV and HCV associated cirrhosis, suggesting different mechanisms of carcinogenesis.