ApoE4 upregulates the activity of mitochondria‐associated ER membranes

In addition to the appearance of senile plaques and neurofibrillary tangles, Alzheimer''s disease (AD) is characterized by aberrant lipid metabolism and early mitochondrial dysfunction. We recently showed that there was increased functionality of mitochondria‐associated endoplasmic reticulum (ER) membranes (MAM), a subdomain of the ER involved in lipid and cholesterol homeostasis, in presenilin‐deficient cells and in fibroblasts from familial and sporadic AD patients. Individuals carrying the ε4 allele of apolipoprotein E (ApoE4) are at increased risk for developing AD compared to those carrying ApoE3. While the reason for this increased risk is unknown, we hypothesized that it might be associated with elevated MAM function. Using an astrocyte‐conditioned media (ACM) model, we now show that ER–mitochondrial communication and MAM function—as measured by the synthesis of phospholipids and of cholesteryl esters, respectively—are increased significantly in cells treated with ApoE4‐containing ACM as compared to those treated with ApoE3‐containing ACM. Notably, this effect was seen with lipoprotein‐enriched preparations, but not with lipid‐free ApoE protein. These data are consistent with a role of upregulated MAM function in the pathogenesis of AD and may help explain, in part, the contribution of ApoE4 as a risk factor in the disease.     

Expression of the voltage-gated sodium channel NaV1.5 in the macrophage late endosome regulates endosomal acidification

Voltage-gated sodium channels expressed on the plasma membrane activate rapidly in response to changes in membrane potential in cells with excitable membranes such as muscle and neurons. Macrophages also require rapid signaling mechanisms as the first line of defense against invasion by microorganisms. In this study, our goal was to examine the role of intracellular voltage-gated sodium channels in macrophage function. We demonstrate that the cardiac voltage-gated sodium channel, NaV1.5, is expressed on the late endosome, but not the plasma membrane, in a human monocytic cell line, THP-1, and primary human monocyte-derived macrophages. Although the neuronal channel, NaV1.6, is also expressed intracellularly, it has a distinct subcellular localization. In primed cells, NaV1.5 regulates phagocytosis and endosomal pH during LPS-mediated endosomal acidification. Activation of the endosomal channel causes sodium efflux and decreased intraendosomal pH. These results demonstrate a functionally relevant intracellular voltage-gated sodium channel and reveal a novel mechanism to regulate macrophage endosomal acidification.     

Attendance at Cervical Cancer Screening and Use of Diagnostic and Therapeutic Procedures on the Uterine Cervix Assessed from Individual Health Insurance Data (Belgium, 2002-2006)

Objective

To assess the coverage for cervical cancer screening as well as the use of cervical cytology, colposcopy and other diagnostic and therapeutic interventions on the uterine cervix in Belgium, using individual health insurance data.

Methods

The Intermutualistic Agency compiled a database containing 14 million records from reimbursement claims for Pap smears, colposcopies, cervical biopsies and surgery, performed between 2002 and 2006. Cervical cancer screening coverage was defined as the proportion of women aged 25–64 that had a Pap smear within the last 3 years.

Results

Cervical cancer screening coverage was 61% at national level, for the target population of women between 25 and 64 years old, in the period 2004–2006. Differences between the 3 regions were small, but varied more substantially between provinces. Coverage was 70% for 25–34 year old women, 67% for those aged 35–39 years, and decreased to 44% in the age group of 60–64 years. The median screening interval was 13 months. The screening coverage varied substantially by social category: 40% and 64%, in women categorised as beneficiary or not-beneficiary of increased reimbursement from social insurance, respectively. In the 3-year period 2004–2006, 3.2 million screen tests were done in the target group consisting of 2.8 million women. However, only 1.7 million women got one or more smears and 1.1 million women had no smears, corresponding to an average of 1.88 smears per woman in three years of time. Colposcopy was excessively used (number of Pap smears over colposcopies = 3.2). The proportion of women with a history of conisation or hysterectomy, before the age of 65, was 7% and 19%, respectively.

Conclusion

The screening coverage increased slightly from 59% in 2000 to 61% in 2006. The screening intensity remained at a high level, and the number of cytological examinations was theoretically sufficient to cover more than the whole target population.     

Rho GTPase-activating bacterial toxins: from bacterial virulence regulation to eukaryotic cell biology

Studies on the interactions of bacterial pathogens with their host have provided an invaluable source of information on the major functions of eukaryotic and prokaryotic cell biology. In addition, this expanding field of research, known as cellular microbiology, has revealed fascinating examples of trans-kingdom functional interplay. Bacterial factors actually exploit eukaryotic cell machineries using refined molecular strategies to promote invasion and proliferation within their host. Here, we review a family of bacterial toxins that modulate their activity in eukaryotic cells by activating Rho GTPases and exploiting the ubiquitin/proteasome machineries. This family, found in human and animal pathogenic Gram-negative bacteria, encompasses the cytotoxic necrotizing factors (CNFs) from Escherichia coli and Yersinia species as well as dermonecrotic toxins from Bordetella species. We survey the genetics, biochemistry, molecular and cellular biology of these bacterial factors from the standpoint of the CNF1 toxin, the paradigm of Rho GTPase-activating toxins produced by urinary tract infections causing pathogenic Escherichia coli. Because it reveals important connections between bacterial invasion and the host inflammatory response, the mode of action of CNF1 and its related Rho GTPase-targetting toxins addresses major issues of basic and medical research and constitutes a privileged experimental model for host-pathogen interaction.     

The Extended Transmembrane Orai1 N-terminal (ETON) Region Combines Binding Interface and Gate for Orai1 Activation by STIM1

STIM1 and Orai1 represent the two molecular key components of the Ca²⁺ release-activated Ca²⁺ channels. Their activation involves STIM1 C terminus coupling to both the N terminus and the C terminus of Orai. Here we focused on the extended transmembrane Orai1 N-terminal (ETON, aa73–90) region, conserved among the Orai family forming an elongated helix of TM1 as recently shown by x-ray crystallography. To identify “hot spot” residues in the ETON binding interface for STIM1 interaction, numerous Orai1 constructs with N-terminal truncations or point mutations within the ETON region were generated. N-terminal truncations of the first four residues of the ETON region or beyond completely abolished STIM1-dependent Orai1 function. Loss of Orai1 function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1 interaction. In a complementary approach, we monitored STIM1-Orai interaction via Orai1 V102A by determining restored Ca²⁺ selectivity as a consequence of STIM1 coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca²⁺ selectivity of Orai1 V102A in the presence of STIM1, demonstrating impairment of STIM1 binding. Hence, the major portion of the ETON region (aa76–90) is essential for STIM1 binding and Orai1 activation. Mutagenesis within the ETON region revealed several hydrophobic and basic hot spot residues that appear to control STIM1 coupling to Orai1 in a concerted manner. Moreover, we identified two basic residues, which protrude into the elongated pore to redound to Orai1 gating. We suggest that several hot spot residues in the ETON region contribute in aggregate to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate.     

Contributions of the EMERALD project to assessing and improving microarray data quality

While minimum information about a microarray experiment (MIAME) standards have helped to increase the value of the microarray data deposited into public databases like ArrayExpress and Gene Expression Omnibus (GEO), limited means have been available to assess the quality of this data or to identify the procedures used to normalize and transform raw data. The EMERALD FP6 Coordination Action was designed to deliver approaches to assess and enhance the overall quality of microarray data and to disseminate these approaches to the microarray community through an extensive series of workshops, tutorials, and symposia. Tools were developed for assessing data quality and used to demonstrate how the removal of poor-quality data could improve the power of statistical analyses and facilitate analysis of multiple joint microarray data sets. These quality metrics tools have been disseminated through publications and through the software package arrayQualityMetrics. Within the framework provided by the Ontology of Biomedical Investigations, ontology was developed to describe data transformations, and software ontology was developed for gene expression analysis software. In addition, the consortium has advocated for the development and use of external reference standards in microarray hybridizations and created the Molecular Methods (MolMeth) database, which provides a central source for methods and protocols focusing on microarray-based technologies.