The E3 Ubiquitin Ligase MARCH6 Degrades Squalene Monooxygenase and Affects 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A Reductase and the Cholesterol Synthesis Pathway

The mevalonate pathway is used by cells to produce sterol and nonsterol metabolites and is subject to tight metabolic regulation. We recently reported that squalene monooxygenase (SM), an enzyme controlling a rate-limiting step in cholesterol biosynthesis, is subject to cholesterol-dependent proteasomal degradation. However, the E3-ubiquitin (E3) ligase mediating this effect was not established. Using a candidate approach, we identify the E3 ligase membrane-associated RING finger 6 (MARCH6, also known as TEB4) as the ligase controlling degradation of SM. We find that MARCH6 and SM physically interact, and consistent with MARCH6 acting as an E3 ligase, its overexpression reduces SM abundance in a RING-dependent manner. Reciprocally, knockdown of MARCH6 increases the level of SM protein and prevents its cholesterol-regulated degradation. Additionally, this increases cell-associated SM activity but is unexpectedly accompanied by increased flux upstream of SM. Prompted by this observation, we found that knockdown of MARCH6 also controls the level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) in hepatocytes and model cell lines. In conclusion, MARCH6 controls abundance of both SM and HMGCR, establishing it as a major regulator of flux through the cholesterol synthesis pathway.     

IL-17A Induces Pendrin Expression and Chloride-Bicarbonate Exchange in Human Bronchial Epithelial Cells

The epithelium plays an active role in the response to inhaled pathogens in part by responding to signals from the immune system. Epithelial responses may include changes in chemokine expression, increased mucin production and antimicrobial peptide secretion, and changes in ion transport. We previously demonstrated that interleukin-17A (IL-17A), which is critical for lung host defense against extracellular bacteria, significantly raised airway surface pH in vitro, a finding that is common to a number of inflammatory diseases. Using microarray analysis of normal human bronchial epithelial (HBE) cells treated with IL-17A, we identified the electroneutral chloride-bicarbonate exchanger Pendrin (SLC26A4) as a potential mediator of this effect. These data were verified by real-time, quantitative PCR that demonstrated a time-dependent increase in Pendrin mRNA expression in HBE cells treated with IL-17A up to 48 h. Using immunoblotting and immunofluorescence, we confirmed that Pendrin protein expression is increased in IL-17 treated HBE cells and that it is primarily localized to the mucosal surface of the cells. Functional studies using live-cell fluorescence to measure intracellular pH demonstrated that IL-17A induced chloride-bicarbonate exchange in HBE cells that was not present in the absence of IL-17A. Furthermore, HBE cells treated with short interfering RNA against Pendrin showed substantially reduced chloride-bicarbonate exchange. These data suggest that Pendrin is part of IL-17A-dependent epithelial changes and that Pendrin may therefore be a therapeutic target in IL-17A-dependent lung disease.     

Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of life and survival of breast cancer patients. In this paper a new approach for the detection of breast cancer is described, based on tracking the mammary architectural elements using diffusion tensor imaging (DTI). The paper focuses on the scanning protocols and image processing algorithms and software that were designed to fit the diffusion properties of the mammary fibroglandular tissue and its changes during malignant transformation. The final output yields pixel by pixel vector maps that track the architecture of the entire mammary ductal glandular trees and parametric maps of the diffusion tensor coefficients and anisotropy indices. The efficiency of the method to detect breast cancer was tested by scanning women volunteers including 68 patients with breast cancer confirmed by histopathology findings. Regions with cancer cells exhibited a marked reduction in the diffusion coefficients and in the maximal anisotropy index as compared to the normal breast tissue, providing an intrinsic contrast for delineating the boundaries of malignant growth. Overall, the sensitivity of the DTI parameters to detect breast cancer was found to be high, particularly in dense breasts, and comparable to the current standard breast MRI method that requires injection of a contrast agent. Thus, this method offers a completely non-invasive, safe and sensitive tool for breast cancer detection.     

The olfactory receptor gene superfamily: data mining, classification, and nomenclature

The vertebrate olfactory receptor (OR) subgenome harbors the largest known gene family, which has been expanded by the need to provide recognition capacity for millions of potential odorants. We implemented an automated procedure to identify all OR coding regions from published sequences. This led us to the identification of 831 OR coding regions (including pseudogenes) from 24 vertebrate species. The resulting dataset was subjected to neighbor-joining phylogenetic analysis and classified into 32 distinct families, 14 of which include only genes from tetrapodan species (Class II ORs). We also report here the first identification of OR sequences from a marsupial (koala) and a monotreme (platypus). Analysis of these OR sequences suggests that the ancestral mammal had a small OR repertoire, which expanded independently in all three mammalian subclasses. Classification of ``fish-like' (Class I) ORs indicates that some of these ancient ORs were maintained and even expanded in mammals. A nomenclature system for the OR gene superfamily is proposed, based on a divergence evolutionary model. The nomenclature consists of the root symbol `OR', followed by a family numeral, subfamily letter(s), and a numeral representing the individual gene within the subfamily. For example, OR3A1 is an OR gene of family 3, subfamily A, and OR7E12P is an OR pseudogene of family 7, subfamily E. The symbol is to be preceded by a species indicator. We have assigned the proposed nomenclature symbols for all 330 human OR genes in the database. A WWW tool for automated name assignment is provided. Received: / Accepted:     

Plant-feeding and non-plant feeding phytoseiids: differences in behavior and cheliceral morphology

In previous studies plant feeding behavior of plant- and non-plant feeding phytoseiids was never examined directly. Moreover, in these studies the cheliceral morphology of phytoseiids was not associated with their ability to feed on plants. In the present study, we monitored the plant-feeding behavior of Euseius scutalis and Amblyseius swirskii. Only E. scutalis was observed penetrating the leaf surface with the movable digit and feeding. Second, using a dye and coloring the gut as an indicator for feeding, we found that E. scutalis pierced an artificial membrane and fed whereas A. swirskii did not. Finally, to identify morphological characteristics typical of plant feeders versus non-plant feeders, we used scanning electron microscopy to examine the adaxial (inner) profile of the chelicerae in 13 phytoseiid species. The only parameter that distinguished between plant- and non-plant feeders was the ratio of the dorsal perimeter length of the fixed digit to the ventral perimeter length of the movable digit. Plant-feeders were characterized by ratio values greater than one whereas the values for non plant-feeders were lower than one. We suggest that a shorter and less curved movable digit, expressed by a high ratio, will facilitate the penetration of the leaf surface. Cheliceral traits proposed here as typical of plant feeders, were observed for five genera, indicating that plant-feeding may be more common in the Phytoseiidae than previously reported. We propose that the ability to feed on plants be added as a cross type trait of phytoseiid life-style types.     

Temperature trends and recent decline in body size of the stone marten Martes foina in Denmark

We studied a sample of 131 skulls of the stone marten Martes foina that were collected in Denmark between 1858 and 1999. Data were available for 37 years, but collection effort was not uniform throughout the study period and annual sample size varied between 1 and 27. We used principal component analysis (PCA) to combine the information of four skull measurements into a single variable (PC1). PC1 was then corrected for factors that significantly affected it (sex and longitude), and residual PC1 was used for further analysis in which we calculated trends in PC1 values during the study period. During the study period there was an increase in mean annual temperature in Denmark, but this increase was not continuous, as there was slight decrease in temperature between 1947 and 1965.We found that skull size (and by implication body size) of the stone marten in Denmark had two periods of decrease and these two periods coincide with the periods of increase in mean annual temperature. These results may indicate that body size of the stone marten is sensitive to the change in ambient temperature, either due to a change in food availability that was caused by the increase in temperature, or decreased its size in accordance with Bergmann's rule.     

Combinatorial and Computational Approaches to Identify Interactions of Macrophage Colony-stimulating Factor (M-CSF) and Its Receptor c-FMS

The molecular interactions between macrophage colony-stimulating factor (M-CSF) and the tyrosine kinase receptor c-FMS play a key role in the immune response, bone metabolism, and the development of some cancers. Because no x-ray structure is available for the human M-CSF·c-FMS complex, the binding epitope for this complex is largely unknown. Our goal was to identify the residues that are essential for binding of the human M-CSF to c-FMS. For this purpose, we used a yeast surface display (YSD) approach. We expressed a combinatorial library of monomeric M-CSF (M-CSF_M) single mutants and screened this library to isolate variants with reduced affinity for c-FMS using FACS. Sequencing yielded a number of single M-CSF_M variants with mutations both in the direct binding interface and distant from the binding site. In addition, we used computational modeling to map the identified mutations onto the M-CSF_M structure and to classify the mutations into three groups as follows: those that significantly decrease protein stability; those that destroy favorable intermolecular interactions; and those that decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSF_M and three variants were produced as soluble proteins; their affinity and structure were analyzed; and very good correlations with both YSD data and computational predictions were obtained. By identifying the M-CSF_M residues critical for M-CSF·c-FMS interactions, we have laid down the basis for a deeper understanding of the M-CSF·c-FMS signaling mechanism and for the development of target-specific therapeutic agents with the ability to sterically occlude the M-CSF·c-FMS binding interface.