The role of sialic acid in human polyomavirus infections

JC virus (JCV) and BK virus (BKV) are human polyomaviruses that infect approximately 85% of the population worldwide [1,2]. JCV is the underlying cause of the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML), a condition resulting from JCV induced lytic destruction of myelin producing oligodendrocytes in the brain [3]. BKV infection of kidneys in renal transplant recipients results in a gradual loss of graft function known as polyomavirus associated nephropathy (PVN) [4]. Following the identification of these viruses as the etiological agents of disease, there has been greater interest in understanding the basic biology of these human pathogens [5,6]. Recent advances in the field have shown that viral entry of both JCV and BKV is dependent on the ability to interact with sialic acid. This review focuses on what is known about the human polyomaviruses and the role that sialic acid plays in determining viral tropism.     

Cyclic hexapeptides related to somatostatin. Conformational analysis employing 1H-NMR and molecular dynamics

We report the conformational analysis of a series of cyclic hexapeptides related to the hormone somatostatin utilizing 1H NMR spectroscopy and NOE restrained molecular dynamics. The conformational preferences and results from biological analysis of these analogs (previous paper) allow for refinement of the current understanding of the structure-activity relationship of somatostatin. For most of the molecules examined, a beta II' turn about the D-tryptophan-lysine residues, postulated to be required for biological activity, was present. From the NOE restrained molecular dynamics, it can be seen that the turn structure is important for the maintenance of the proper orientation of the side chains of the adjacent phenylalanine, tryptophan and lysine. The biologically active analogs have the side chains of lysine and D-tryptophan extended away from the 18-membered ring in close proximity to each other for a significant portion of the dynamic simulations. Although other conformations are accessible and monitored during the simulations, we believe this is important for biological recognition. The absence of the beta II' turn at the D-tryptophan-lysine disrupts this side chain array producing inactive molecules. The role of the bridging region, the Phe-Pro dipeptide, is to stabilize the beta II' turn and help maintain the proper orientation of the biologically important side chains.     

Reference concentrations of cholecalciferol in animals: a basis for establishing non-target exposure

Abstract

Cholecalciferol (vitamin D₃) is widely used as a vertebrate pesticide in New Zealand. However, cholecalciferol also occurs naturally in animals. Therefore, when trying to determine whether a non-target animal has been exposed to cholecalciferol baits, knowledge of the baseline cholecalciferol concentrations in the animal's plasma and tissue is required. We analysed cattle, sheep, pig, deer, dog and cat plasma and liver samples for the vitamin D₃ metabolite 25-hydroxycholecalciferol (25-OHD), a sensitive biomarker for cholecalciferol. Based on these data and a literature search we present 25-OHD reference ranges. We also examined the literature for 25-OHD concentrations in poisoned animals and compared these to the reference ranges. Where plasma and liver samples have 25-OHD concentrations at least four times higher than our reference ranges it is likely that the animal has been exposed to cholecalciferol baits. 25-OHD concentrations 10 times higher than the reference range indicate ingestion of abnormally high amounts of cholecalciferol.     

End-of-life care issues in advanced dementia

Appropriate management of advanced dementia requires it to be recognised as a terminal condition that needs palliative care. Interventions during this stage should be carefully chosen to ensure the improvement or maintenance of the quality of life of the person with dementia. Advanced care planning is an important aspect of dementia care. Carers and relatives should be educated and encouraged to actively participate in discussions related to artificial nutrition, cardiopulmonary resuscitation (CPR) and other medical interventions.     

Cancer cells regulate biomechanical properties of human microvascular endothelial cells

Metastasis is a key event of malignant tumor progression. The capability to metastasize depends on the ability of the cancer cell to migrate into connective tissue, adhere, and possibly transmigrate through the endothelium. Previously we reported that the endothelium does not generally act as barrier for cancer cells to migrate in three-dimensional extracellular matrices (3D-ECMs). Instead, the endothelium acts as an enhancer or a promoter for the invasiveness of certain cancer cells. How invasive cancer cells diminish the endothelial barrier function still remains elusive. Therefore, this study investigates whether invasive cancer cells can decrease the endothelial barrier function through alterations of endothelial biomechanical properties. To address this, MDA-MB-231 breast cancer cells were used that invade deeper and more numerous into 3D-ECMs when co-cultured with microvascular endothelial cells. Using magnetic tweezer measurements, MDA-MB-231 cells were found to alter the mechanical properties of endothelial cells by reducing endothelial cell stiffness. Using spontaneous bead diffusion, actin cytoskeletal remodeling dynamics were shown to be increased in endothelial cells co-cultured with MDA-MB-231 cells compared with mono-cultured endothelial cells. In addition, knockdown of the α5 integrin subunit in highly transmigrating α5β1(high) cells derived from breast, bladder, and kidney cancer cells abolished the endothelial invasion-enhancing effect comparable with the inhibition of myosin light chain kinase. These results indicate that the endothelial invasion-enhancing effect is α5β1 integrin-dependent. Moreover, inhibition of Rac-1, Rho kinase, MEK kinase, and PI3K reduced the endothelial invasion-enhancing effect, indicating that signaling via small GTPases may play a role in the endothelial facilitated increased invasiveness of cancer cells. In conclusion, decreased stiffness and increased cytoskeletal remodeling dynamics of endothelial cells may account for the breakdown of endothelial barrier function, suggesting that biomechanical alterations are sufficient to facilitate the transmigration and invasion of invasive cancer cells into 3D-ECMs.     

Targeting CAL as a negative regulator of DeltaF508-CFTR cell-surface expression: an RNA interference and structure-based mutagenetic approach

PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated DeltaF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL.CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.     

Anchorage of Vinculin to Lipid Membranes Influences Cell Mechanical Properties

The focal adhesion protein vinculin (1066 residues) can be separated into a 95-kDa head and a 30-kDa tail domain. Vinculin's lipid binding sites localized on the tail, helix 3 (residues 944-978) and the unstructured C-terminal arm (residues 1052-1066, the so-called lipid anchor), influence focal adhesion turnover and are important for cell migration and adhesion. Using magnetic tweezers, we characterized the cell mechanical behavior in mouse embryonic fibroblast (MEF)-vin(−/−) cells transfected with EGFP-linked-vinculin deficient of the lipid anchor (vinΔC, residues 1-1051). MEF-vinΔC cells incubated with fibronectin-coated paramagnetic beads were less stiff, and more beads detached during these experiments compared to MEF-rescue cells. Cells expressing vinΔC formed fewer focal contacts as determined by confocal microscopy. Two-dimensional traction measurements showed that MEF-vinΔC cells generate less force compared to rescue cells. Attenuated traction forces were also found in cells that expressed vinculin with point mutations (R1060 and K1061 to Q) of the lipid anchor that impaired lipid binding. However, traction generation was not diminished in cells that expressed vinculin with impaired lipid binding caused by point mutations on helix 3. Mutating the src-phosphorylation site (Y1065 to F) resulted in reduced traction generation. These observations show that both the lipid binding and the src-phosphorylation of vinculin's C-terminus are important for cell mechanical behavior.     

Chimeric exchanges within the bradykinin B2 receptor intracellular face with the prostaglandin EP2 receptor as the donor: importance of the second intracellular loop for cAMP synthesis

The prostaglandin E2 (PGE(2)) EP2 receptor (EP2R) type is G protein coupled (GPCR) and links to Galphas. Through this receptor PGE(2) activates cAMP production. The bradykinin (BK) B2 receptor (BKB2R) is also a GPCR but links to Galphaq and Galphai and does not activate cAMP production in response to bradykinin. In an attempt to convert the BKB2R into a Galphas-linked adenylate cyclase-activating receptor we proceeded to make global and discrete motif replacements of the intracellular (IC) face of the BKB2R with the corresponding regions of the human EP2R. With this approach we produced hybrid receptors which, when stably transfected into wild type (WT) Rat-1 cells, bound BK but produced cAMP. Replacement of the second loop (IC2), third loop (IC3), the entire C terminus, and the distal C terminus resulted in receptors which bound BK. However, only the IC2 and IC3 exchanges resulted in cAMP-producing receptors. Of these two regions, the IC2 exchange was by far the better cAMP-generating receptor, producing cAMP at approximately 6.6-fold above WT BKB2R or approximately one fourth the amount produced by WT EP2R-transfected Rat-1 cells. Both human and rat EP2R and human beta2-adrenergic receptor exchanges of the IC2 produced equal quantities of cAMP. Focusing on the rBKB2R/hEP2R IC2 chimeras, the region consisting of residues 136-147 (BKB2R residue numbering) proved to contain a cAMP-generating motif. Within this region, the proximal six amino acids from the EP2R (HPYFYQ) at position 136-141 proved crucial for cAMP production (10-fold over WT BKB2R). The distal part of this region, the six residues at 142-147, played no role in cAMP production. On the other hand, the ALV motif of the BKB2R IC2, residues 133-135, proved important with respect to phosphatydilinositol (PI) turnover. Replacing the entire IC2 of BKB2R resulted in poor PI turnover, while including the AVL of BKB2R retained approximately half of the WT PI turnover. With respect to receptor uptake, all the IC2 mutants endocytosed as WT BKB2R (60% in 1h). However, the exchange of the distal and the whole C termini resulted in a marked drop in endocytosis (30% in 1h). These results demonstrate that the construction of a cAMP-producing BKB2/EP2 receptor hybrid is possible, with the IC2 region distal to DRYLALV proving important to Galphas linkage and the LALV motif within the IC2 of BKB2R and the region proximal to it proving important for Galphaq and Galphai linkage. Additionally, our results confirm the importance of the distal C terminus in determining receptor uptake.