The relation between cartilage biomarkers (C2C,C1,2C,CS846, and CPII) and the long-term outcome of rheumatoid arthritis patients within the CAMERA trial

Introduction  

The aim of this study was to investigate whether serum biomarker levels of C2C, C1,2C, CS846, and CPII can predict the long-term course of disease activity and radiographic progression early in the disease course of rheumatoid arthritis (RA).     

Membrane Trafficking of Large Conductance Calcium-activated Potassium Channels Is Regulated by Alternative Splicing of a Transplantable, Acidic Trafficking Motif in the RCK1-RCK2 Linker

Trafficking of the pore-forming α-subunits of large conductance calcium- and voltage-activated potassium (BK) channels to the cell surface represents an important regulatory step in controlling BK channel function. Here, we identify multiple trafficking signals within the intracellular RCK1-RCK2 linker of the cytosolic C terminus of the channel that are required for efficient cell surface expression of the channel. In particular, an acidic cluster-like motif was essential for channel exit from the endoplasmic reticulum and subsequent cell surface expression. This motif could be transplanted onto a heterologous nonchannel protein to enhance cell surface expression by accelerating endoplasmic reticulum export. Importantly, we identified a human alternatively spliced BK channel variant, hSloΔ_579–664, in which these trafficking signals are excluded because of in-frame exon skipping. The hSloΔ_579–664 variant is expressed in multiple human tissues and cannot form functional channels at the cell surface even though it retains the putative RCK domains and downstream trafficking signals. Functionally, the hSloΔ_579–664 variant acts as a dominant negative subunit to suppress cell surface expression of BK channels. Thus alternative splicing of the intracellular RCK1-RCK2 linker plays a critical role in determining cell surface expression of BK channels by controlling the inclusion/exclusion of multiple trafficking motifs.     

Increased large conductance calcium-activated potassium (BK) channel expression accompanied by STREX variant downregulation in the developing mouse CNS

Background  

Large conductance calcium- and voltage activated potassium (BK) channels are important determinants of neuronal excitability through effects on action potential duration, frequency and synaptic efficacy. The pore- forming subunits are encoded by a single gene, KCNMA1, which undergoes extensive alternative pre mRNA splicing. Different splice variants can confer distinct properties on BK channels. For example, insertion of the 58 amino acid stress-regulated exon (STREX) insert, that is conserved throughout vertebrate evolution, encodes channels with distinct calcium sensitivity and regulation by diverse signalling pathways compared to the insertless (ZERO) variant. Thus, expression of distinct splice variants may allow cells to differentially shape their electrical properties during development. However, whether differential splicing of BK channel variants occurs during development of the mammalian CNS has not been examined.     

Distinct acyl protein transferases and thioesterases control surface expression of calcium-activated potassium channels

Protein palmitoylation is rapidly emerging as an important determinant in the regulation of ion channels, including large conductance calcium-activated potassium (BK) channels. However, the enzymes that control channel palmitoylation are largely unknown. Indeed, although palmitoylation is the only reversible lipid modification of proteins, acyl thioesterases that control ion channel depalmitoylation have not been identified. Here, we demonstrate that palmitoylation of the intracellular S0-S1 loop of BK channels is controlled by two of the 23 mammalian palmitoyl-transferases, zDHHC22 and zDHHC23. Palmitoylation by these acyl transferases is essential for efficient cell surface expression of BK channels. In contrast, depalmitoylation is controlled by the cytosolic thioesterase APT1 (LYPLA1), but not APT2 (LYPLA2). In addition, we identify a splice variant of LYPLAL1, a homolog with ～30% identity to APT1, that also controls BK channel depalmitoylation. Thus, both palmitoyl acyltransferases and acyl thioesterases display discrete substrate specificity for BK channels. Because depalmitoylated BK channels are retarded in the trans-Golgi network, reversible protein palmitoylation provides a critical checkpoint to regulate exit from the trans-Golgi network and thus control BK channel cell surface expression.     

Hierarchal type III secretion of translocators and effectors from Escherichia coli O157:H7 requires the carboxy terminus of SepL that binds to Tir

Type III secretion (T3S) from enteric bacteria is a co-ordinated process with a hierarchy of secreted proteins. In enteropathogenic and enterohaemorrhagic Escherichia coli , SepL and SepD are essential for translocator but not effector protein export, but how they function to control this differential secretion is not known. This study has focused on the different activities of SepL including membrane localization, SepD binding, EspD export and Tir secretion regulation. Analyses of SepL truncates demonstrated that the different functions associated with SepL can be separated. In particular, SepL with a deletion of 11 amino acids from the C-terminus was able to localize to the bacterial membrane, export translocon proteins but not regulate Tir or other effector protein secretion. From the repertoire of effector proteins only Tir was shown to bind directly to full-length SepL and the C-terminal 48 amino acids of SepL was sufficient to interact with Tir. By synchronizing induction of T3S, it was evident that the Tir-binding capacity of SepL is important to delay the release of effector proteins while the EspADB translocon is secreted. The interaction between Tir and SepL is therefore a critical step that controls the timing of T3S in attaching and effacing pathogens.     

Dex-ras1 and Serum- and Glucocorticoid-inducible Protein Kinase 1: Regulation of Expression by Dexamethasone in HEK293 Cells

The molecular and cellular basis of the psychotropic actions of adrenal corticosteroids is poorly understood. Previously, we reported that modulation of large conductance Ca²⁺-activated potassium channel (BK-channel) function by glucocorticoids can be recapitulated in human embryonic kidney293 (HEK293) cells (J Physiol 537:57, 2001). In the present paper, we examined the effect of dexamethasone on the expression of candidate mediator proteins of glucocorticoid action, dex-ras1 and serum and glucocorticoid inducible protein kinase 1 (SGK), in HEK293 cells. Dex-ras1 mRNA was readily detectable under basal conditions however, no changes of dex-ras1 mRNA expression occurred upon exposure to 100 nM of dexamethasone for 2 h. In contrast, a 2.5-fold increase of SGK mRNA was found under similar conditions. Total levels of cellular SGK protein were unaltered upon exposure to dexamethasone, but a marked increase of SGK in a Triton-X100 insoluble fraction was observed. BK-channel α-subunits could not be co-immunoprecipitated with SGK. In summary, SGK, but not dex-ras1, mRNA is rapidly induced by glucocorticoid stimulation in HEK293 cells. However, there appears to be no direct protein-protein interaction between SGK and BK-channel α-subunits. Presented to mark the 70th birthday of Professor George Fink. Special issue article in honor of George Fink.     

Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based ‘lab on a chip’ technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO₂ wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO₂ regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.     

Effect of temperature on seed production in the invasive grass Glyceria maxima (Hartm.) Holmb. (Poaceae) in South Africa

Temperature is one of the main factors that determine sexual reproduction in terrestrial and emergent aquatic plant species. The effect of temperature on sexual reproduction and seed production of Glyceria maxima (Hartm.) Holmb. in the southern hemisphere is unknown. Glyceria maxima collections in February 2010 at three isolated infestations in KwaZulu-Natal failed to yield a single seed, only empty panicles. Laboratory experiments showed that vernalisation had no consistent effect on seed production. Field- and laboratory-grown plants produced seeds in the 2010/2011 season, because of having sufficient time at optimum temperatures required for seed production (1 491 and 1 585 hours, respectively), compared to a shorter period (1 352 hours) of suitable temperatures during the 2009/2010 growing season. An inadequate period of optimum temperatures (15–25°C) during seed production resulted in the lack of seeds in the field in the 2009/2010 growing season. This study showed that temperature and duration of exposure thereto during the seed-production period play vital roles in G. maxima sexual reproduction.