CaMKK-CaMK1a,a New Post-Traumatic Signalling Pathway Induced in Mouse Somatosensory Neurons

Neurons innervating peripheral tissues display complex responses to peripheral nerve injury. These include the activation and suppression of a variety of signalling pathways that together influence regenerative growth and result in more or less successful functional recovery. However, these responses can be offset by pathological consequences including neuropathic pain. Calcium signalling plays a major role in the different steps occurring after nerve damage. As part of our studies to unravel the roles of injury-induced molecular changes in dorsal root ganglia (DRG) neurons during their regeneration, we show that the calcium calmodulin kinase CaMK1a is markedly induced in mouse DRG neurons in several models of mechanical peripheral nerve injury, but not by inflammation. Intrathecal injection of NRTN or GDNF significantly prevents the post-traumatic induction of CaMK1a suggesting that interruption of target derived factors might be a starter signal in this de novo induction. Inhibition of CaMK signalling in injured DRG neurons by pharmacological means or treatment with CaMK1a siRNA resulted in decreased velocity of neurite growth in vitro. Altogether, the results suggest that CaMK1a induction is part of the intrinsic regenerative response of DRG neurons to peripheral nerve injury, and is thus a potential target for therapeutic intervention to improve peripheral nerve regeneration.     

Improved Diagnosis of the Transition to JAK2 V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms

Most cases of BCR-ABL1-negative myeloproliferative neoplasms (MPNs), essential thrombocythemia, polycythemia vera and primary myelofibrosis are associated with JAK2 ^V617F mutations. The outcomes of these cases are critically influenced by the transition from JAK2 ^V617F heterozygosity to homozygosity. Therefore, a technique providing an unbiased assessment of the critical allele burden, 50% JAK2 ^V617F, is highly desirable. In this study, we present an approach to assess the JAK2 ^V617F burden from genomic DNA (gDNA) and complementary DNA (cDNA) using one-plus-one template references for allele-specific quantitative-real-time-PCR (qPCR). Plasmidic gDNA and cDNA constructs encompassing one PCR template for JAK2 ^V617F spaced from one template for JAK2^{Wild Type} were constructed by multiple fusion PCR amplifications. Repeated assessments of the 50% JAK2^V617F burden within the dynamic range of serial dilutions of gDNA and cDNA constructs resulted in 52.53±4.2% and 51.46±4.21%, respectively. The mutation-positive cutoff was estimated to be 3.65% (mean +2 standard deviation) using 20 samples from a healthy population. This qPCR approach was compared with the qualitative ARMS-PCR technique and with two standard methods based on qPCR, and highly significant correlations were obtained in all cases. qPCR assays were performed on paired gDNA/cDNA samples from 20 MPN patients, and the JAK2 ^V617F expression showed a significant correlation with the allele burden. Our data demonstrate that the qPCR method using one-plus-one template references provides an improved assessment of the clinically relevant transition of JAK2 ^V617F from heterozygosity to homozygosity.     

Rearrangements in the Relative Orientation of Cytoplasmic Domains Induced by a Membrane-anchored Protein Mediate Modulations in Kv Channel Gating

Interdomain interactions between intracellular N and C termini have been described for various K⁺ channels, including the voltage-gated Kv2.1, and suggested to affect channel gating. However, no channel regulatory protein directly affecting N/C interactions has been demonstrated. Most Kv2.1 channel interactions with regulatory factors occur at its C terminus. The vesicular SNARE that is also present at a high concentration in the neuronal plasma membrane, VAMP2, is the only protein documented to affect Kv2.1 gating by binding to its N terminus. As its binding target has been mapped near a site implicated in Kv2.1 N/C interactions, we hypothesized that VAMP2 binding to the N terminus requires concomitant conformational changes in the C terminus, which wraps around the N terminus from the outside, to give VAMP2 access. Here, we first determined that the Kv2.1 N terminus, although crucial, is not sufficient to convey functional interaction with VAMP2, and that, concomitant to its binding to the “docking loop” at the Kv2.1 N terminus, VAMP2 binds to the proximal part of the Kv2.1 C terminus, C1a. Next, using computational biology approaches (ab initio modeling, docking, and molecular dynamics simulations) supported by molecular biology, biochemical, electrophysiological, and fluorescence resonance energy transfer analyses, we mapped the interaction sites on both VAMP2 and Kv2.1 and found that this interaction is accompanied by rearrangements in the relative orientation of Kv2.1 cytoplasmic domains. We propose that VAMP2 modulates Kv2.1 inactivation by interfering with the interaction between the docking loop and C1a, a mechanism for gating regulation that may pertain also to other Kv channels.Interdomain interactions between intracellularly located N and C termini have been described for various K⁺ channels, including inwardly rectifying Kir2.3 and Kir6.2 (1, 2), small conductance Ca²⁺-activated (hSK3) (3), and voltage-gated Kv2.1 (4) and Kv4.1 (5) channels. In the case of Kv2.1, two modes of interaction have been proposed: an association of the distal part of Kv2.1 C terminus (termed CTA domain; amino acids (aa) 741–853)⁴ with aa 67 and 75 of the Kv2.1 N terminus (4); or an association between the proximal part of the Kv2.1 C terminus (aa 444–477) and the predicted loop structure (aa 55–71) in the N-terminal T1 domain (6). In addition, involvement of the S4-S5 linker in this interaction has been suggested (7). Although these studies propose two different C-terminal sites, they indicate a specific loop in the N terminus of Kv2.1 (6, 8), which could be functionally related to the Shaker and Shal docking loops in the lateral part of their T1 domains (9, 10). These latter loops are responsible for the subfamily-specific association with β-subunits (Kvβ and KChIP, respectively). Further, the interaction between the N and C cytoplasmic termini (N/C interaction) of Kv2.1 has been shown to be dynamic and voltage-dependent and to involve structural rearrangements between these domains, which could affect both activation and inactivation gating of the channel (4, 6, 7). These rearrangements can be clearly detected with fluorescence resonance energy transfer (FRET) (11). A similar N/C interaction has been shown to affect gating of the closely related Kv4.1 channel (5, 12).It is conceivable that the specific packaging of Kv2.1 cytoplasmic termini (a relatively long C terminus (>400 aa) wrapping the N terminus (<190 aa) from the outside (4)) not only supports multiple interactions between the termini but also reflects the fact that most of the interactions of the channel with intracellular and membrane-bound regulatory factors occur at the C terminus, including channel phosphorylation (13–15), clustering through a unique proxinal restriction and clustering signal (16), and protein-protein interactions with both the plasma membrane SNAREs, syntaxin 1A and SNAP-25 (17–19), and the MiRP2 (KCNE3) peptide (20). For the Kv2.1 N terminus, on the other hand, there are only two examples of protein-protein interactions: a transient association with KChAP (21), which does not affect channel function; and an interaction with the vesicular SNARE partner VAMP2 (synaptobrevin 2), which is also present at a high concentration in the neuronal plasma membrane and enhances channel inactivation (8). Specifically, VAMP2 has been shown to associate with the extension of a docking loop in the lateral part of the T1 domain (8) near the site of interaction with the C terminus (4, 6). Thus, it is reasonable to hypothesize that interaction with VAMP2 will affect the N/C interaction, similar to proton-mediated Kir2.3 (1) and Kir1.1 (22) N/C interactions or the ATP-dependent Kir6.2 (2) N/C interaction. To date, no protein molecule that directly affects N/C interactions in a K⁺ channel has been demonstrated. Because VAMP2 was the first protein documented to affect Kv2.1 channel gating by binding to a specific N-terminal site, which is probably masked by the C terminus, we have put forward the idea that its interaction with the Kv2.1 N terminus requires conformational changes in the C terminus that will enable its access to the N terminus.Here we endeavored to gain a mechanistic and structural understanding of the Kv2.1-VAMP2 interaction. Based on our evidence, we propose that VAMP2 modulates Kv2.1 gating by interfering with the Kv2.1 cytoplasmic N/C interaction.     

GALNT3, a gene associated with hyperphosphatemic familial tumoral calcinosis, is transcriptionally regulated by extracellular phosphate and modulates matrix metalloproteinase activity

GALNT3 encodes UDP-N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyl-transferarase 3 (ppGalNacT3), a glycosyltransferase which has been suggested to prevent proteolysis of FGF23, a potent phosphaturic protein. Accordingly, loss-of-function mutations in GALNT3 cause hyperphosphatemic familial tumoral calcinosis (HFTC), a rare autosomal recessive disorder manifesting with increased kidney reabsorption of phosphate, resulting in severe hyperphosphatemia and widespread ectopic calcifications. Although these findings definitely attribute a role to ppGalNacT3 in the regulation of phosphate homeostasis, little is currently known about the factors regulating GALNT3 expression. In addition, the effect of decreased GALNT3 expression in peripheral tissues has not been explored so far. In the present study, we demonstrate that GALNT3 expression is under the regulation of a number of factors known to be associated with phosphate homeostasis, including inorganic phosphate itself, calcium and 1,25-dihydroxyvitamin D(3). In addition, we show that decreased GALNT3 expression in human skin fibroblasts leads to increased expression of FGF7 and of matrix metalloproteinases, which have been previously implicated in the pathogenesis of ectopic calcification. Thus, the present data suggest that ppGalNacT3 may play a role in peripheral tissues of potential relevance to the pathogenesis of disorders of phosphate metabolism.     

Evaluation of clonality in enterococci isolated in Brazil carrying Tn1546-like elements associated with vanA plasmids

Fifty-one vancomycin-resistant enterococci samples isolated from different geographic regions in Brazil were studied. All the isolates harboured the vanA gene as demonstrated by PCR analysis, and in a majority of strains the gene was associated with a transferable plasmid of 70 kb. A single variant of the prototype Tn1546 associated with common transferable vanA-containing plasmids has spread among the enterococcal strains circulating in Brazil. The VanA element integrity in these enterococci strains and the different pulsed-field gel electrophoresis patterns suggest horizontal transmission of the vancomycin resistance transposon in Brazilian strains.     

CXCL8-induced FAK phosphorylation via CXCR1 and CXCR2: cytoskeleton- and integrin-related mechanisms converge with FAK regulatory pathways in a receptor-specific manner

CXCL8 is a potent chemokine, inducing focal adhesion kinase (FAK) phosphorylation, and migration via a FAK-mediated pathway. Since, unlike growth factors, chemokines directly control integrins and cytoskeleton rearrangements, we determined whether these elements regulate CXCL8-induced FAK phosphorylation. The analysis intentionally dissociated between the CXCL8 receptors CXCR1 and CXCR2. In both CXCR1- and CXCR2-expressing cells, actin and microtubules were required for CXCL8-induced FAK phosphorylation, and CXCL8-induced cell spreading was accompanied by concordant re-localization of FAK with actin and beta-tubulin. The phosphorylation of five FAK sites depended on intact actin filaments and microtubules. While in CXCR2-expressing cells FAK phosphorylation was adhesion-dependent and was stimulated by fibronectin, in CXCR1-expressing cells FAK phosphorylation was adhesion-independent. Of note, even in the absence of integrin stimulation, the CXCL8-induced phosphorylation of FAK in CXCR1-expressing cells required cytoskeletal elements. CXCL8-induced migration in both cell types was highly reliant on actin filaments, but only the migration of CXCR1-expressing cells was fully dependent on microtubules. Overall, several aspects of CXCL8-induced FAK phosphorylation and migration are regulated in a receptor-specific manner. These observations lay the basis for future investigation of the equilibrium between CXCR1 and CXCR2 in cells expressing both receptors together, such as neutrophils, endothelial cells and tumor cells.     

DIVERSIFICATION OF THE AFRICAN GENUS PROTEA (PROTEACEAE) IN THE CAPE BIODIVERSITY HOTSPOT AND BEYOND: EQUAL RATES IN DIFFERENT BIOMES

The Cape region of South Africa is a hotspot of flowering plant biodiversity. However, the reasons why levels of diversity and endemism are so high remain obscure. Here, we reconstructed phylogenetic relationships among species in the genus Protea, which has its center of species richness and endemism in the Cape, but also extends through tropical Africa as far as Eritrea and Angola. Contrary to previous views, the Cape is identified as the ancestral area for the radiation of the extant lineages: most species in subtropical and tropical Africa are derived from a single invasion of that region. Moreover, diversification rates have been similar within and outside the Cape region. Migration out of the Cape has opened up vast areas, but those lineages have not diversified as extensively at fine spatial scales as lineages in the Cape. Therefore, higher net rates of diversification do not explain the high diversity and endemism of Protea in the Cape. Instead, understanding why the Cape is so diverse requires an explanation for how Cape species are able to diverge and persist at such small spatial scales.     

Developmentally regulated lectin from embryonic chick pectoral muscle. Purification by affinity chromatography.

A lectin, whose specific activity in soluble extracts of embryonic chick pectoral muscle increases strikingly between 8 and 16 days of development, has been purified by affinity chromatography on derivatized Sepharose 4B coupled to p-aminophenyl-beta-D-lactoside. After affinity chromatography the lectin is pure except for minor contamination with another protein possibly representing a second muscle lectin. The latter can be completely removed by preparative isoelectric focusing. The purified lectin has an apparent molecular weight of 30,000 and an apparent subunit molecular weight of 15,000. Its isoelectric point is 4.0. The most potent saccharide inhibitors tested were thiodigalactoside and lactose. An antibody has been raised to the pure lectin. Studies with this antibody indicate that the lectin is present both on the surface of and within myoblasts.     

Syntaxin 1A binds to the cytoplasmic C terminus of Kv2.1 to regulate channel gating and trafficking

Voltage-gated K(+) (Kv) 2.1 is the dominant Kv channel that controls membrane repolarization in rat islet beta-cells and downstream insulin exocytosis. We recently showed that exocytotic SNARE protein SNAP-25 directly binds and modulates rat islet beta-cell Kv 2.1 channel protein at the cytoplasmic N terminus. We now show that SNARE protein syntaxin 1A (Syn-1A) binds and modulates rat islet beta-cell Kv2.1 at its cytoplasmic C terminus (Kv2.1C). In HEK293 cells overexpressing Kv2.1, we observed identical effects of channel inhibition by dialyzed GST-Syn-1A, which could be blocked by Kv2.1C domain proteins (C1: amino acids 412-633, C2: amino acids 634-853), but not the Kv2.1 cytoplasmic N terminus (amino acids 1-182). This was confirmed by direct binding of GST-Syn-1A to the Kv2.1C1 and C2 domains proteins. These findings are in contrast to our recent report showing that Syn-1A binds and modulates the cytoplasmic N terminus of neuronal Kv1.1 and not by its C terminus. Co-expression of Syn-1A in Kv2.1-expressing HEK293 cells inhibited Kv2.1 surfacing, which caused a reduction of Kv2.1 current density. In addition, Syn-1A caused a slowing of Kv2.1 current activation and reduction in the slope factor of steady-state inactivation, but had no affect on inactivation kinetics or voltage dependence of activation. Taken together, SNAP-25 and Syn-1A mediate secretion not only through its participation in the exocytotic SNARE complex, but also by regulating membrane potential and calcium entry through their interaction with Kv and Ca(2+) channels. In contrast to Ca(2+) channels, where these SNARE proteins act on a common synprint site, the SNARE proteins act not only on distinct sites within a Kv channel, but also on distinct sites between different Kv channel families.