Putative functions of extracellular matrix glycoproteins in secondary palate morphogenesis

Cleft palate is a common birth defect in humans. Elevation and fusion of paired palatal shelves are coordinated by growth and transcription factors, and mutations in these can cause malformations. Among the effector genes for growth factor signaling are extracellular matrix (ECM) glycoproteins. These provide substrates for cell adhesion (e.g., fibronectin, tenascins), but also regulate growth factor availability (e.g., fibrillins). Cleft palate in Bmp7 null mouse embryos is caused by a delay in palatal shelf elevation. In contrast, palatal shelves of Tgf-β3 knockout mice elevate normally, but a cleft develops due to their failure to fuse. However, nothing is known about a possible functional interaction between specific ECM proteins and Tgf-β/Bmp family members in palatogenesis. To start addressing this question, we studied the mRNA and protein distribution of relevant ECM components during secondary palate development, and compared it to growth factor expression in wildtypewild type and mutant mice. We found that fibrillin-2 (but not fibrillin-1) mRNA appeared in the mesenchyme of elevated palatal shelves adjacent to the midline epithelial cells, which were positive for Tgf-β3 mRNA. Moreover, midline epithelial cells started expressing fibronectin upon contact of the two palatal shelves. These findings support the hypothesis that fibrillin-2 and fibronectin are involved in regulating the activity of Tgf-β3 at the fusing midline. In addition, we observed that tenascin-W (but not tenascin-C) was misexpressed in palatal shelves of Bmp7-deficient mouse embryos. In contrast to tenascin-C, tenascin-W secretion was strongly induced by Bmp7 in embryonic cranial fibroblasts in vitro. These results are consistent with a putative function for tenascin-W as a target of Bmp7 signaling during palate elevation. Our results indicate that distinct ECM proteins are important for morphogenesis of the secondary palate, both as downstream effectors and as regulators of Tgf-β/Bmp activity.

Drug target identification in intracellular and extracellular protozoan parasites

The increasing demand for novel anti-parasitic drugs due to resistance formation to well-established chemotherapeutically important compounds has increased the demands for a better understanding of the mechanism(s) of action of existing drugs and of drugs in development. While different approaches have been developed to identify the targets and thus mode of action of anti-parasitic compounds, it has become clear that many drugs act not only on one, but possibly several parasite molecules or even pathways. Ideally, these targets are not present in any cells of the host. In the case of apicomplexan parasites, the unique apicoplast, provides a suitable target for compounds binding to DNA or ribosomal RNA of prokaryotic origin. In the case of intracellular pathogens, a given drug might not only affect the pathogen by directly acting on parasite-associated targets, but also indirectly, by altering the host cell physiology. This in turn could affect the parasite development and lead to parasite death. In this review, we provide an overview of strategies for target identification, and present examples of selected drug targets, ranging from proteins to nucleic acids to intermediary metabolism.

Eosinophil extracellular DNA traps: molecular mechanisms and potential roles in disease

Eosinophil extracellular traps (EETs) are part of the innate immune response and are seen in multiple infectious, allergic, and autoimmune eosinophilic diseases. EETs are composed of a meshwork of DNA fibers and eosinophil granule proteins, such as major basic protein (MBP) and eosinophil cationic protein (ECP). Interestingly, the DNA within the EETs appears to have its origin in the mitochondria of eosinophils, which had released most their mitochondrial DNA, but were still viable, exhibiting no evidence of a reduced life span. Multiple eosinophil activation mechanisms are represented, whereby toll-like, cytokine, chemokine, and adhesion receptors can all initiate transmembrane signal transduction processes leading to the formation of EETs. One of the key signaling events required for DNA release is the activation of the NADPH oxidase. Here, we review recent progress made in the understanding the molecular mechanisms involved in DNA and granule protein release, discuss the presence of EETs in disease, speculate on their potential role(s) in pathogenesis, and compare available data on other DNA-releasing cells, particularly neutrophils.

Thymic stromal lymphopoietin stimulates the formation of eosinophil extracellular traps

Thymic stromal lymphopoietin (TSLP) that is released by epithelial cells upon certain environmental triggers activates cells of the innate and adaptive immune system resulting in a preferential T helper 2 immune response. By releasing eosinophil extracellular traps (EETs), eosinophils achieve an efficient extracellular bacterial killing. Eosinophil extracellular traps release, however, has been observed in both infectious and noninfectious eosinophilic diseases. Here, we aim to investigate whether eosinophils generate functional EETs as a direct response to TSLP, and further to study the extra- and intracellular mechanisms involved in this process as well as TSLP receptor (TSLPR) expression by eosinophils in vitro and in vivo.

Cardiac G-protein-coupled receptor kinase 2 ablation induces a novel Ca2+ handling phenotype resistant to adverse alterations and remodeling after myocardial infarction

G-protein-coupled receptor kinase 2 (GRK2) is a primary regulator of β-adrenergic signaling in the heart. G-protein-coupled receptor kinase 2 ablation impedes heart failure development, but elucidation of the cellular mechanisms has not been achieved, and such elucidation is the aim of this study.

Regulation of Cx45 hemichannels mediated by extracellular and intracellular calcium

Connexin45 (Cx45) hemichannels (HCs) open in the absence of Ca(2+) and close in its presence. To elucidate the underlying mechanisms, we examined the role of extra- and intracellular Ca(2+) on the electrical properties of HCs. Experiments were performed on HeLa cells expressing Cx45 using electrical (voltage clamp) and optical (Ca(2+) imaging) methods. HCs exhibit a time- and voltage-dependent current (I(hc)), activating with depolarization and inactivating with hyperpolarization. Elevation of [Ca(2+)](o) from 20 nM to 2 μM reversibly decreases I(hc), decelerates its rate of activation, and accelerates its deactivation. Our data suggest that [Ca(2+)](o) modifies the channel properties by adhering to anionic sites in the channel lumen and/or its outer vestibule. In this way, it blocks the channel pore and reversibly lowers I(hc) and modifies its kinetics. Rapid lowering of [Ca(2+)](o) from 2 mM to 20 nM, achieved early during a depolarizing pulse, led to an outward I(hc) that developed with virtually no delay and grew exponentially in time paralleled by unaffected [Ca(2+)](i). A step increase of [Ca(2+)](i) evoked by photorelease of Ca(2+) early during a depolarizing pulse led to a transient decrease of I(hc) superimposed on a growing outward I(hc); a step decrease of [Ca(2+)](i) elicited by photoactivation of a Ca(2+) scavenger provoked a transient increase in I(hc). Hence, it is tempting to assume that Ca(2+) exerts a direct effect on Cx45 hemichannels.

Transient rhythmic network activity in the somatosensory cortex evoked by distributed input in vitro

The initiation and maintenance of physiological and pathophysiological oscillatory activity depends on the synaptic interactions within neuronal networks. We studied the mechanisms underlying evoked transient network oscillation in acute slices of the adolescent rat somatosensory cortex and modeled its underpinning mechanisms. Oscillations were evoked by brief spatially distributed noisy extracellular stimulation, delivered via bipolar electrodes. Evoked transient network oscillation was detected with multi-neuron patch-clamp recordings under different pharmacological conditions. The observed oscillations are in the frequency range of 2-5 Hz and consist of 4-12 mV large, 40-150 ms wide compound synaptic events with rare overlying action potentials. This evoked transient network oscillation is only weakly expressed in the somatosensory cortex and requires increased [K+]o of 6.25 mM and decreased [Ca2+]o of 1.5 mM and [Mg2+]o of 0.5 mM. A peak in the cross-correlation among membrane potential in layers II/III, IV and V neurons reflects the underlying network-driven basis of the evoked transient network oscillation. The initiation of the evoked transient network oscillation is accompanied by an increased [K+]o and can be prevented by the K+ channel blocker quinidine. In addition, a shift of the chloride reversal potential takes place during stimulation, resulting in a depolarizing type A GABA (GABAA) receptor response. Blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA), N-methyl-D-aspartate (NMDA), or GABA(A) receptors as well as gap junctions prevents evoked transient network oscillation while a reduction of AMPA or GABA(A) receptor desensitization increases its duration and amplitude. The apparent reversal potential of -27 mV of the evoked transient network oscillation, its pharmacological profile, as well as the modeling results suggest a mixed contribution of glutamatergic, excitatory GABAergic, and gap junctional conductances in initiation and maintenance of this oscillatory activity. With these properties, evoked transient network oscillation resembles epileptic afterdischarges more than any other form of physiological or pathophysiological neocortical oscillatory activity.

Spine Ca2+ signaling in spike-timing-dependent plasticity

Calcium is a second messenger, which can trigger the modification of synaptic efficacy. We investigated the question of whether a differential rise in postsynaptic Ca2+ ([Ca2+]i) alone is sufficient to account for the induction of long-term potentiation (LTP) and long-term depression (LTD) of EPSPs in the basal dendrites of layer 2/3 pyramidal neurons of the somatosensory cortex. Volume-averaged [Ca2+]i transients were measured in spines of the basal dendritic arbor for spike-timing-dependent plasticity induction protocols. The rise in [Ca2+]i was uncorrelated to the direction of the change in synaptic efficacy, because several pairing protocols evoked similar spine [Ca2+]i transients but resulted in either LTP or LTD. The sequence dependence of near-coincident presynaptic and postsynaptic activity on the direction of changes in synaptic strength suggested that LTP and LTD were induced by two processes, which were controlled separately by postsynaptic [Ca2+]i levels. Activation of voltage-dependent Ca2+ channels before metabotropic glutamate receptors (mGluRs) resulted in the phospholipase C-dependent (PLC-dependent) synthesis of endocannabinoids, which acted as a retrograde messenger to induce LTD. LTP required a large [Ca2+]i transient evoked by NMDA receptor activation. Blocking mGluRs abolished the induction of LTD and uncovered the Ca2+-dependent induction of LTP. We conclude that the volume-averaged peak elevation of [Ca2+]i in spines of layer 2/3 pyramids determines the magnitude of long-term changes in synaptic efficacy. The direction of the change is controlled, however, via a mGluR-coupled signaling cascade. mGluRs act in conjunction with PLC as sequence-sensitive coincidence detectors when postsynaptic precede presynaptic action potentials to induce LTD. Thus presumably two different Ca2+ sensors in spines control the induction of spike-timing-dependent synaptic plasticity.

Extracellular nucleotides induce migration of renal mesangial cells by upregulating sphingosine kinase-1 expression and activity

BACKGROUND AND PURPOSE: Extracellular nucleotides act as potent mitogens for renal mesangial cells (MC). In this study we determined whether extracellular nucleotides trigger additional responses in MCs and the mechanisms involved. EXPERIMENTAL APPROACH: MC migration was measured after nucleotide stimulation in an adapted Boyden-chamber. Sphingosine kinase-1 (SK-1) protein expression was detected by Western blot analysis and mRNA expression quantified by real-time PCR. SK activity was measured by an in vitro kinase assay using sphingosine as substrate. KEY RESULTS: Nucleotide stimulation caused biphasic activation of SK-1, but not SK-2. The first peak occurred after minutes of stimulation and was followed by a second delayed peak after 4-24 h of stimulation. The delayed activation of SK-1 is due to increased SK-1 mRNA steady-state levels and de novo synthesis of SK-1 protein, and depends on PKC and the classical MAPK cascade. To see whether nucleotide-stimulated cell responses require SK-1, we selectively depleted SK-1 from cells by using small-interference RNA (siRNA). MC migration is highly stimulated by ATP and UTP; this is mimicked by exogenously added S1P. Depletion of SK-1 by siRNA drastically reduced the effect of ATP and UTP on cell migration but not on cell proliferation. Furthermore, MCs isolated from SK-1-deficient mice were completely devoid of nucleotide-induced migration. CONCLUSIONS AND IMPLICATIONS: These data show that extracellular nucleotides besides being mitogenic also trigger MC migration and this cell response critically requires SK-1 activity. Thus, pharmacological intervention of SK-1 may have impacts on situations where MC migration is important such as during inflammatory kidney diseases.