Calcium Calmodulin Research Articles

Lens GABA receptors are a target of GABA-related agonists that mitigate experimental myopia

Coordinated growth of eye tissues is required to achieve visual acuity. However, visual experience also guides this process. Experimental myopia can be produced by altering light entering the eye, but also by changing light/dark regimens. Drug discovery studies demonstrated that γ-aminobutyric acid (GABA)-related agonists (e.g., baclofen) will mitigate experimental myopia, and are also drugs studied for their capacity to affect neurodevelopmental disorders that include Fragile X Syndrome and related autism spectrum disorders. GABA receptors thought to mediate these responses in the eye have been studied in the neural retina as well as the cornea and sclera which are both innervated tissues. In addition to neurons, lenses express GAD25/65/67 GABA metabolic enzymes and at least 13 GABA receptor subunits with developmental expression profiles that match neural development. Evidence that lens GABA receptors are expressed in a cell environment comparable to neurons is seen in the lens expression of AMPA and NMDA glutamate receptors together with an unexpectedly comprehensive array of associated signaling proteins that include post-synaptic-density 95 (PSD95), calcium calmodulin kinase IIα (CaMKIIα), Fragile X Syndrome mental retardation protein (FMRP), ephrin receptors, Ca(V)1.2, 1.3 channels, cyclin-dependent kinase 5 (Cdk5), and neuronal C-src among others. Moreover, lens cells share fundamental molecular regulatory mechanisms that integrate the regulation and function of these genes at the DNA, RNA, and protein levels in neurons. GABA has trophic, growth promoting effects early in neuron development and later assumes its classic inhibitory role in the adult neural system. We hypothesize that the extensive parallels between GABA and glutamate receptor biology in lens and brain identifies the lens as a site of GABA agonist drug action affecting experimental myopia, acting through lens GABA receptors to similarly affect growth in both elongated cell types.

Antineuronal antibodies in a heterogeneous group of youth and young adults with tics and obsessive-compulsive disorder.

Antineuronal antibodies have been implicated in tic and obsessive compulsive disorders (OCD) associated with group A streptococcal infections. We investigated antineuronal autoantibody levels as well as antibody-mediated neuronal cell signaling activity, as previously reported for Sydenham chorea and pediatric autoimmune neuropsychiatric disorder associated with streptococci (PANDAS), to determine immunological profiles for a large cohort of children with tics and/or OCD. Study participants (n=311; ages 4-27 years, 66% male) were selected from a larger group of individuals with self-reported neuropsychiatric symptoms (n=742) and included only those with accurate knowledge of group A streptococcal infection status, except for four individuals in whom streptococcal infection status was unknown. Healthy control samples (n=16; ages 5-14 years, 81% male), came from the National Institute of Mental Health and Yale University. In addition to serum donations, participants and/or legal guardians provided neuropsychiatric and related medical histories of symptoms that had lasted >1 year. Antineuronal immunoglobulin G (IgG) titers were measured by standard enzyme-linked immunosorbent assay (ELISA) and compared with mean titers of normal age-matched sera against lysoganglioside, tubulin, and dopamine receptors (D1R and D2R). Antibody-mediated signaling of calcium calmodulin dependent protein kinase II (CaMKII) activity in a human neuronal cell line (SK-N-SH) was tested in serum. Of 311 individuals, 222 (71%) had evidence of group A streptococcal infection, which was associated with tics and/or OCD status (p=0.0087). Sera from individuals with tics and/or OCD (n=261) had evidence of elevated serum IgG antibodies against human D1R (p<0.0001) and lysoganglioside (p=0.0001), and higher serum activation of CaMKII activity (p<0.0001) in a human neuronal cell line compared with healthy controls (n=16). Furthermore, patients with tics and OCD had significantly increased activation of CaMKII activity compared with patients with only tics or only OCD (p<0.033 for each). Our study suggested a significant correlation of streptococcal-associated tics and OCD with elevated anti-D1R and antilysoganglioside antineuronal antibodies in serum concomitant with higher activation of CaMKII in human neuronal cells. Youth and young adults with chronic tics and OCD may have underlying infectious/immunologic etiology.

Sublethal toxicity of chlorpyrifos to salmonid olfaction after hypersaline acclimation

Salmonid habitats can be impacted by several environmental factors, such as salinization, which can also affect salmonid tolerance to anthropogenic stressors, such as pesticides. Previous studies have shown that hypersaline acclimation enhances the acute toxicity of certain organophosphate and carbamate pesticides to euryhaline fish; however, sublethal impacts have been far less studied. The current study aims to determine how hypersaline acclimation and exposure to the organophosphate chlorpyrifos (CPF) impact salmonid olfaction. Combined acclimation and exposure to CPF was shown to impact rainbow trout olfaction at the molecular, physiological, and behavioral levels. Concurrent exposure to hypersalinity and 0.5μg/L CPF upregulated four genes (chloride intracellular channel 4, G protein zgc:101761, calcium calmodulin dependent protein kinase II delta, and adrenergic alpha 2C receptor) that inhibit olfactory signal transduction. At the physiological level, hypersalinity and chlorpyrifos caused a decrease in sensory response to the amino acid l-serine and the bile salt taurocholic acid. Combined acclimation and exposure also negatively impacted behavior and reduced the avoidance of a predator cue (l-serine). Thus, acclimation to hypersaline conditions and exposure to environmentally relevant concentrations of chlorpyrifos caused an inhibition of olfactory signal transduction leading to a decreased response to odorants and impairment of olfactory mediated behaviors.

“Moonlighting” GAPDH Protein Localizes with AMPA Receptor GluA2 and L1 Axonal Cell Adhesion Molecule at Fiber Cell Borders in the Lens

Purpose: The canonical role of glyceraldehyde phosphate dehydrogenase (GAPDH) is as an enzyme in glycolysis. GAPDH is also a principal “moonlighting” protein with additional roles at diverse sites in a variety of cells. Surface GAPDH on mammalian, yeast, and bacterial cells acts as a receptor and also mediates cell contacts. In neurons, extracellular GAPDH localizes at synapses. Two GAPDH binding partners at synapses are α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor (AMPA) GluA2 subunit at dendritic spines and L1 cell adhesion molecule at pre-synaptic membranes, and both proteins are also expressed in lenses. Fiber cell membrane protrusions and dendritic spines have similar size, shape, and spacing, contain F-actin, and express clathrin/AP-2 Adaptor at their surfaces linked with Tyr-phosphatase STEP-regulated endocytosis of AMPA/GluA2 receptors. AMPA receptors work with NMDA (N-methyl-d-aspartate) and GABA (γ-aminobutyric acid) receptors, calcium calmodulin kinase II (CaMKIIα), channel proteins, STEP, and ephrin receptors, which are also expressed in lenses. In neurons, coordinate AMPA/GluA2 receptor endocytosis with GAPDH is linked with disease. GAPDH was previously characterized as a fiber cell membrane protein and shown to decrease substantially in interior fiber cells in human age-related cataract. Here, we examined GAPDH spatial expression in healthy lenses in two vertebrate species.Methods: In situ methods were used to examine GAPDH expression in lenses of healthy young adult rabbits and chickens. Immunoblots were used to detect L1 in lenses.Results: The present study demonstrated that GAPDH is present at fiber cell borders in adult rabbit and chicken lenses with evidence of focal concentrations along the fiber cell perimeter, and overlapped with detection of p-Tyr-GluA2, L1, STEP, actin and clathrin. We observed that L1-140 kDa was the prominent form in lens.Conclusions: Our findings indicate investigations into GAPDH “moonlighting” activities similar to its role in cell–cell interactions at neuron surfaces are warranted in the lens.

Inhibition of protein kinase (PK) Cδ attenuates methamphetamine-induced dopaminergic toxicity via upregulation of phosphorylation of tyrosine hydroxylase at Ser40 by modulation of protein phosphatase 2A and PKA.

Recently, we proposed that inhibition of protein kinase (PK) Cδ may be a useful target for protection against methamphetamine (MA)-induced dopaminergic toxicity. We demonstrated that treatment with MA resulted in a significant decrease in phosphorylation of tyrosine hydroxylase (TH) at Ser(40) in the striatum, but not in the phosphorylation of TH at Ser(31) . In the present study, treatment with rottlerin (1.5 or 3.0 μg, i.c.v, once a day for 5 days), a PKCδ inhibitor, or a PKCδ antisense oligonucleotide (ASO; 2.5 μg/μl, i.c.v., 3 times) significantly attenuated MA-induced reductions in the phosphorylation of TH at Ser(40) and in the expression of PKA in the striatum of mice. This attenuation was significantly counteracted by H89 (10 or 30 ng, i.c.v., 1 h after the last MA administration), a PKA inhibitor. Treatment with rottlerin or ASO significantly attenuated the MA-induced increase in protein phosphatase (PP) 2A activity. FTY720 (1 or 5 mg/kg, i.p., 1 h after the last MA administration), a PP2A activator, significantly reversed the recovery in TH phosphorylation mediated by inhibition of PKCδ after MA treatment. Both H89 and FTY720 counteracted the recovery of MA-induced behavioural impairments induced by PKCδ inhibition. The effects, mediated by rottlerin or ASO in MA-treated wild-type mice were comparable with those in MA-treated PKCδ(-/-) mice. However, neither inhibition of the mitogen-activated protein kinase subfamily (extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38) nor inhibition of calcium calmodulin kinase II significantly altered PKCδ inhibition-mediated attenuation of MA-induced impairment of TH phosphorylation. The results suggest that genetic or pharmacological inhibition of PKCδ requires modulation of PKA expression and/or PP2A activity to attenuate the impairment of TH phosphorylation at Ser(40) and behavioural activity induced by MA.

Tetrahydrohyperforin: a neuroprotective modified natural compound against Alzheimer's disease.

According to the World Health Organization (WHO), a total of 35.6 million cases of dementia were estimated in 2010, with close to 7.7 million new cases each year. In 2008, WHO declared dementia a priority condition. 90% of all dementia cases are considered to be Alzheimer's disease (AD). Therefore, great effort was made to understand the etiology of the disease and stop or slow down AD progression.

Distinct action of the α-glucosidase inhibitor miglitol on SGLT3, enteroendocrine cells, and GLP1 secretion.

Oral ingestion of carbohydrate triggers glucagon-like peptide 1 (GLP1) secretion, but the molecular mechanism remains elusive. By measuring GLP1 concentrations in murine portal vein, we found that the ATP-sensitive K+ (KATP) channel is not essential for glucose-induced GLP1 secretion from enteroendocrine L cells, while the sodium-glucose co-transporter 1 (SGLT1) is required, at least in the early phase (5 min) of secretion. By contrast, co-administration of the α-glucosidase inhibitor (α-GI) miglitol plus maltose evoked late-phase secretion in a glucose transporter 2-dependent manner. We found that GLP1 secretion induced by miglitol plus maltose was significantly higher than that by another α-GI, acarbose, plus maltose, despite the fact that acarbose inhibits maltase more potently than miglitol. As miglitol activates SGLT3, we compared the effects of miglitol on GLP1 secretion with those of acarbose, which failed to depolarize the Xenopus laevis oocytes expressing human SGLT3. Oral administration of miglitol activated duodenal enterochromaffin (EC) cells as assessed by immunostaining of phosphorylated calcium–calmodulin kinase 2 (phospho-CaMK2). In contrast, acarbose activated much fewer enteroendocrine cells, having only modest phospho-CaMK2 immunoreactivity. Single administration of miglitol triggered no GLP1 secretion, and GLP1 secretion by miglitol plus maltose was significantly attenuated by atropine pretreatment, suggesting regulation via vagal nerve. Thus, while α-GIs generally delay carbohydrate absorption and potentiate GLP1 secretion, miglitol also activates duodenal EC cells, possibly via SGLT3, and potentiates GLP1 secretion through the parasympathetic nervous system.

Nociceptin/orphanin FQ-induced inhibition of delayed rectifier potassium currents by calcium/calmodulin-dependent protein kinase type II.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been shown to inhibit delayed rectifier potassium current (IK) in acutely dissociated rat parietal cortical neurons. However, the detailed mechanism of N/OFQ-induced inhibition on IK is not clear. This study is the first to explore an involvement of calcium/calmodulin (CaM)-dependent protein kinase type II (CaMKII) in mediating N/OFQ-induced responses. Utilizing pharmacological inhibitors of CaM and CaMKII, we have investigated the contribution of CaMKII in N/OFQ-induced effects using the whole-cell patch-clamp technique. In whole-cell voltage clamp, W-7 (100 μM), an antagonist of CaM, as well as KN-62, an inhibitor of CaMKII activity, attenuated the inhibitory effects of N/OFQ on IK. Activation and inactivation analysis indicated that the kinetics of IK were altered by N/OFQ, with decreased activation and promoted inactivation of IK. W-7 and KN-62 (10 μM) partly abolished the activation and inactivation curves shift of IK induced by N/OFQ. These findings show that CaMKII plays a critical role in N/OFQ-induced inhibition of IK in acutely dissociated rat parietal cortical neurons.

Cutaneous expression of calcium/calmodulin-dependent protein kinase II in rats with type 1 and type 2 diabetes

Changes in calcium–calmodulin protein kinase II (CaMKII) have been well demonstrated in nervous tissue of diabetic animal models. Skin shares the same ectodermal origin as nervous tissue and it is often affected in diabetic patients. The goal of this study was to analyze expression of CaMKII in rat foot pad 2 weeks and 2 months after induction of diabetes type 1 and 2.Forty-two Sprague-Dawley rats were used. Diabetes mellitus type 1 (DM1) was induced with intraperitoneally (i.p.) injected 55mg/kg of streptozotocin (STZ) and diabetes mellitus type 2 (DM2) with a combination of high-fat diet (HFD) and i.p. injection of low-dose STZ (35mg/kg). Two weeks and two months following diabetes induction rats were sacrificed and skin samples from plantar surface of the both hind paws were removed. Immunohistochemistry was performed for detection of total CaMKII (tCaMKII) and its alpha isoform (pCaMKIIα). For detection of intraepidermal nerve fibers polyclonal antiserum against protein gene product 9.5 (PGP 9.5) was used.The results showed that CaMKII was expressed in the skin of both diabetic models. Total CaMKII was uniformly distributed throughout the epidermis and pCaMKIIα was limited to stratum granulosum. The tCaMKII and pCaMKIIα were not expressed in intraepidermal nerve fibers. Two weeks after induction of diabetes in rats there were no significant differences in expression of tCaMKII and pCaMKIIα between DM1 and DM2 compared to respective controls. In the 2-month experiments, significant increase in epidermal expression of tCaMKII and pCaMKIIα was observed in DM1 animals compared to controls, but not in DM2 animals.This study is the first description of cutaneous CaMKII expression pattern in a diabetic model. CaMKII could play a role in transformation of skin layers and contribute to cutaneous diabetic changes. Further research on physiological role of CaMKII in skin and its role in cutaneous diabetic complications should be undertaken in order to elucidate its function in epidermis.

Protein kinase LKB1 promotes RAB7-mediated neuropilin-1 degradation to inhibit angiogenesis.

After internalization, transmembrane receptors (TMRs) are typically recycled back to the cell surface or targeted for degradation. Efficient TMR trafficking is critical for regulation of several processes, including signal transduction pathways, development, and disease. Here, we determined that trafficking of the angiogenic receptor neuropilin-1 (NRP-1) is abrogated by the liver kinase B1 (LKB1), a serine-threonine kinase of the calcium calmodulin family. We found that aberrant NRP-1 expression in tumor cells from patients with lung adenocarcinoma is associated with decreased levels of LKB1. In cultured lung cells, LKB1 accentuated formation of a complex between NRP-1 and RAB7 in late endosomes. LKB1 specifically bound GTP-bound RAB7, but not a dominant-negative GDP-bound form of RAB7, promoting rapid transfer and lysosome degradation of NRP-1. siRNA-mediated depletion of RAB7 disrupted the transfer of NRP-1 to the lysosome, resulting in recovery of the receptor as well as increased tumor growth and angiogenesis. Together, our findings indicate that LKB1 functions as a RAB7 effector and suppresses angiogenesis by promoting the cellular trafficking of NRP-1 from RAB7 vesicles to the lysosome for degradation. Furthermore, these data suggest that LKB1 and NRP-1 have potential as therapeutic targets for limiting tumorigenesis.

Effect size of memory deficits in mice with adult-onset P301L tau expression

Transgenic mice expressing mutations in tau have yielded essential discoveries for Alzheimer's disease. One of the most commonly used tau mouse models is the tet-off Tg(tauP301L)4510 model that expresses P301L human tau driven by the calcium–calmodulin kinase IIα (CaMKIIα) promoter system. Tau expression in this model is regulatable, allowing for suppression of mutant tau expression until adulthood and prevention of possible developmental alterations resulting from P301L tau expression during development. Here, we compared the effect and sample sizes needed for three learning and memory tasks in mice with adult-onset P301L tau expression. Our findings indicate that the Incremental Repeated Acquisition (IRA) and trace fear conditioning tasks, neither of which have previously been published with these mice, were highly sensitive to P301L tau expression, whereas the Morris water maze, the most commonly used task with this model, was the least sensitive. Memory deficits were observed at a time when tau pathology was subtle and prior to readily detectable neuronal loss. Thus, we provide essential information (effect and sample sizes needed) for establishing experimental designs at a time point when memory deficits are likely to go undetected if inadequate sample sizes are used. Our work also suggests the tet-off Tg4510 model provides a way to avoid mutant tau expression during the perinatal and early postnatal stages, thereby preventing possible developmental alterations unrelated to Alzheimer's disease.

Two domains of the smoothelin-like 1 protein bind apo- and calcium–calmodulin independently

The smoothelin-like 1 protein (SMTNL1) is a modulator of smooth and skeletal muscle contractility and can bind to calmodulin and tropomyosin. Calmodulin is the major calcium sensor of eukaryotic cells and it can cycle between calcium-free (apo-CaM) and calcium-bound (Ca-CaM) forms. Bioinformatic screening of the SMTNL1 sequence predicted a second CaM-binding region (CBD1) that is located N-terminal to the previously defined apo-CaM-binding site (CBD2). Pull-down assays, surface plasmon resonance, isothermal calorimetry and NMR techniques were used to determine that CBD1 associated preferentially to Ca-CaM while CBD2 bound preferentially to apo-CaM. Mutation of hydrophobic residues abolished Ca-CaM-binding to CBD1 while acidic residues in CBD2 were necessary for apo-CaM-binding to CBD2. The dissociation constant (Kd) for Ca-CaM-binding to a CBD1 peptide was 26∗10−6M while the value for binding to a longer protein construct was 0.5∗10−6M. The binding of SMTNL1 to both apo-CaM and Ca-CaM suggests that endogenous CaM is continuously associated with SMTNL1 to allow for quick response to changes in intracellular calcium levels. We also found that the intrinsically disordered N-terminus of SMTNL1 can reduce binding to apo-CaM and increase binding to Ca-CaM. This finding suggests that an additional CaM-binding region may exist and/or that intramolecular interactions between the N-terminus and the folded C-terminus reduce apo-CaM-binding to CBD2. Intriguingly, CBD1 is located close to the SMTNL1 phosphorylation site and tropomyosin-binding region. We discuss the possibility that all three signals are integrated at the region surrounding CBD1.

Ca²⁺ signals promote GLUT4 exocytosis and reduce its endocytosis in muscle cells.

Elevating cytosolic Ca(2+) stimulates glucose uptake in skeletal muscle, but how Ca(2+) affects intracellular traffic of GLUT4 is unknown. In tissue, changes in Ca(2+) leading to contraction preclude analysis of the impact of individual, Ca(2+)-derived signals. In L6 muscle cells stably expressing GLUT4myc, the Ca(2+) ionophore ionomycin raised cytosolic Ca(2+) and caused a gain in cell surface GLUT4myc. Extra- and intracellular Ca(2+) chelators (EGTA, BAPTA-AM) reversed this response. Ionomycin activated calcium calmodulin kinase II (CaMKII), AMPK, and PKCs, but not Akt. Silencing CaMKIIδ or AMPKα1/α2 partly reduced the ionomycin-induced gain in surface GLUT4myc, as did peptidic or small molecule inhibitors of CaMKII (CN21) and AMPK (Compound C). Compared with the conventional isoenzyme PKC inhibitor Gö6976, the conventional plus novel PKC inhibitor Gö6983 lowered the ionomycin-induced gain in cell surface GLUT4myc. Ionomycin stimulated GLUT4myc exocytosis and inhibited its endocytosis in live cells. siRNA-mediated knockdown of CaMKIIδ or AMPKα1/α2 partly reversed ionomycin-induced GLUT4myc exocytosis but did not prevent its reduced endocytosis. Compared with Gö6976, Gö6983 markedly reversed the slowing of GLUT4myc endocytosis triggered by ionomycin. In summary, rapid Ca(2+) influx into muscle cells accelerates GLUT4myc exocytosis while slowing GLUT4myc endocytosis. CaMKIIδ and AMPK stimulate GLUT4myc exocytosis, whereas novel PKCs reduce endocytosis. These results identify how Ca(2+)-activated signals selectively regulate GLUT4 exocytosis and endocytosis in muscle cells.

Homeostasis and secretion of calcium in the oviductal mucosa of toad Rhinella arenarum.

The presence of a calcium pump, calbindin D-28KD, and calmodulin in the secretory cells (SC) of the oviductal pars convoluta (PC) of Rhinella arenarum was established for the first time in amphibians using immunohistochemical techniques. Marked variations were observed in the localization and degree of expression of these proteins according to the duct segment and the period of the sexual cycle analyzed. During the preovulatory and ovulatory periods the calcium pump colocalized with calbindin D-28KD can be seen mainly in the apical border of the SC, which are located in the first zones of PC and synthesize and secrete the components of the inner jelly coat layers. These envelopes, which surround the oocytes, contain the molecules indispensable for fertilization, probably inducing the sperm acrosome reaction (AR). Our results suggest that calmodulin, colocalized with the calcium pump at the SC cytoplasmic level, would be involved in the active transport of the cation inside the secretory granules, maintaining adequate levels of intracellular Ca(2+) . During the postreproductive period, a calcium pump colocalized with calbindin D-28KD appears for the first time in the cycle in the basal zones of the SC. This system may be related to the replenishing of intracellular Ca(2+) stores. In contrast, in R. arenarum the Ca(2+) present in the jelly coats that surround the oocytes participates in the AR during fertilization, suggesting that this secretion system of the cation provided by the oviductal mucosa is functionally more active during the reproductive period of this species.

Erratum to: Cross-Talk Between Calcium–Calmodulin and Brassinolide for Fungal Endophyte-Induced Volatile Oil Accumulation of Atractylodes lancea Plantlets

Erratum to: Cross-Talk Between Calcium–Calmodulin and Brassinolide for Fungal Endophyte-Induced Volatile Oil Accumulation of Atractylodes lancea Plantlets

The amino terminus of high-voltage-activated calcium channels

Voltage-gated calcium channels (VGCCs), calmodulin (CaM), and calmodulin kinase II (CaMKII) are essential for various nervous system functions. CaM and CaMKII differentially regulate calcium dependent facilitation (CDF) and calcium dependent inactivation (CDI) of the Cav1 and Cav2 families of VGCCs. It is generally accepted that conserved structures in the C-terminus of these channels regulate CDF and CDI, and yet recent evidence indicates that other intracellular regions may be involved. We recently discovered that N-terminal sequences in Cav1.2 bind CaM and CaMKII, and function to regulate CDI as well as surface expression and open probability, respectively. Cav1 and Cav2 share significant portions of N-terminal sequence and therefore we explored whether homologous binding sites might exist in Cav2.1. Here, we show that like the proximal N-terminus of Cav1.2, the homologous region of Cav2.1 contains sequences which interact either directly or indirectly with CaM.

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.

Peroxisomal plant nitric oxide synthase (NOS) protein is imported by peroxisomal targeting signal type 2 (PTS2) in a process that depends on the cytosolic receptor PEX7 and calmodulin.

Nitric oxide (NO) production in plant peroxisomes by l-arginine-dependent NO synthase activity has been proven. The PEX5 and PEX7 PTS receptors, which recognize PTS1- and PTS2-containing proteins, are localized in the cytosol. Using AtPex5p and AtPex7p knockdown in Arabidopsis by RNA interference (RNAi) designated as pex5i and pex7i, we found that the l-arginine-dependent protein responsible for NO generation in peroxisomes appears to be imported through an N-terminal PTS2. Pharmacological analyzes using a calcium channel blocker and calmodulin (CaM) antagonist show that the import of the peroxisomal NOS protein also depends on calcium and calmodulin.

Abstract 528: MicroRNA-30 Family Inhibits Neointima Hyperplasia by Targeting CaMKIIδ

miRNA is one class of small and non-coding RNAs, which regulate coding gene expressions. miR-30 family members are abundantly expressed in the vascular smooth muscle layer in the artery walls. However, the roles of miR-30 in vascular smooth muscle and vascular diseases are little unknown. Here, we report that the expression of miR-30 family members is dramatically reduced in the vascular walls upon vascular injury. Rescuing down-regulated miR-30 by overexpression using lenti-vitral delivery in injured rat carotid artery significantly inhibited injury-induced neointima formation. In vitro, the expression of miR-30 family members in the dedifferentiated cultured vascular smooth muscle cells (VSMCs) was approximately 50% lower than it in the intact vascular smooth muscle. Western blot analysis and luciferase assay showed that miR-30 directly targeted 3' UTR of calcium calmodulin dependent protein kinase II δ isoform (CaMKIIδ) and inhibited CaMKIIδ expression in the culture VSMCs. VSMC proliferation was significantly inhibited by overexpressing miR-30 and this inhibitory effect was partially recovered by CaMKIIδ rescue. These data suggest that miR-30 is a strong and novel regulatory factor in neointima formation, and may be developed to be a new therapeutic target to prevent or reduce vascular diseases.

Eukaryotic elongation factor 2 kinase, an unusual enzyme with multiple roles.

Eukaryotic elongation factor 2 kinase (eEF2K) is a member of the small group of atypical 'α-kinases'. It phosphorylates and inhibits eukaryotic elongation factor 2, to slow down the elongation stage of protein synthesis, which normally consumes a great deal of energy and amino acids. The activity of eEF2K is normally dependent on calcium ions and calmodulin. eEF2K is also regulated by a plethora of other inputs, including inhibition by signalling downstream of anabolic signalling pathways such as the mammalian target of rapamycin complex 1. Recent data show that eEF2K helps to protect cancer cells against nutrient starvation and is also cytoprotective in other settings, including hypoxia. Growing evidence points to roles for eEF2K in neurological processes such as learning and memory and perhaps in depression.