Oligostilbenes from the seeds of Paeonia lactiflora as potent GLP-1 secretagogues targeting TGR5 receptor.

One unusual stilbene trimer-flavonoid hybrid, paeonilactiflobenoid (1), together with six known stilbenes (2-7) were isolated from the seeds of Paeonia lactiflora. The structure of 1 was elucidated with the aid of HRESIMS, 1D and 2D NMR, [α]D spectroscopic data and ECD calculation. Compounds 2-7 showed stimulative effects on GLP-1 secretion with promoting rates of 79.8%-880.4% (25 µM) and 217.6%-1089.4% (50 µM), more potent than the positive control, oleoylethanolamide (250.2% at 50 µM). Moreover, compounds 4 and 6 exhibited agonistic activity on the G protein-coupled receptor (GPCR) TGR5 with stimulative ratios of 40.2% and 40.5% at 50 µM, and 54.2% and 49.1% at 100 µM, respectively. Docking study manifested that 6 well located in the catalytic pocket of TGR5 by hydrogen-bond and hydrophobic interactions. The GLP-1 promotion of 6 could be attenuated by IP3, Ca2+/CaMKII and MEK/ERK pathway inhibitors, suggesting that these pathways played important roles in GLP-1 secretion. Thus, stilbenes in peony seeds maybe regarded as potential GLP-1 secretagogues through TGR5-IP3-Ca2+/CaMKII-MEK/ERK pathways.

CaMKII: a central molecular organizer of synaptic plasticity, learning and memory.

Calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is the most abundant protein in excitatory synapses and is central to synaptic plasticity, learning and memory. It is activated by intracellular increases in calcium ion levels and triggers molecular processes necessary for synaptic plasticity. CaMKII phosphorylates numerous synaptic proteins, thereby regulating their structure and functions. This leads to molecular events crucial for synaptic plasticity, such as receptor trafficking, localization and activity; actin cytoskeletal dynamics; translation; and even transcription through synapse-nucleus shuttling. Several new tools affording increasingly greater spatiotemporal resolution have revealed the link between CaMKII activity and downstream signalling processes in dendritic spines during synaptic and behavioural plasticity. These technologies have provided insights into the function of CaMKII in learning and memory.

CaMKII activation persistently segregates postsynaptic proteins via liquid phase separation.

Transient information input to the brain leads to persistent changes in synaptic circuits, contributing to the formation of memory engrams. Pre- and postsynaptic structures undergo coordinated functional and structural changes during this process, but how such changes are achieved by their component molecules remains largely unknown. We found that activated CaMKII, a central player of synaptic plasticity, undergoes liquid-liquid phase separation with the NMDA-type glutamate receptor subunit GluN2B. Due to CaMKII autophosphorylation, the condensate stably persists even after Ca2+ is removed. The selective binding of activated CaMKII with GluN2B cosegregates AMPA receptors and the synaptic adhesion molecule neuroligin into a phase-in-phase assembly. In this way, Ca2+-induced liquid-liquid phase separation of CaMKII has the potential to act as an activity-dependent mechanism to crosslink postsynaptic proteins, which may serve as a platform for synaptic reorganization associated with synaptic plasticity.

CaMKII Modulates the Cardiac Transient Outward K+ Current through its Association with Kv4 Channels in Non-Caveolar Membrane Rafts.

BACKGROUND/AIMS: To test whether the physiological regulation of the cardiac Kv4 channels by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is restricted to lipid rafts and whether the interactions observed in rat cardiomyocytes also occur in the human ventricle. METHODS: Ventricular myocytes were freshly isolated from Sprague-Dawley rats. Ito was recorded by the whole-cell Patch-Clamp technique. Membrane rafts were isolated by centrifugation in a discontinuous sucrose density gradient. The presence of the proteins of interest was analysed by western blot. Immunogold staining and electron microscopy of heart vibrosections was performed to localize Kv4.2/Kv4.3 and CaMKII proteins. Protein-protein interactions were determined by co-immunoprecipitation experiments in rat and human ventricular mycoytes. RESULTS: Patch-Clamp recordings in control conditions and after lipid raft or caveolae disruption show that the CaMKII-Kv4 channel complex must associate in non-caveolar lipid rafts to be functional. Separation in density gradients, co-immunoprecipitation and electron microscopy show that there are two Kv4 channel populations: one located in caveolae, that is CaMKII independent, and another one located in planar membrane rafts, which is bound to CaMKII. CONCLUSION: CaMKII regulates only the Kv4 channel population located in non-caveolar lipid rafts. Thus, the regulation of cardiac Kv4 channels in rat and human ventricle depends on their subcellular localization.

Neuroprotective Effect of Astragalus Polysacharin on Streptozotocin (STZ)-Induced Diabetic Rats.

BACKGROUND In the recent years, there has been increasing interest in traditional Chinese medicine as a neuroprotective nutrient in the management of chronic neurodegenerative disease, such as diabetic cognitive decline. Astragalus polysacharin (APS), a Chinese herb extract, is a biologically active treatment for neurodegenerative diseases. Therefore, in the present study, we investigated the neuroprotective effects of APS (20 mg/kg) on diabetes-induced memory impairments in Sprague-Dawley (SD) rats and explored its underlying mechanisms of action. MATERIAL AND METHODS Thirty SD rats were randomly divided into a control group (CON group, n=10), a diabetic model (DM) group (n=10), and an APS group (n=10). We administered 55 mg/kg streptozotocin (STZ, Sigma) by intraperitoneal injection to induce a diabetic model. Food and water intake, body weight, and blood fasting plasma glucose (FPG) were measured. The Morris water maze test (MWM) was used to assess learning and memory ability, and we measured levels of N-methyl-D-aspartate receptor (NMDA), calcium/calmodulin-dependent protein kinase II (CaMKII), and cAMP response element-binding protein (CREB) in the hippocampus. RESULTS APS (20 mg/kg) administration decreased the rats' fasting plasma glucose (FPG) levels and body weight. APS (20 mg/kg) administration improved the cognitive performance of diabetes-induced rats in the Morris water maze test. APS (20 mg/kg) administration reduced the number of dead cells in the CA1 region of the hippocampus. Furthermore, APS (20 mg/kg) administration obviously upregulated the phosphorylation levels CREB, NMDA, and CaMK II. CONCLUSIONS These results suggest that APS has the neuroprotective effects, and it may be a candidate for treatment of neurodegenerative diseases such as diabetic cognitive impairment.

A simple end-point assay for calcium channel activity.

Cellular calcium signaling events are transient. Hence they are observed in real time using fluorescence imaging or electrophysiological methods that require sophisticated instrumentation and specialized skills. For high throughput assays simple and inexpensive techniques are desirable. Many calcium channels that serve as drug targets have subtypes arising from diverse subunit combinations. These need to be targeted selectively for achieving efficacy and for avoiding side effects in therapies. This in turn increases the number of calcium channels that act as drug targets. We report a novel method for intracellular calcium sensing that utilizes the calcium dependent stable interaction between CaM kinase II (CaMKII) and its ligands such as the NMDA receptor subunit GluN2B. The CaMKII-GluN2B complex formed persists as a memory of the transient increase in calcium. In a cell-based assay system GFP-α-CaMKII expressed in the cytosol responds to calcium by translocating towards GluN2B sequence motif exogenously expressed on mitochondria or endoplasmic reticulum. The resulting punctate fluorescence pattern serves as the signal for intracellular calcium release. The pattern is stable, unaffected by sample processing and is observable without real time imaging. The activities of calcium channel proteins heterologously expressed in HEK-293 cells were detected with specificity using this technique. A calcium sensor vector and a calcium sensor cell line were developed as tools to perform this technique. This technique being simple and less expensive could significantly facilitate high throughput screening in calcium channel drug discovery.

Preparation of polysomal fractions from mouse brain synaptoneurosomes and analysis of polysomal-bound mRNAs.

BACKGROUND: Here we describe a detailed, reliable protocol for isolation of polysomal fractions from mouse brain synaptoneurosomes. This method is an important tool to study local protein synthesis in neurons. NEW METHOD: We combined rapid preparation of synaptoneurosomes by filtration with polysome profiling. We provide a detailed protocol highlighting difficulties and critical steps of: i) preparation of synaptoneurosomes; ii) polyribosome fractionation from synaptoneurosomes; iii) extraction of proteins and RNA from sucrose gradient fractions. RESULTS: and Comparison with Existing Methods We fractionated polyribosomes from synaptoneurosomes and detected the association of Mmp9, Camk2a and Stx1B mRNA with polysomes in the unstimulated conditions. Synaptic stimulation led to increased levels of Mmp9 and Camk2a mRNA in the heavy polysomal fractions. We compared our protocol with existing methods CONCLUSIONS: We have developed a reliable, effective method to prepare polyribosomal fractions from synaptoneurosomes to study polyribosomal binding of mRNAs as an aspect of synaptic translation in vitro.

Neonatal exposure to a moderate dose of ionizing radiation causes behavioural defects and altered levels of tau protein in mice.

Medical use of ionizing radiation (IR) has great benefits for treatment and diagnostic imaging, but procedures as computerized tomography (CT) may deliver a significant radiation dose to the patient. Recently, awareness has been raised about possible non-cancer consequences from low dose exposure to IR during critical phases of perinatal and/or neonatal brain development. In the present study neonatal NMRI mice were whole body irradiated with a single dose of gamma radiation (0; 350 and 500 mGy) on postnatal day 10 (PND 10). At 2 and 4 months of age, mice of both sexes were observed for spontaneous behaviour in a novel home environment. The neuroproteins CaMKII, GAP-43, synaptophysin and total tau in male mouse cerebral cortex and hippocampus were analysed 24h post-irradiation and in adults at 6 months of age exposed to 0 or 500 mGy on PND 10. A significantly dose-response related deranged spontaneous behaviour in 2- and 4-month-old mice was observed, where both males and females displayed a modified habituation, indicating reduced cognitive function. The dose of 350 mGy seems to be a tentative threshold. Six-month-old male mice showed a significantly increased level of total tau in cerebral cortex after irradiation to 500 mGy compared to controls. This demonstrates that a single moderate dose of IR, given during a defined critical period of brain development, is sufficient to cause persistently reduced cognitive function. Moreover, an elevation of tau protein was observed in male mice displaying reduced cognitive function.

Protein changes contributing to right ventricular cardiomyocyte diastolic dysfunction in pulmonary arterial hypertension.

BACKGROUND: Right ventricular (RV) diastolic function is impaired in patients with pulmonary arterial hypertension (PAH). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca(2+) sensitivity underlie diastolic dysfunction in PAH. This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH. METHODS AND RESULTS: RV samples from PAH patients undergoing heart-lung transplantation were compared to non-failing donors (Don). Titin stiffness contribution to RV diastolic dysfunction was determined by Western-blot analyses using antibodies to protein-kinase-A (PKA), Cα (PKCα) and Ca(2+)/calmoduling-dependent-kinase (CamKIIδ) titin and phospholamban (PLN) phosphorylation sites: N2B (Ser469), PEVK (Ser170 and Ser26), and PLN (Thr17), respectively. PKA and PKCα sites were significantly less phosphorylated in PAH compared with donors (P<0.0001). To test the functional relevance of PKA-, PKCα-, and CamKIIδ-mediated titin phosphorylation, we measured the stiffness of single RV cardiomyocytes before and after kinase incubation. PKA significantly decreased PAH RV cardiomyocyte diastolic stiffness, PKCα further increased stiffness while CamKIIδ had no major effect. CamKIIδ activation was determined indirectly by measuring PLN Thr17phosphorylation level. No significant changes were found between the groups. Myofilament Ca(2+) sensitivity is mediated by sarcomeric troponin I (cTnI) phosphorylation. We observed increased unphosphorylated cTnI in PAH compared with donors (P<0.05) and reduced PKA-mediated cTnI phosphorylation (Ser22/23) (P<0.001). Finally, alterations in Ca(2+)-handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca(2+) clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors (P<0.05). CONCLUSIONS: Increased titin stiffness, reduced cTnI phosphorylation, and altered levels of phosphorylation of Ca(2+) handling proteins contribute to RV diastolic dysfunction in PAH.

Multiple spatial and kinetic subpopulations of CaMKII in spines and dendrites as resolved by single-molecule tracking PALM.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is essential for synaptic plasticity underlying memory formation. Some functions of CaMKII are mediated by interactions with synaptic proteins, and activity-triggered translocation of CaMKII to synapses has been heavily studied. However, CaMKII actions away from the postsynaptic density (PSD) remain poorly understood, in part because of the difficulty in discerning where CaMKII binds in live cells. We used photoactivated localization microscopy (PALM) in rat hippocampal neurons to track single molecules of CaMKIIα, mapping its spatial and kinetic heterogeneity at high resolution. We found that CaMKIIα exhibits at least three kinetic subpopulations, even within individual spines. Latrunculin application or coexpression of CaMKIIß carrying its actin-binding domain strongly modulated CaMKII diffusion, indicating that a major subpopulation is regulated by the actin cytoskeleton. CaMKII in spines was typically more slowly mobile than in dendrites, consistent with presence of a higher density of binding partners or obstacles. Importantly, NMDA receptor stimulation that triggered CaMKII activation prompted the immobilization and presumed binding of CaMKII in spines not only at PSDs but also at other points up to several hundred nanometers away, suggesting that activated kinase does not target only the PSD. Consistent with this, single endogenous activated CaMKII molecules detected via STORM immunocytochemistry were concentrated in spines both at the PSD and at points quite distant from the synapse. Together, these results indicate that CaMKII mobility within spines is determined by association with multiple interacting proteins, even outside the PSD, suggesting diverse mechanisms by which CaMKII may regulate synaptic transmission.

Melatonin reverses the decreases in hippocampal protein serine/threonine kinases observed in an animal model of autism.

Lower global cognitive function scores are a common symptom of autism spectrum disorders (ASDs). This study investigates the effects of melatonin on hippocampal serine/threonine kinase signaling in an experimental ASD model. We found that chronic melatonin (1.0 or 5.0 mg/kg/day, 28 days) treatment significantly rescued valproic acid (VPA, 600 mg/kg)-induced decreases in CaMKII (Thr286), NMDAR1 (Ser896), and PKA (Thr197) phosphorylation in the hippocampus without affecting total protein levels. Compared with control rats, the immunostaining of pyramidal neurons in the hippocampus revealed a decrease in immunolabeling intensity for phospho-CaMKII (Thr286) in the hippocampus of VPA-treated rats, which was ameliorated by chronic melatonin treatment. Consistent with the elevation of CaMKII/PKA/PKC phosphorylation observed in melatonin-treated rat, long-term potentiation (LTP) was enhanced after chronic melatonin (5.0 mg/kg) treatment, as reflected by extracellular field potential slopes that increased from 56 to 60 min (133.4 ± 3.9% of the baseline, P < 0.01 versus VPA-treated rats) following high-frequency stimulation (HFS) in hippocampal slices. Accordingly, melatonin treatment also significantly improved social behavioral deficits at postnatal day 50 in VPA-treated rats. Taken together, the increased phosphorylation of CaMKII/PKA/PKC signaling might contribute to the beneficial effects of melatonin on autism symptoms.

Expression of phospho-Ca(2+) /calmodulin-dependent protein kinase II in the pre-Bötzinger complex of rats.

The pre-Bötzinger complex (pre-BötC) in the ventrolateral medulla oblongata is a presumed kernel of respiratory rhythmogenesis. Ca(2+) -activated non-selective cationic current is an essential cellular mechanism for shaping inspiratory drive potentials. Ca(2+) /calmodulin-dependent protein kinase II (CaMKII), an ideal 'interpreter' of diverse Ca(2+) signals, is highly expressed in neurons in mediating various physiological processes. Yet, less is known about CaMKII activity in the pre-BötC. Using neurokinin-1 receptor as a marker of the pre-BötC, we examined phospho (P)-CaMKII subcellular distribution, and found that P-CaMKII was extensively expressed in the region. P-CaMKII-ir neurons were usually oval, fusiform, or pyramidal in shape. P-CaMKII immunoreactivity was distributed within somas and dendrites, and specifically in association with the post-synaptic density. In dendrites, most synapses (93.1%) examined with P-CaMKII expression were of asymmetric type, occasionally with symmetric type (6.9%), whereas in somas, 38.1% were of symmetric type. P-CaMKII asymmetric synaptic identification implicates that CaMKII may sense and monitor Ca(2+) activity, and phosphorylate post-synaptic proteins to modulate excitatory synaptic transmission, which may contribute to respiratory modulation and plasticity. In somas, CaMKII acts on both symmetric and asymmetric synapses, mediating excitatory and inhibitory synaptic transmission. P-CaMKII was also localized to the perisynaptic and extrasynaptic regions in the pre-BötC.

Immunolocalization of CaMKII and NR2B in hippocampal subregions of rat during postnatal development.

Although the expression of CaMKII and synaptic-associated proteins has been widely studied, the temporospatial distribution of CaMKII and NMDAR subunits in different hippocampal subregions during postnatal development still lacks detailed information. In this study, we used immunofluorescent staining to assess CaMKII and NR2B expressions and the relationship between them in CA1, CA3, and DG of rat hippocampus on postnatal (P) days: P0, P4, P7, P10, P14, P21, P28, and P56. The results showed that from P0 to P56, CaMKII expression increased gradually, while NR2B expression decreased gradually, and the time points of their expression peak differed in CA1, CA3, and DG during postnatal development. Although the expression of CaMKII was negatively correlated with NR2B in CA1 and DG, the coexpression of CaMKII with NR2B existed in CA1, CA3, and DG during postnatal development. Interestingly, after P21, CaMKII expression and the coexpression of CaMKII with NR2B became obvious in the Stratum lucidum of CA3. The specific temporospatial distribution pattern of CaMKII with NR2B might be related to the different physiological functions during postnatal development. Discovery of the alteration of the relationship between expression of CaMKII and NMDAR subunits may be helpful for understanding mechanisms and therapy of neurodegenerative diseases.

On the mechanism of synaptic depression induced by CaMKIIN, an endogenous inhibitor of CaMKII.

Activity-dependent synaptic plasticity underlies, at least in part, learning and memory processes. NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) is a major synaptic plasticity model. During LTP induction, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is activated, autophosphorylated and persistently translocated to the postsynaptic density, where it binds to the NMDAR. If any of these steps is inhibited, LTP is disrupted. The endogenous CaMKII inhibitor proteins CaMKIINα,ß are rapidly upregulated in specific brain regions after learning. We recently showed that transient application of peptides derived from CaMKIINα (CN peptides) persistently depresses synaptic strength and reverses LTP saturation, as it allows further LTP induction in previously saturated pathways. The treatment disrupts basal CaMKII-NMDAR interaction and decreases bound CaMKII fraction in spines. To unravel CaMKIIN function and to further understand CaMKII role in synaptic strength maintenance, here we more deeply investigated the mechanism of synaptic depression induced by CN peptides (CN-depression) in rat hippocampal slices. We showed that CN-depression does not require glutamatergic synaptic activity or Ca(2+) signaling, thus discarding unspecific triggering of activity-dependent long-term depression (LTD) in slices. Moreover, occlusion experiments revealed that CN-depression and NMDAR-LTD have different expression mechanisms. We showed that CN-depression does not involve complex metabolic pathways including protein synthesis or proteasome-mediated degradation. Remarkably, CN-depression cannot be resolved in neonate rats, for which CaMKII is mostly cytosolic and virtually absent at the postsynaptic densities. Overall, our results support a direct effect of CN peptides on synaptic CaMKII-NMDAR binding and suggest that CaMKIINα,ß could be critical plasticity-related proteins that may operate as cell-wide homeostatic regulators preventing saturation of LTP mechanisms or may selectively erase LTP-induced traces in specific groups of synapses.

Expression profiling of Ca(2+)/calmodulin-dependent signaling molecules in the rat dorsal and ventral hippocampus after acute lead exposure.

The septal and temporal poles of the hippocampus differ markedly in their anatomical organization, but whether these distinct regions exhibit differential neurochemical profiles underlying lead (Pb(2+)) neurotoxicity remains to be determined. In the present study, we examined changes in the expression of Ca(2+)/calmodulin-dependent enzymes, including calpain, calcineurin, phospho-CaMKII (Thr286) and neuronal nitric oxide synthase (nNOS), in the rat dorsal and ventral hippocampus (DH and VH) after acute Pb(2+) exposure. Five days after Pb(2+) exposure, we observed constitutively active forms of calcineurin (45 kDa and 48 kDa) in ventral portions of the hippocampus, a result consistent with the observed calpain activation that is indicated by the breakdown of spectrin in this region. Our data demonstrate that nNOS expression is significantly higher in the ventral region of the hippocampus when compared to the dorsal region, whereas phosphorylation of CaMKII (Thr286) is less pronounced in the ventral portion of the hippocampus and more pronounced in dorsal regions after acute Pb(2+) exposure. Thus, it appears likely that the ventral region of hippocampus is more vulnerable to the neurotoxic effects of Pb(2+) than the dorsal region. Taken together, the present data suggest that acute lead exposure leads to differential expression patterns of Ca(2+)/calmodulin-dependent enzymes along the dorsoventral axis of the hippocampus.

Measuring CaMKII concentration in dendritic spines.

Here, we present a method for measuring the concentration of endogenous protein in cellular compartments. Importantly, the method is applicable to compartments such as dendritic spines with dimensions often close to the resolution limit of optical microscopy. To our knowledge, a method with such capabilities has not yet been described. The method utilizes overexpression of the protein of interest, which is tagged with fluorescent protein. This is followed by immunostaining of both overexpressed and endogenous proteins. Expression of a volume marker is also required. We applied this method to measure the concentration of Ca/calmodulin kinase II (CaMKII) in different cellular compartments of hippocampal pyramidal neurons. It was found that the concentrations of CaMKIIα subunits in cell bodies, proximal dendrites, and spines on these dendrites are 71, 46, and 103 µM, respectively. Considering the 3:1 ratio of α to ß CaMKII subunits in the hippocampus, the concentrations of total (α+ß) CaMKII subunits in these compartments are 94, 61, and 138 µM, respectively.

Rapid development of genetically encoded FRET reporters.

To meet the demand on genetically encoded reporter molecules for live cell imaging, we introduce a new facile combined cloning and FRET reporter analysis strategy. The versatile and fully orthogonal cloning approach involves a set of up to 36 vectors featuring a variety of fluorescent protein FRET pairs and different length linkers. The construct set was successfully applied to two calmodulin-binding proteins, the death-associated protein kinase 1 (DAPK1) and calcium/calmodulin-dependent protein kinase II α (Camk2a). Clone analysis and reporter validation was performed by printing plasmid DNA arrays and subsequent semiautomated microscopy of reversely transfected cells. Characterization of the best performing DAPK1 and Camk2a reporters revealed significant differences in translating calcium signals into kinase responses despite the close functional and structural similarity.

Omega-3 fatty acids deprivation affects ontogeny of glutamatergic synapses in rats: relevance for behavior alterations.

Essential omega-3 polyunsaturated fatty acids (omega3) are crucial to brain development and function, being relevant for behavioral performance. In the present study we examined the influence of dietary omega3 in the development of the glutamatergic system and on behavior parameters in rats. Female rats received isocaloric diets, either with omega3 (omega3 group) or a omega3 deficient diet (D group). In ontogeny experiments of their litters, hippocampal immunocontent of ionotropic NMDA and AMPA glutamatergic receptors subunits (NR2 A\B and GluR1, respectively) and the alpha isoform of the calcium-calmodulin protein kinase type II (alphaCaMKII) were evaluated. Additionally, hippocampal [(3)H]glutamate binding and uptake were assessed. Behavioral performance was evaluated when the litters were adult (60 days old), through the open-field, plus-maze, inhibitory avoidance and flinch-jump tasks. The D group showed decreased immunocontent of all proteins analyzed at 02 days of life (P2) in comparison with the omega3 group, although the difference disappeared at 21 days of life (except for alphaCaMKII, which content normalized at 60 days old). The same pattern was found for [(3)H]glutamate binding, whereas [(3)H]glutamate uptake was not affected. The D group also showed memory deficits in the inhibitory avoidance, increased in the exploratory pattern in open-field, and anxiety-like behavior in plus-maze. Taken together, our results suggest that dietary omega3 content is relevant for glutamatergic system development and for behavioral performance in adulthood. The putative correlation among the neurochemical and behavioral alterations caused by dietary omega3 deficiency is discussed.

Effects of sodium fluoride on hyperactivation and Ca2+ signaling pathway in sperm from mice: an in vivo study.

Sperm hyperactivation is crucial for a successful fertilization; however, the influence of fluoride (F) to hyperactivation is still in its infancy. The purpose of this study was to investigate the effect of sodium fluoride (NaF) on sperm hyperactivation, Ca2+/CALM-CAMK2 signaling, and CatSper1 and CatSper2 mRNA expression in mice sperm. Adult male Kunming mice were administrated with 30, 70, and 150 mg NaF/l (corresponding to 2.84 +/- 0.29, 6.28 +/- 0.61, and 14.18 +/- 1.00 mg F/kg body weight per day) through drinking water for 49 days. The results showed that NaF reduced the sperm hyperactivated motility in a dose-dependent manner. Compared with the controls, intracellular Ca2+ concentration and CAMK2 protein were significantly decreased in mice treated with 70 and 150 mg NaF/l, while no effect on CALM was determined in all treatment groups. Furthermore, decreased sperm CatSper1 mRNA expression was also observed in response to middle and higher doses of NaF (70, 150 mg/l) with comparison to the control group, whereas no change in the mRNA expression of CatSper2 was detected in NaF administrated groups. Treatment with 30 mg NaF/l exhibited slight effects on the above indexes with no statistical difference. These findings indicated that exposure to 70 and 150 mg/l NaF for 49 days could result in low hyperactivation via alteration of Ca2+ signaling pathway involving CatSper1 in sperm from mice.

Proteomic profiling of PrP27-30-enriched preparations extracted from the brain of hamsters with experimental scrapie.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders characterized by the accumulation in the CNS of a pathological conformer (PrP(TSE)) of the host-encoded cellular prion protein (PrP(C)). PrP(TSE) has a central role in the pathogenesis of the disease but other factors are likely involved in the pathological process. In this work we employed a multi-step proteomic approach for the identification of proteins that co-purify with the protease-resistant core of PrP(TSE) (PrP27-30) extracted from brains of hamsters with experimental scrapie. We identified ferritin, calcium/calmodulin-dependent protein kinase alpha type II, apolipoprotein E, and tubulin as the major components associated with PrP27-30 but also trace amounts of actin, cofilin, Hsp90alpha, the gamma subunit of the T-complex protein 1, glyceraldehyde 3-phosphate dehydrogenase, histones, and keratins. Whereas some of these proteins (tubulin and ferritin) are known to bind PrP, other proteins (calcium/calmodulin-dependent protein kinase alpha type II, Hsp90alpha) may associate with PrP(TSE) fibrils during disease. Apolipoprotein E and actin have been previously observed in association with PrP(TSE), whereas cofilin and actin were shown to form abnormal rods in the brain of patients with Alzheimer disease. The roles of these proteins in the development of brain lesions are still unclear and further work is needed to explain their involvement in the pathogenesis of TSEs.