Dietary Repletion with ω3 Fatty Acid or with COX Inhibition Reverses Cognitive Effects in F3 ω3 Fatty-Acid–Deficient Mice

Dietary deficiency of ω3 fatty acid during development leads to impaired cognitive function. However, the effects of multiple generations of ω3 fatty-acid deficiency on cognitive impairment remain unclear. In addition, we sought to test the hypothesis that the cognitive impairments of ω3 fatty-acid–deficient mice are mediated through the arachidonic acid–cyclooxygenase (COX) pathway. To address these issues, C57BL/6J mice were bred for 3 generations and fed diets either deficient (DEF) or sufficient (SUF) in ω3 fatty acids. At postnatal day 21, the F3 offspring remained on the dam''s diet or were switched to the opposite diet, creating 4 groups. In addition, 2 groups that remained on the dam''s diet were treated with a COX inhibitor. At 19 wk of age, spatial-recognition memory was tested on a Y-maze. Results showed that 16 wk of SUF diet reversed the cognitive impairment of F3 DEF mice. However, 16 wk of ω3 fatty-acid–deficient diet impaired the cognitive performance of the F3 SUF mice, which did not differ from that of the F3 DEF mice. These findings suggest that the cognitive deficits after multigenerational maintenance on ω3 fatty-acid–deficient diet are not any greater than are those after deficiency during a single generation. In addition, treatment with a COX inhibitor prevented spatial-recognition deficits in F3 DEF mice. Therefore, cognitive impairment due to dietary ω3 fatty-acid deficiency appears to be mediated by the arachidonic acid–COX pathway and can be prevented by 16 wk of dietary repletion with ω3 fatty acids or COX inhibition.Abbreviations: AA, arachidonic acid; COX, cyclooxygenase; DEF, ω3 fatty-acid–deficient; DHA, docosahexaenoic acid; MWM, Morris water maze; SUF, ω3 fatty-acid–sufficientDietary deficiency of ω3 fatty acid is associated with impaired cognitive function. For example, rats on ω3 fatty-acid–deficient diet took significantly longer to locate the platform during the swimming test in the Morris water-maze (MWM) test.⁶ Previous studies report that dietary ω3 fatty-acid deficiency led to significantly shorter latencies in the passive-avoidance test in rats³ and increased time in the Barnes circular test in mice.⁷ However, ω3 fatty-acid–deficient diet increased the time and number of entries in the maze-learning task in a single generation of mice.²² These findings may suggest that ω3 fatty-acid–deficient diet influences cognitive function in animals by impairing their performance in spatial-recognition memory tasks.Previous studies have shown that rodents raised on an ω3 fatty-acid–deficient diet over 2 or 3 generations have impaired learning performance in the MWM task.^17,25 Dietary ω3 fatty-acid deficiency in the F2 and F3 rats prolonged the escape latency and delayed acquisition of the MWM task compared with those of rats fed an ω3 fatty-acid–sufficient diet for both generations.¹⁷ In a subsequent study, F3 rats fed on ω3 fatty-acid–deficient diet since birth or at weaning had a lower mean swimming speed to locate the platform during the MWM task.¹⁸ Previous studies show that feeding mice an ω3 fatty-acid–deficient diet for 3 generations reduced swimming performance in the MWM test.²⁵ Interestingly, feeding rats suboptimal levels of docosahexaenoic acid (DHA) for four generations significantly prolonged latencies in the MWM task compared those of rats fed higher levels of DHA.¹² These results suggest that multigenerational feeding of an ω3 fatty-acid–deficient diet impairs performance in tests of spatial-recognition memory.Importantly, after several generations of ω3 fatty-acid deficiency, switching rats to a sufficient diet at birth restored their performance on the spatial-recognition task to normal.¹⁸ Similarly, cognitive impairment in the brightness-discrimination test in mice after 2 generations of dietary ω3 fatty-acid deficiency was reversed by providing ω3 fatty-acid–sufficient diet after weaning.⁹ Cognitive performance in the MWM test did not differ in mice provided an ω3 fatty-acid–sufficient diet only and those switched at 7 wk of age from an ω3 fatty-acid–deficient diet to a sufficient diet.⁴ Overall, these findings indicate that the cognitive impairments due to ω3 fatty-acid deficiency are reversed by providing a diet containing sufficient amounts of ω3 fatty acids.Previous studies have been shown that the cognitive and memory deficits of a transgenic mouse model are due to increased prostaglandin activity from formation of cyclooxygenase (COX).¹³ Dietary ω3 fatty acid deficiency has been suggested to increase prostaglandin activity in animals.¹⁶ Therefore, the administration of a COX inhibitor may protect against cognitive impairment in the elevated plus-maze task by inhibiting the synthesis of prostaglandin.¹¹ Treatment with a COX inhibitor improved open-field exploration in mice by inhibiting the synthesis of prostaglandin.²³ Similarly, COX inhibitors such as celecoxib inhibit prostaglandin E2 levels and consequently improve cognitive performance in rats as assessed by the elevated plus-maze test.⁵ In addition, the administration of naproxen, another COX inhibitor, was protective against motor and cognitive impairment in rats by decreasing oxidative stress.¹⁴ Moreover, naproxen reduced oxidative stress levels and prevented neurologic disorders, especially memory deficits, in an animal model of excitotoxic neuronal injury.²⁰ Clearly, these findings suggest that COX inhibitors may protect against cognitive and memory deficits in animals by inhibiting prostaglandin activity.Clarifying the differences in cognitive function between the first and third generations of mice likely would improve our understanding of the factors contributing to differences in cognitive deficits due to dietary ω3 fatty-acid deficiency. To this end, we raised and maintained third-generation mice on a diet either sufficient or deficient in ω3 fatty acids or on a cross-over diet. Spatial-recognition memory in the F3 mice was tested by using the Y-maze. The aim of our transgenerational studies was to determine whether dietary ω3 fatty-acid deficiency causes severe cognitive impairment in F3 mice. In addition, these studies examined the hypothesis that the cognitive impairment of F3 mice on an ω3 fatty-acid deficient diet results from increased prostaglandin activity due to eicosanoid production from the arachidonic acid (AA)–COX pathway. Furthermore, we hypothesized that treatment with naproxen, a COX inhibitor, would improve cognitive function as a result of inhibiting prostaglandin activity.     

Calpain regulates N-terminal interaction of GSK-3β with 14-3-3ζ, p53 and PKB but not with axin

Calpain produces a truncation of GSK3β that removes the N-terminal inhibitory domain. Here we analyze the effect of that truncation on protein-protein interaction. We pulled down GST-tagged proteins in the presence of full length GSK-3β and calpain-cleaved GSK-3β. Commercial GSK-3β was first incubated with calpain for 2.5 min in vitro, and then with GST-tagged proteins in the presence of calpeptin, a synthetic calpain inhibitor. Western blot analyses were performed to determine if there is an interaction between these GST-tagged proteins and truncated GSK-3β. Using axin GST-tagged, we pulled down the protein in the presence of full length GSK-3β and calpain-cleaved GSK-3β. Western blot analyses showed full length GSK-3β in the pellet as well GSK-3β cleaved by calpain. Thus axin was able to bind GSK-3β without the N-terminal end. When the same experiment was carried out with GST-tagged 14-3-3ζ, p53 and PKB, full length GSK-3β was observed in the pellet, but GSK-3β truncated by calpain was not pulled down demonstrating that GSK-3β N-terminal end is necessary to interact with these three proteins. Our data demonstrate that N-terminal end is necessary for 14-3-3ζ, p53 and PKB interaction. However, the interaction of GSK3β with axin is not altered by calpain. These data support a physiological role for GSK3β truncation mediated by calpain.     

Expression of 14-3-3γ in patients with breast cancer: Correlation with clinicopathological features and prognosis

AimProtein 14-3-3γ is an important member of the 14-3-3 family that play important roles in the regulation of various cellular processes. The aim of the study is to investigate the association between 14-3-3γ expression and the clinicopathological features of patients with breast cancer.MethodsThe expression of 14-3-3γ was detected by Western blot in both foci of breast cancer and adjacent non-cancerous tissues. In addition, 14-3-3γ expression was analyzed by immunohistochemistry in 60 clinicopathologically characterized breast cancer cases. The association of 14-3-3γ expression with survival of the patients were analyzed.ResultsThe expression level of 14-3-3γ protein in breast cancer were significantly higher than that in non-cancerous mammary gland tissues. Moreover, high expression of 14-3-3γ correlated with tumor size and tumor grade (all P < 0.05). Patients with high 14-3-3γ expression had worse overall survival rate than that with low expression (P < 0.05). Furthermore, multivariate analysis showed that 14-3-3γ expression was an independent predictor of overall survival (HR, 0.196; 95%CI, 0.043–0.892; P = 0.035).ConclusionsOur data suggest for the first time that the increased expression of 14-3-3γ in breast cancer is associated significantly with tumor progression and poor prognosis. 14-3-3γ may be a novel and potential prognostic marker for breast cancer.     

M3 Muscarinic Receptor Interaction with Phospholipase C β3 Determines Its Signaling Efficiency

Phospholipase Cβ (PLCβ) enzymes are activated by G protein-coupled receptors through receptor-catalyzed guanine nucleotide exchange on Gαβγ heterotrimers containing G_q family G proteins. Here we report evidence for a direct interaction between M3 muscarinic receptor (M3R) and PLCβ3. Both expressed and endogenous M3R interacted with PLCβ in coimmunoprecipitation experiments. Stimulation of M3R with carbachol significantly increased this association. Expression of M3R in CHO cells promoted plasma membrane localization of YFP-PLCβ3. Deletion of the PLCβ3 C terminus or deletion of the PLCβ3 PDZ ligand inhibited coimmunoprecipitation with M3R and M3R-dependent PLCβ3 plasma membrane localization. Purified PLCβ3 bound directly to glutathione S-transferase (GST)-fused M3R intracellular loops 2 and 3 (M3Ri2 and M3Ri3) as well as M3R C terminus (M3R/H8-CT). PLCβ3 binding to M3Ri3 was inhibited when the PDZ ligand was removed. In assays using reconstituted purified components in vitro, M3Ri2, M3Ri3, and M3R/H8-CT potentiated Gα_q-dependent but not Gβγ-dependent PLCβ3 activation. Disruption of key residues in M3Ri3N and of the PDZ ligand in PLCβ3 inhibited M3Ri3-mediated potentiation. We propose that the M3 muscarinic receptor maximizes the efficiency of PLCβ3 signaling beyond its canonical role as a guanine nucleotide exchange factor for Gα.     

Accumbal 14-3-3ζ protein downregulation is associated with cocaine-conditioned memory

Cocaine-conditioned memory has been known to cause cocaine craving and relapse, while its underlying mechanisms remain unclear. We explored accumbal protein candidates responsible for a cocaine-conditioned memory, cocaine-induced conditioned place preference (CPP). Two-dimensional gel electrophoresis in conjunction with liquid chromatography mass spectrometry analysis was utilized to identify accumbal protein candidates involved in the retrieval of cocaine-induced CPP. Among the identified candidate proteins, a downregulated 14-3-3ζ protein was chosen and confirmed by Western immunoblotting. A polymer-mediated plasmid DNA delivery system was then used to overexpress 14-3-3 protein in mouse nucleus accumbens before the CPP retrieval tests. Overexpression of accumbal 14-3-3ζ protein was found to diminish conditioned cue/context-mediated cocaine-induced CPP. In contrast, another isoform of 14-3-3 protein, 14-3-3ε protein, did not affect conditioned cue/context-mediated cocaine-induced CPP. Overexpression of accumbal 14-3-3ζ protein did not produce motor activity-impairing effect or alter local dopamine metabolism. Moreover, overexpression of accumbal 14-3-3ζ protein did not affect food-induced CPP. These results, taken together, indicated that overexpressed accumbal 14-3-3ζ protein specifically decreased conditioned cue/context-mediated cocaine memory. Further understanding of the function of accumbal 14-3-3ζ protein may shed light on the treatment of cocaine craving and relapse.     

Solvent effect on cation–π interactions with Al3+

Cation-π interactions are known to be one of the strongest noncovalent forces in the gas phase, but they rarely occur in a fully solvated environment. The present work used two different ab initio molecular dynamics-based approaches to describe the correlation between the strength of the cation-π interactions and the number of water molecules surrounding the cation. Five different complexes between an aluminum cation and different molecules containing aromatic rings were studied, and the degree of hydration of each complex was varied. Results indicated that cation-π interactions vanish when the aluminum cation is surrounded by more than three water molecules. The results also highlighted the influence of -OH ligands on the interaction strength.     

Calsyntenin-3 Molecular Architecture and Interaction with Neurexin 1α

Calsyntenin 3 (Cstn3 or Clstn3), a recently identified synaptic organizer, promotes the development of synapses. Cstn3 localizes to the postsynaptic membrane and triggers presynaptic differentiation. Calsyntenin members play an evolutionarily conserved role in memory and learning. Cstn3 was recently shown in cell-based assays to interact with neurexin 1α (n1α), a synaptic organizer that is implicated in neuropsychiatric disease. Interaction would permit Cstn3 and n1α to form a trans-synaptic complex and promote synaptic differentiation. However, it is contentious whether Cstn3 binds n1α directly. To understand the structure and function of Cstn3, we determined its architecture by electron microscopy and delineated the interaction between Cstn3 and n1α biochemically and biophysically. We show that Cstn3 ectodomains form monomers as well as tetramers that are stabilized by disulfide bonds and Ca²⁺, and both are probably flexible in solution. We show further that the extracellular domains of Cstn3 and n1α interact directly and that both Cstn3 monomers and tetramers bind n1α with nanomolar affinity. The interaction is promoted by Ca²⁺ and requires minimally the LNS domain of Cstn3. Furthermore, Cstn3 uses a fundamentally different mechanism to bind n1α compared with other neurexin partners, such as the synaptic organizer neuroligin 2, because Cstn3 does not strictly require the sixth LNS domain of n1α. Our structural data suggest how Cstn3 as a synaptic organizer on the postsynaptic membrane, particularly in tetrameric form, may assemble radially symmetric trans-synaptic bridges with the presynaptic synaptic organizer n1α to recruit and spatially organize proteins into networks essential for synaptic function.     

Mammalian meiosis involves DNA double-strand breaks with 3′ overhangs

Meiotic recombination in yeast is initiated at DNA double-strand breaks (DSBs), processed into 3′ single-strand overhangs that are active in homology search, repair and formation of recombinant molecules. Are 3′ overhangs recombination intermediaries in mouse germ cells too? To answer this question we developed a novel approach based on the properties of the Klenow enzyme. We carried out two different, successive in situ Klenow enzyme-based reactions on sectioned preparations of testicular tubules. Signals showing 3′ overhangs were observed during wild-type mouse spermatogenesis, but not in Spo11 ^?/? males, which lack meiotic DSBs. In Atm ^?/? mice, abundant positively stained spermatocytes were present, indicating an accumulation of non-repaired DSBs, suggesting the involvement of ATM in repair of meiotic DSBs. Thus the processing of DSBs into 3′ overhangs is common to meiotic cells in mammals and yeast, and probably in all eukaryotes.     

Inactivation of Buckwheat α-Glucosidase with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

The amino acid residue(s) involved in the activity of buckwheat α-glucosidase was modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester. The modification resulted in the decrease in the hydrolytic activity of the enzyme following pseudo-first order kinetics. Competitive inhibitors, such as Tris and turanose, protected the enzyme against the inactivation. Protection was provided also by alkali metal, alkaline-earth metal and ammonium ions, though these cations are non-essential for the activity of the enzyme. Turanose or K⁺ protected one carboxyl group per enzyme from the modification with carbodiimide and glycine ethyl ester. Free sulfhydryl group of the enzyme was also partially modified with carbodiimide, but the inactivation was considered to be mainly attributed to the modification of essential carboxyl group rather than to that of free sulfhydryl group.     

Regulation of autophagic activity by 14-3-3ζ proteins associated with class III phosphatidylinositol-3-kinase

14-3-3s are binding proteins with survival functions in cells by interaction with proteins involved in the regulation of cell fate. The role of 14-3-3 during autophagy was investigated, thus, a forced expression of 14-3-3ζ reduces C2-ceramide-induced autophagy, whereas depletion of 14-3-3ζ promotes autophagy. The 14-3-3 role in autophagyc-related proteins was also investigated. The human vacuolar protein sorting 34 (hVps34), the class III phosphatidylinositol-3-kinase mediates multiple vesicle-trafficking processes such as endocytosis and autophagy, its activation being a requirement for autophagy initiation. Using chromatography techniques, hVps34 were eluted from a 14-3-3 affinity column, showing also a direct interaction with 14-3-3 proteins under physiological condition. Further analysis suggests that hVps34/14-3-3 association is a phorbol-12-myristate-13-acetate-dependent phosphorylated mechanism promoting a strong inhibition of the hVps34 lipid kinase activity, proteins kinase C being the likely kinase involved in phosphorylation and 14-3-3 binding of hVps34 under physiological conditions. Meanwhile, stimulation of autophagy leads to the dissociation of the 14-3-3/hVps34 complex enhancing hVps34 lipid kinase activity. Forced expression of 14-3-3ζ reduces hVps34 kinase activity and depletion of 14-3-3ζ promotes upregulation of this activity. In this study, 14-3-3ζ proteins are shown as a negative regulator of autophagy through regulation of a key component of early stages of the autophagy pathway, such as hVps34.     

Interaction of 3’-O-caffeoyl D-quinic acid with multisubunit protein helianthinin

Chlorogenic acid, 3’-O-caffeoyl D-quinic acid, is an inherent ligand present inHelianthus annuus L. The effect of pH on chlorogenic acid binding to helianthinin suggests that maximum binding occurs at pH 6.0. The protein-polyphenol complex precipitates as a function of time. The association constant of the binding of chlorogenic acid to helianthinin, determined by equilibrium dialysis, at 31°C has a value of 3.5 ± 0.1 × 10⁴M^−-1 resulting in a ΔG value of − 6.32 ± 0.12 kcal /mol. The association constantK _ais 1.0 ± 0.1 × 10⁴M⁻¹ as determined by ultraviolet difference spectral titration at 25°C with ΔG° of -5.46 ± 0.06 kcal/mol. From fluorescence spectral titration at 28°C, theK _avalue is 1.38 ± 0.1 × 1 0 4M⁻¹ resulting in a ΔG of − 5.70 ± 0.05 kcal/mol. The total number of binding sites on the protein are 420 ± 50 as calculated from equilibrium dialysis. Microcalorimetric data of the ligand-protein interaction at 23°C suggests mainly two classes of binding. The thermal denaturation temperature,T _mof the protein decreases from 76°C to 72°C at 1 × 10⁻³M chlorogenic acid concentration upon complexation. This suggests that the complexation destabilizes the protein. The effect of temperature onK _aof chlorogenic acid shows a nonlinear increase from 10.2°C to 45°C. Chemical modification of both lysyl and tryptophanyl residues of the protein decreases the strength of binding of chlorogenic acid. Lysine, tryptophan and tyrosine of protein are shown to be present at the binding site. Based on the above data, it is suggested that charge-transfer complexation and entropically driven hydrophobic interaction are the predominant forces that are responsible for binding of chlorogenic acid to the multisubunit protein, helianthinin. Publication No. 324.     

Enantioselective Hydrolysis of α-Cyano-3-phenoxybenzyl Acetate with Arthvobacter Lipase

Lipase-catalyzed enantioselective hydrolysis of the acetic ester of racemic α-cyano-3-phenoxybenzyl alcohol (CPBA) was examined to prepare (S)-CPBA. Most of the lipases tested hydrolyzed (S)-CPBA acetate preferentially, while Candida cylindracea lipase favored (R)-CPBA acetate. Enantioselective hydrolysis by Arthrobacter lipase gave the optically pure (S)-CPBA in the reaction mixture of pH 4.0. The kinetic studies showed that (R)-CPBA acetate reacted as a competitive inhibitor. The Arthrobacter lipase solution in the water/oil biphasic reaction system could be used repeatedly. The lipase immobilized to resins had insufficient activity or low operational stability for the repeated batch reaction. The unhydrolyzed (R)-CPBA acetate was racemized by heating with triethylamine and could be reused as the substrate of the enzymatic hydrolysis. A chemico-enzymatic process for the preparation of (S)-CPBA was developed based on these studies.     

Proteinase 3 carries small unusual carbohydrates and associates with αlpha-defensins

The neutrophil granulocyte is an important first line of defense against intruding pathogens and it contains a range of granules armed with antibacterial peptides and proteins. Proteinase 3 (PR3) is one among several serine proteases of the azurophilic granules in neutrophil granulocytes. Here, we characterize the glycosylation of PR3 and its association with antimicrobial human neutrophil peptides (HNPs, α-defensins) and the effect of these on the mechanism of inhibition of the major plasma inhibitor of PR3, α1-antitrypsin. The glycosylation of purified, mature PR3 showed some heterogeneity with carbohydrates at Asn 102 and 147 carrying unusual small moieties indicating heavy processing. Mass spectrometric analysis and immuno blotting revealed strong association of highly purified PR3 with α-defensins and oligomers hereof. Irreversible inhibition of PR3 by α1-antitrypsin did not affect its association with defensins. Other proteins from neutrophil granules were also found to be associated with defensins, whereas purified plasma proteins did not carry defensins. These results point to a role of defensins in controlling and targeting the activity of neutrophil granule proteins.     

Increased Association of Dynamin Ⅱ with Myosin Ⅱ in Ras Transformed NIH3T3 Cells

Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin Ⅱ has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin Ⅱ and myosin Ⅱ. The dynamin Ⅱ expression level was higher when co-expressed with myosin Ⅱ in Ras transformed NIH3T3 cells than in normal NIH3T3 cells. Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins. The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin Ⅱ inter- acted with myosin Ⅱ. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway, was found to be associated with dynamin Ⅱ and myosin Ⅱ gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin Ⅱ acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.     

Effect of phosphorylation on interaction of human tau protein with 14-3-3ζ

Interaction of the shortest isoform of tau protein (τ3) with human 14-3-3ζ was analyzed by means of native gel electrophoresis, chemical crosslinking and size-exclusion chromatography. Phosphorylation by cAMP-dependent protein kinase (up to 2 mole of phosphate per mole of τ3) strongly enhanced interaction of τ3 with 14-3-3. Apparent K_D of the complexes formed by phosphorylated τ3 and 14-3-3 was close to 2 μM, whereas the corresponding constant for unphosphorylated τ3 was at least 10 times higher. The stoichiometry of the complexes formed by phosphorylated τ3 and 14-3-3 was variable and was different from 1:1. 14-3-3 decreased the probability of formation of chemically crosslinked large homooligomers of phosphorylated τ3 and at the same time induced formation of crosslinked heterooligomeric complexes of τ3 and 14-3-3 with an apparent molecular mass of 120–140 kDa.