A two-state model for Ca2+/CaM-dependent protein kinase II (alphaCaMKII) in response to persistent Ca2+ stimulation in hippocampal neurons

Persistent elevation of the intracellular free Ca(2+) concentration [Ca(2+)](i) is neurotoxic and therefore it is important to understand how it affects downstream components of the Ca(2+) signaling pathway. The response of calmodulin (CaM) and alphaCa(2+)/CaM-dependent protein kinase II (alphaCaMKII), to intracellular Ca(2+) overload in hippocampal neurons is studied by confocal imaging of fluorescently tagged proteins. Transient and persistent redistribution of CaM and alphaCaMKII together is seen from the cytosol to dendritic and somatic punctae. Typical persistent redistribution occurs following a lag of 138+/-(S.E.M.) 12 s and is complete at 460+/-(S.E.M.) 34 s (n=18), lack of Thr(286)-autophosphorylation of alphaCaMKII however promotes the formation of early transient punctae (peak at 40 s). In contrast, the T286D-mimick of phospho-Thr(286)-alphaCaMKII forms punctae with a delay >10 min, indicating that Thr(286)-autophosphorylation is antagonistic to CaMKII clustering. A two-state model is proposed in which phospho-Thr(286)-alphaCaMKII, formed immediately upon Ca(2+) stimulation, is primarily responsible for target interactions and memory functions of alphaCaMKII. However, a distinct clustering form denoted alphaCaMKII(c), generated upon persistent intracellular free Ca(2+) elevation, is deposited in the punctae which are made of self-interacting CaM/CaMKII complexes. Punctate deposition disables both the interactions and the activity of CaMKII.     

Copper(II) binding to two novel histidine-containing model hexapeptides: evidence for a metal ion driven turn conformation

The solution conformation and the copper(II) binding properties have comparatively been investigated for the two novel hexapeptides Ac-HPSGHA-NH₂ (P2) and Ac-HGSPHA-NH₂ (P4). The study has been carried out by means of CD, NMR, EPR and UV-Vis spectroscopic techniques in addition to potentiometric measurements to determine the stability constants of the different copper(II) complex species formed in the pH range 3-11. The peptides contain two histidine residues as anchor sites for the metal ion and differ only for the exchanged position of the proline residue with glycine. CD and NMR results for the uncomplexed peptide ligands suggest a predominantly unstructured peptide chain in aqueous solution. Potentiometric and spectroscopic data (UV-Vis, CD and EPR) show that both peptides strongly interact with copper(II) ions by forming complexes with identical stoichiometries but different structures. Furthermore, Far-UV CD experiments indicate that the conformation of the peptides is dramatically affected following copper(II) complexation with the P4 peptide adopting a β-turn-like conformation.     

5-HT<Subscript>6/7</Subscript> Receptor Antagonists Facilitate Dopamine Release in the Cochlea via a GABAergic Disinhibitory Mechanism

In humans, serotonin (5-HT) has been implicated in numerous physiological and pathological processes in the peripheral auditory system. Dopamine (DA), another transmitter of the lateral olivocochlear (LOC) efferents making synapses on cochlear nerve dendrites, controls auditory nerve activation and protects the sensory nerve against overactivation. Using in vitro microvolume superfusion techniques we tested 5-HT₆ and 5-HT₇ receptor antagonists whether they can influence dopamine (DA) release from the guinea-pig cochlea in control and in ischemic conditions using currently available and new 5-HT₆ and 5-HT₇ antagonists and mixed antagonists, which were synthesized and characterized for the current study. While the 5-HT₇ antagonist SB-258719 was ineffective, SB-271046, which blocks the 5-HT₆ receptor, caused a significant increase in cochlear DA release what is contradictory with the excitatory nature of this type of receptor. Moreover, the mixed 5-HT_6/7 antagonist EGIS-12233 induced an even more pronounced increase in the resting DA release. To understand why the block of an excitatory receptor results in an increase instead of a decrease in function, we investigated the possible involvement of an indirect neural mechanism through an inhibitory system. In the presence of the GABA_A receptor blocker bicuculline, EGIS-12233 failed to increase the release of DA, suggesting that the serotonin receptor modulation of DA release from the lateral olivocochlear efferents in the cochlea was produced indirectly by decreasing the GABAergic inhibitory tone on dopaminergic nerve endings. The mixed 5-HT₇/D₄ receptor antagonist EGIS-11983 significantly increased both the stimulation-evoked and the resting DA release, while the selective D4 blocker L-741,741 alone had no significant effect. Ischemia, simulated by oxygen and glucose deprivation from the perfusion solution had no action on the effect of the drugs. Drugs that can increase the release of DA from LOC terminals in the cochlea may have a role in the treatment of sensorineural hearing loss.     

Changes of organic matter quality along the longitudinal axis of a large shallow lake (Lake Balaton)

Hydrobiologia - Dissolved organic matter (DOM) is quantitatively the most significant pool of organic matter in lakes. Within DOM, the pool of dissolved organic carbon (DOC) is dominated...     

A Cucumber Mosaic Virus Based Expression System for the Production of Porcine Circovirus Specific Vaccines

Potential porcine circovirus type 2 (PCV2) capsid protein epitopes, suitable for expression on the surface of cucumber mosaic virus (CMV) particles were determined by a thorough analysis of the predicted PCV capsid protein structure. The ab initio protein structure prediction was carried out with fold recognition and threading methods. The putative PCV epitopes were selected on the basis of PCV virion models and integrated into the plant virus coat protein, after amino acid position 131. The recombinants were tested for infectivity and stability on different Nicotiana species and stable recombinant virus particles were purified. The particles were tested for their ability to bind to PCV induced porcine antibodies and used for specific antibody induction in mice and pigs. The results showed that PCV epitopes expressed on the CMV surface were recognized by the porcine antibodies and they were also able to induce PCV specific antibody response. Challenge experiment with PCV2 carried out in immunized pigs showed partial protection against the infection. Based on these results it was concluded that specific antiviral vaccine production for the given pathogen was feasible, offering an inexpensive way for the mass production of such vaccines.     

Genetic control of the quantitative variation of erythrocytic glyoxalase-1 (GLO-1) in mice

Quantitative genetic variation in the glyoxalase-1 content (QGlo-1) of red cells of mice is described. Its genetic control is shown to be exerted by either the Glo-1 locus or a closely linked gene to the left of H-2K. At least six alleles, designated QGlo-1 ^a through QGlo-1 ^f, can be found in different inbred strains of mice.This work was supported in part by Grants HL 0911 and AI 15413 of the National Institutes of Health and by a grant from the Sally and Alma Solomon Foundation.     

Role of Guanine Nucleotide Exchange Factor-H1 in Complement-mediated RhoA Activation in Glomerular Epithelial Cells

Visceral glomerular epithelial cells (GEC), also known as podocytes, are vital for the structural and functional integrity of the glomerulus. The actin cytoskeleton plays a central role in maintaining GEC morphology. In a rat model of experimental membranous nephropathy (passive Heymann nephritis (PHN)), complement C5b-9-induced proteinuria was associated with the activation of the actin regulator small GTPase, RhoA. The mechanisms of RhoA activation, however, remained unknown. In this study, we explored the role of the epithelial guanine nucleotide exchange factor, GEF-H1, in complement-induced RhoA activation. Using affinity precipitation to monitor GEF activity, we found that GEF-H1 was activated in glomeruli isolated from rats with PHN. Complement C5b-9 also induced parallel activation of GEF-H1 and RhoA in cultured GEC. In GEC in which GEF-H1 was knocked down, both basal and complement-induced RhoA activity was reduced. On the other hand, GEF-H1 knockdown augmented complement-mediated cytolysis, suggesting a role for GEF-H1 and RhoA in protecting GEC from cell death. The MEK1/2 inhibitor, U0126, and mutation of the ERK-dependent phosphorylation site (T678A) prevented complement-induced GEF-H1 activation, indicating a role for the ERK pathway. Further, complement induced GEF-H1 and microtubule accumulation in the perinuclear region. However, both the perinuclear accumulation and the activation of GEF-H1 were independent of microtubules and myosin-mediated contractility, as shown using drugs that interfere with microtubule dynamics and myosin II activity. In summary, we have identified complement-induced ERK-dependent GEF-H1 activation as the upstream mechanism of RhoA stimulation, and this pathway has a protective role against cell death.     

Subgroups of Paediatric Acute Lymphoblastic Leukaemia Might Differ Significantly in Genetic Predisposition to Asparaginase Hypersensitivity

L-asparaginase (ASP) is a key element in the treatment of paediatric acute lymphoblastic leukaemia (ALL). However, hypersensitivity reactions (HSRs) to ASP are major challenges in paediatric patients. Our aim was to investigate genetic variants that may influence the risk to Escherichia coli-derived ASP hypersensitivity. Sample and clinical data collection was carried out from 576 paediatric ALL patients who were treated according to protocols from the Berlin—Frankfurt—Münster Study Group. A total of 20 single nucleotide polymorphisms (SNPs) in GRIA1 and GALNT10 genes were genotyped. Patients with GRIA1 rs4958351 AA/AG genotype showed significantly reduced risk to ASP hypersensitivity compared to patients with GG genotype in the T-cell ALL subgroup (OR = 0.05 (0.01–0.26); p = 4.70E-04), while no such association was found in pre-B-cell ALL. In the medium risk group two SNPs of GRIA1 (rs2055083 and rs707176) were associated significantly with the occurrence of ASP hypersensitivity (OR = 0.21 (0.09–0.53); p = 8.48E-04 and OR = 3.02 (1.36–6.73); p = 6.76E-03, respectively). Evaluating the genders separately, however, the association of rs707176 with ASP HSRs was confined only to females. Our results suggest that genetic variants of GRIA1 might influence the risk to ASP hypersensitivity, but subgroups of patients can differ significantly in this respect.