Tetrahedral intermediates in thiamin diphosphate-dependent decarboxylations exist as a 1',4'-imino tautomeric form of the coenzyme, unlike the michaelis complex or the free coenzyme

Two circular dichroism signals observed on thiamin diphosphate (ThDP)-dependent enzymes, a positive band in the 300-305 nm range and a negative one in the 320-330 nm range, were investigated on yeast pyruvate decarboxylase (YPDC) and on the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1). Addition of the tetrahedral ThDP-acetaldehyde adduct, 2-alpha-hydroxyethylThDP, to PDHc-E1 generates the positive band at 300 nm, consistent with the formation of the 1',4'-iminopyrimidine tautomer, as also demonstrated for phosphonolactylthiamin diphosphate, a stable analogue of the tetrahedral ThDP-pyruvate adduct 2-alpha-lactylThDP (Jordan, F. et al. (2003) J. Am. Chem. Soc. 125, 12732-12738). Therefore, we suggest that all tetrahedral ThDP-bound covalent complexes will also prefer this tautomer, and that the 4'-aminopyrimidine of ThDP participates in multiple steps of acid-base catalysis on ThDP enzymes. Studies with YPDC and PDHc-E1, and their active center variants, in conjunction with chemical models, enabled assignment of the negative band at 330 nm to a charge-transfer transition between the 4'-aminopyrimidine tautomer (presumed electron donor) and the thiazolium ring (presumed electron acceptor) of ThDP, with no significant contributions from any amino acid side chain of the proteins. However, in both YPDC and PDHc-E1, the presence of substrate or substrate surrogate was required to enable detection, suggesting that the band at 320-330 nm be used as a reporter for the Michaelis complex, involving the amino tautomer, on both enzymes. As the positive band near 300 nm reports on the 1',4'-imino tautomer of ThDP, methods are now available for kinetic monitoring of both tautomeric forms.     

Retrovirus-polymer complexes: study of the factors affecting the dose response of transduction

We have previously shown that complexes of Polybrene (PB), chondroitin sulfate C (CSC), and retrovirus transduce cells more efficiently than uncomplexed virus because the complexes are large and sediment, reaching the cells more rapidly than by diffusion. Transduction reaches a peak at equal weight concentrations of CSC and PB and declines when the dose of PB is higher or lower than CSC. We hypothesized that the nonlinear dose response of transduction was a complex function of the molecular characteristics of the polymers, cell viability, and the number of viruses incorporated into the complexes. To test this hypothesis, we formed complexes using an amphotropic retrovirus and several pairs of oppositely charged polymers and used them to transduce murine fibroblasts. We examined the effect of the type and concentration of polymers used on cell viability, the size and charge of the complexes, the number of viruses incorporated into the complexes, and virus binding and transduction. Transduction was enhanced (2.5- to 5.5-fold) regardless of which polymers were used and was maximized when the number of positive charge groups was in slight excess (15-28%) of the number of negative charge groups. Higher doses of cationic polymer were cytotoxic, whereas complexes formed with lower doses were smaller, contained fewer viruses, and sedimented more slowly. These results show that the dose response of transduction by virus-polymer complexes is nonlinear because excess cationic polymer is cytotoxic, whereas excess anionic polymer reduces the number of active viruses that are delivered to the cells.     

Divergent host responses during primary simian immunodeficiency virus SIVsm infection of natural sooty mangabey and nonnatural rhesus macaque hosts

To understand how natural sooty mangabey hosts avoid AIDS despite high levels of simian immunodeficiency virus (SIV) SIVsm replication, we inoculated mangabeys and nonnatural rhesus macaque hosts with an identical inoculum of uncloned SIVsm. The unpassaged virus established infection with high-level viral replication in both macaques and mangabeys. A species-specific, divergent immune response to SIV was evident from the first days of infection and maintained in the chronic phase, with macaques showing immediate and persistent T-cell proliferation, whereas mangabeys displayed little T-cell proliferation, suggesting subdued cellular immune responses to SIV. Importantly, only macaques developed (CD4+)-T-cell depletion and AIDS, thus indicating that in mangabeys limited immune activation is a key mechanism to avoid immunodeficiency despite high levels of SIVsm replication. These studies demonstrate that it is the host response to infection, rather than properties inherent to the virus itself, that causes immunodeficiency in SIV-infected nonhuman primates.     

Molecular Implications of Repeated Aggression: Th,Dat1, Snca and Bdnf Gene Expression in the VTA of Victorious Male Mice

Background

It is generally recognized that recurrent aggression can be the result of various psychiatric disorders. The aim of our study was to analyze the mRNA levels, in the ventral tegmental area (VTA) of the midbrain, of the genes that may possibly be associated with aggression consistently shown by male mice in special experimental settings.

Methodology/Principal Findings

The genes were Th, Dat1, Snca and Bdnf; the male mice were a group of animals that had each won 20 daily encounters in succession and a group of animals that had the same winning track record followed by a no-fight period for 14 days. Increased Th, Dat1 and Snca mRNA levels were in the fresh-from-the-fight group as compared to the controls. Increased Th and Dat1 mRNA levels were in the no-fight winners as compared to the controls. Significant positive correlations were found between the level of aggression and Th and Snca mRNA levels.

Conclusions

Repeated positive fighting experience enhances the expression of the Th, Dat1 and Snca genes, which are associated with brain dopaminergic systems. The expression of the Th and Dat1 genes stays enhanced for a long time.     

From synaptic spines to nuclear signaling: nuclear and synaptic actions of the amyloid precursor protein

Despite intensive studies of the secretase‐mediated processing of the amyloid precursor protein (APP) to form the amyloid β‐peptide (Aβ), in relation to Alzheimer's disease (AD), no new therapeutic agents have reached the clinics based on reducing Aβ levels through the use of secretase inhibitors or immunotherapy. Furthermore, the normal neuronal functions of APP and its various metabolites still remain under‐investigated and unclear. Here, we highlight emerging areas of APP function that may provide new insights into synaptic development, cognition, and gene regulation. By modulating expression levels of endogenous APP in primary cortical neurons, the frequency and amplitude of calcium oscillations is modified, implying a key role for APP in maintaining neuronal calcium homeostasis essential for synaptic transmission. Disruption of this homeostatic mechanism predisposes to aging and AD. Synaptic spine loss is a feature of neurogeneration resulting in learning and memory deficits, and emerging evidence indicates a role for APP, probably mediated via one or more of its metabolites, in spine structure and functions. The intracellular domain of APP (AICD) has also emerged as a key epigenetic regulator of gene expression controlling a diverse range of genes, including APP itself, the amyloid‐degrading enzyme neprilysin, and aquaporin‐1. A fuller understanding of the physiological and pathological actions of APP and its metabolic network could provide new opportunities for therapeutic intervention in AD.     

(E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily

Snapdragon flowers emit two monoterpene olefins, myrcene and (E)-beta-ocimene, derived from geranyl diphosphate, in addition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene synthases (ama1e20 and ama0c15) and an (E)-beta-ocimene synthase (ama0a23). Although the two myrcene synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. (E)-beta-Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly (E)-beta-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-beta-ocimene and myrcene. These newly isolated snapdragon monoterpene synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx(8)W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene synthases, suggesting that the reaction mechanism of Tps-g monoterpene synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S-linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production.     

с-Met receptor can be activated by extracellular alkaline medium

Receptor tyrosine kinase (RTK) Met or c-Met is a target of hepatocyte growth factor (HGF) and it plays an important role under normal and pathological conditions. Activation of Met signaling pathway is associated with several cellular processes, such as proliferation, survival, motility, angiogenesis, invasion, and metastasis. In this article, we describe the ability of Met to activate upon a mild alkali treatment. To identify potential alkali-regulated proteins, CAKI-1 cells were treated with alkaline media and further tested for protein phosphorylation changes. By anti-phosphotyrosine antibody precipitation and lectin chromatography, we identified Met as a major cytoplasmic membrane protein that responded to pH changes by its phosphorylation. The activation of Met by alkali occurred at pH >8.0 and was dose-dependent. Specificity of the Met response to alkali was confirmed by the treatment with Met kinase inhibitor SU11274 and also by Met receptor knockout using CRISPR/CAS9 genome editing system. Both approaches completely blocked the Met phosphorylation response in CAKI-1 cells. Similar pH-dependent Met activation was observed in the HeLa cell line. Our data suggest existence of ligand-independent mechanism of Met receptor activation.     

Brain Intraventricular Injection of Amyloid-β in Zebrafish Embryo Impairs Cognition and Increases Tau Phosphorylation,Effects Reversed by Lithium

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder with no effective treatment and commonly diagnosed only on late stages. Amyloid-β (Aβ) accumulation and exacerbated tau phosphorylation are molecular hallmarks of AD implicated in cognitive deficits and synaptic and neuronal loss. The Aβ and tau connection is beginning to be elucidated and attributed to interaction with different components of common signaling pathways. Recent evidences suggest that non-fibrillary Aβ forms bind to membrane receptors and modulate GSK-3β activity, which in turn phosphorylates the microtubule-associated tau protein leading to axonal disruption and toxic accumulation. Available AD animal models, ranging from rodent to invertebrates, significantly contributed to our current knowledge, but complementary platforms for mechanistic and candidate drug screenings remain critical for the identification of early stage biomarkers and potential disease-modifying therapies. Here we show that Aβ1–42 injection in the hindbrain ventricle of 24 hpf zebrafish embryos results in specific cognitive deficits and increased tau phosphorylation in GSK-3β target residues at 5dpf larvae. These effects are reversed by lithium incubation and not accompanied by apoptotic markers. We believe this may represent a straightforward platform useful to identification of cellular and molecular mechanisms of early stage AD-like symptoms and the effects of neuroactive molecules in pharmacological screenings.