Systemic Neurochemical Alterations in Schizophrenic Brain: Glutamate Metabolism in Focus

We have used a systemic approach to establish a relationship between enzyme measures of glial glutamate and energy metabolism (glutamine synthetase and glutamine synthetase-like protein, glutamate dehydrogenase isoenzymes, brain isoform creatine phosphokinase) and two major glial proteins (glial fibrillary acidic protein and myelin basic protein) in autopsied brain samples taken from patients with schizophrenia (SCH) and mentally healthy subjects (23 and 22 cases, respectively). These biochemical parameters were measured in tissue extracts in three brain areas (prefrontal cortex, caudate nucleus, and cerebellum). Significant differences in the level of at least one of the glutamate metabolizing enzymes were observed between two studied groups in all studied brain areas. Different patterns of correlative links between the biochemical parameters were found in healthy and schizophrenic brains. These findings give a new perspective to our understanding of the impaired regulation of enzyme levels in the brain in SCH.     

Ubiquitination Increases Parkin Activity to Promote Autophagic α-Synuclein Clearance

Parkinson’s disease (PD) is a movement disorder associated with genetic and age related causes. Although autosomal recessive early onset PD linked to parkin mutations does not exhibit α-Synuclein accumulation, while autosomal dominant and sporadic PD manifest with α-Synuclein inclusions, loss of dopaminergic substantia nigra neurons is a common denominator in PD. Here we show that decreased parkin ubiquitination and loss of parkin stability impair interaction with Beclin-1 and alter α-Synuclein degradation, leading to death of dopaminergic neurons. Tyrosine kinase inhibition increases parkin ubiquitination and interaction with Beclin-1, promoting autophagic α-Synuclein clearance and nigral neuron survival. However, loss of parkin via deletion increases α-Synuclein in the blood compared to the brain, suggesting that functional parkin prevents α-Synuclein release into the blood. These studies demonstrate that parkin ubiquitination affects its protein stability and E3 ligase activity, possibly leading to α-Synuclein sequestration and subsequent clearance.     

Prenatal Stress in Maternal Hyperhomocysteinemia: Impairments in the Fetal Nervous System Development and Placental Function

Biochemistry (Moscow) - The article presents current views on maternal hyperhomocysteinemia (HHcy) as an important factor causing prenatal stress and impaired nervous system development in fetuses...     

Concordance of Epileptic Networks Associated with Epileptic Spikes Measured by High-Density EEG and Fast fMRI

Objective

The present study aims to investigate whether a newly developed fast fMRI called MREG (magnetic resonance encephalography) measures metabolic changes related to interictal epileptic discharges (IED). For this purpose BOLD changes are correlated with the IED distribution and variability.

Methods

Patients with focal epilepsy underwent EEG-MREG using a 64 channel cap. IED voltage maps were generated using 32 and 64 channels and compared regarding their correspondence to the BOLD response. The extents of IEDs (defined as number of channels with >50% of maximum IED negativity) were correlated with the extents of positive and negative BOLD responses. Differences in inter-spike variability were investigated between interictal epileptic discharges (IED) sets with and without concordant positive or negative BOLD responses.

Results

17 patients showed 32 separate IED types. In 50% of IED types the BOLD changes could be confirmed by another independent imaging method. The IED extent significantly correlated with the positive BOLD extent (p = 0.04). In 6 patients the 64-channel EEG voltage maps better reflected the positive or negative BOLD response than the 32-channel EEG; in all others no difference was seen. Inter-spike variability was significantly lower in IED sets with than without concordant positive or negative BOLD responses (with p = 0.04).

Significance

Higher density EEG and fast fMRI seem to improve the value of EEG-fMRI in epilepsy. The correlation of positive BOLD and IED extent could suggest that widespread BOLD responses reflect the IED network. Inter-spike variability influences the likelihood to find IED concordant positive or negative BOLD responses, which is why single IED analysis may be promising.     

The C-Terminal Part of Microcin B Is Crucial for DNA Gyrase Inhibition and Antibiotic Uptake by Sensitive Cells

Microcin B (McB) is a ribosomally synthesized antibacterial peptide. It contains up to nine oxazole and thiazole heterocycles that are introduced posttranslationally and are required for activity. McB inhibits the DNA gyrase, a validated drug target. Previous structure-activity analyses indicated that two fused heterocycles located in the central part of McB are important for antibacterial action and gyrase inhibition. Here, we used site-specific mutagenesis of the McB precursor gene to assess the functional significance of the C-terminal part of McB that is located past the second fused heterocycle and contains two single heterocycles as well as an unmodified four-amino-acid C-terminal tail. We found that removal of unmodified C-terminal amino acids of McB, while having no effect on fused heterocycles, has a very strong negative effect on activity in vivo and in vitro. In fact, even nonconservative point substitutions in the last McB amino acid have a very strong effect by simultaneously decreasing uptake and ability to inhibit the gyrase. The results highlight the importance of unmodified McB amino acids for function and open the way for creation of recombinant McB derivatives with an altered or expanded spectrum of antibacterial action.     

Macropinocytosis in Shiga toxin 1 uptake by human intestinal epithelial cells and transcellular transcytosis

Shiga toxin 1 and 2 production is a cardinal virulence trait of enterohemorrhagic Escherichia coli infection that causes a spectrum of intestinal and systemic pathology. However, intestinal sites of enterohemorrhagic E. coli colonization during the human infection and how the Shiga toxins are taken up and cross the globotriaosylceramide (Gb3) receptor-negative intestinal epithelial cells remain largely uncharacterized. We used samples of human intestinal tissue from patients with E. coli O157:H7 infection to detect the intestinal sites of bacterial colonization and characterize the distribution of Shiga toxins. We further used a model of largely Gb3-negative T84 intestinal epithelial monolayers treated with B-subunit of Shiga toxin 1 to determine the mechanisms of non-receptor-mediated toxin uptake. We now report that E. coli O157:H7 were found at the apical surface of epithelial cells only in the ileocecal valve area and that both toxins were present in large amounts inside surface and crypt epithelial cells in all tested intestinal samples. Our in vitro data suggest that macropinocytosis mediated through Src activation significantly increases toxin endocytosis by intestinal epithelial cells and also stimulates toxin transcellular transcytosis. We conclude that Shiga toxin is taken up by human intestinal epithelial cells during E. coli O157:H7 infection regardless of the presence of bacterial colonies. Macropinocytosis might be responsible for toxin uptake by Gb3-free intestinal epithelial cells and transcytosis. These observations provide new insights into the understanding of Shiga toxin contribution to enterohemorrhagic E. coli-related intestinal and systemic diseases.     

Staphylococcus epidermidis polysaccharide intercellular adhesin activates complement

Staphylococcus epidermidis is a frequent cause of nosocomial infections. The central virulence factor of S. epidermidis is biofilm formation. Polysaccharide intercellular adhesin (PIA) constitutes the major biofilm matrix-component. PIA and biofilm have been implicated in S. epidermidis evasion of host immune defence. We examined the effects of S. epidermidis PIA on the inflammatory response with focus on complement activation. We used a human whole-blood ex vivo model of infection and compared the effects of a PIA-positive S. epidermidis strain (SE1457) and its PIA-negative isogenic mutant (M10). The independent effect of purified PIA on complement activation was investigated. In glucose-rich media, the mutant formed a proteinacious DNA-rich biofilm, whereas SE1457 formed a thick PIA-biofilm. In biofilm growth, SE1457 induced a stronger activation of the complement system compared with M10. We verified that purified PIA was independently responsible for a strong activation of the complement system. In contrast, M10 induced higher granulocyte activation by expression of CD11b and higher secretion of cytokines. We conclude that PIA has potent pro-inflammatory properties by activating the complement system. However, in a complex balance of the immune response, the decreased activation of granulocytes and cytokines by a PIA biofilm may limit host eradication of S. epidermidis.     

Life at the border: Adaptation of proteins to anisotropic membrane environment

This review discusses main features of transmembrane (TM) proteins which distinguish them from water‐soluble proteins and allow their adaptation to the anisotropic membrane environment. We overview the structural limitations on membrane protein architecture, spatial arrangement of proteins in membranes and their intrinsic hydrophobic thickness, co‐translational and post‐translational folding and insertion into lipid bilayers, topogenesis, high propensity to form oligomers, and large‐scale conformational transitions during membrane insertion and transport function. Special attention is paid to the polarity of TM protein surfaces described by profiles of dipolarity/polarizability and hydrogen‐bonding capacity parameters that match polarity of the lipid environment. Analysis of distributions of Trp resides on surfaces of TM proteins from different biological membranes indicates that interfacial membrane regions with preferential accumulation of Trp indole rings correspond to the outer part of the lipid acyl chain region—between double bonds and carbonyl groups of lipids. These “midpolar” regions are not always symmetric in proteins from natural membranes. We also examined the hydrophobic effect that drives insertion of proteins into lipid bilayer and different free energy contributions to TM protein stability, including attractive van der Waals forces and hydrogen bonds, side‐chain conformational entropy, the hydrophobic mismatch, membrane deformations, and specific protein–lipid binding.     

Salvaging Pyrococcus furiosus Protein Targets at SECSG

Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.     

HIM1, a new yeast Saccharomyces cerevisiae gene playing a role in control of spontaneous and induced mutagenesis

We have identified a new Saccharomyces cerevisiae gene, HIM1, mapped on the right arm of the chromosome IV (ORF YDR317w), mutations in which led to an increase in spontaneous mutation rate and elevated the frequencies of mutations, induced by UV-light, nitrous acid, ethylmethane sulfonate and methylmethane sulfonate. At the same time, him1 mutation did not result in the increase of the sensitivity to the lethal action of these DNA-damaging agents. We tested the induced mutagenesis in double mutants carrying him1 mutation and mutations in other repair genes: apn1, blocking base excision repair; rad2, rev3, and rad54, blocking three principal DNA repair pathways; pms1, blocking mismatch repair; hsm2 and hsm3 mutations, which lead to a mutator effect. Epistatic analysis showed a synergistic interaction of him1 with pms1, apn1, and rad2 mutations, and epistasis with the rev3, the rad54, the hsm2, and the hsm3. To elucidate the role of the HIM1 in control of spontaneous mutagenesis, we checked the repair of DNA mispaired bases in the him1 mutant and discovered that it was not altered in comparison to the wild-type strain. In our opinion, our results suggest that HIM1 gene participates in the control of processing of mutational intermediates appearing during error-prone bypass of DNA damage.