Significance of the degree of synaptic Zn<Superscript>2+</Superscript> signaling in cognition

Zinc is a trace nutrient for the brain and a signal factor to serve for brain function. A portion of zinc is released from glutamatergic (zincergic) neuron terminals in the brain. Synaptic Zn²⁺ signaling is involved in synaptic plasticity such as long-term potentiaion (LTP), which is a cellular mechanism of memory. The block and/or loss of synaptic Zn²⁺ signaling in the hippocampus and amygdala with Zn²⁺ chelators affect cognition, while the role of synaptic Zn²⁺ signal is poorly understood, because zinc-binding proteins are great in number and multi-functional. Chronic zinc deficiency also affects cognition and cognitive decline induced by zinc deficiency might be associated with the increase in plasma glucocorticoid rather than the decrease in synaptic Zn²⁺ signaling. On the other hand, excess glutamatergic (zincergic) neuron activity induces excess influx of extracellular Zn²⁺ into hippocampal neurons, followed by cognitive decline. Intracellular Zn²⁺ dynamics, which is linked to presynaptic glutamate release, is critical for LTP and cognitive performance. This paper deals with insight into cognition from zinc as a nutrient and signal factor.     

Phylogenetics of family Enterobacteriaceae and proposal to reclassify Escherichia hermannii and Salmonella subterranea as Atlantibacter hermannii and Atlantibacter subterranea gen. nov., comb. nov.

Multilocus sequence analysis based on hypervariable housekeeping proteins was utilized to differentiate closely related species in the family Enterobacteriaceae. Of 150 housekeeping proteins, the top 10 hypervariable proteins were selected and concatenated to obtain distance data. Distances between concatenated proteins within the family were 0.9–41.2%, whereas the 16S rRNA and atpD‐gyrB‐infB‐rpoB concatenated sequence (4MLSA) distances were 0.8–6.0% and 0.9–22.1%, respectively. These data indicate that phylogenetic analysis by concatenation of hypervariable proteins is a powerful tool for discriminating species in the family Enterobacteriaceae. To confirm the discriminatory power of the 10 chosen concatenated hypervariable proteins (C10HKP), phylogenetic trees based on C10HKP, 4MLSA, and the 16S rRNA gene were constructed. Comparison of average bootstrap values among C10HKP, 4MLSA and 16S rRNA genes indicated that the C10HKP tree was the most reliable. Location via the C10HKP tree was consistent with existing assignments for almost all species in the family Enterobacteriaceae. However, the C10HKP tree suggested that several species (including Enterobacter massiliensis, Escherichia vulneris, Escherichia hermannii, and Salmonella subterranea) should be reassigned to different clusters than those defined in previous analyses. Furthermore, E. hermannii and S. subterranea appeared to fall onto a branch independent from those occupied by the other Enterobacteriaceae. Therefore, we propose Atlantibacter gen. nov., such that E. hermannii and S. subterranea would be transferred to genus Atlantibacter as Atlantibacter hermannii, comb. nov. and Atlantibacter subterranea. comb. nov., respectively.     

Classification of ligand molecules in PDB with graph match-based structural superposition

The fast heuristic graph match algorithm for small molecules, COMPLIG, was improved by adding a structural superposition process to verify the atom–atom matching. The modified method was used to classify the small molecule ligands in the Protein Data Bank (PDB) by their three-dimensional structures, and 16,660 types of ligands in the PDB were classified into 7561 clusters. In contrast, a classification by a previous method (without structure superposition) generated 3371 clusters from the same ligand set. The characteristic feature in the current classification system is the increased number of singleton clusters, which contained only one ligand molecule in a cluster. Inspections of the singletons in the current classification system but not in the previous one implied that the major factors for the isolation were differences in chirality, cyclic conformations, separation of substructures, and bond length. Comparisons between current and previous classification systems revealed that the superposition-based classification was effective in clustering functionally related ligands, such as drugs targeted to specific biological processes, owing to the strictness of the atom–atom matching.     

Involvement of the Type IX Secretion System in Capnocytophaga ochracea Gliding Motility and Biofilm Formation

Capnocytophaga ochracea is a Gram-negative, rod-shaped bacterium that demonstrates gliding motility when cultured on solid agar surfaces. C. ochracea possesses the ability to form biofilms; however, factors involved in biofilm formation by this bacterium are unclear. A type IX secretion system (T9SS) in Flavobacterium johnsoniae was shown to be involved in the transport of proteins (e.g., several adhesins) to the cell surface. Genes orthologous to those encoding T9SS proteins in F. johnsoniae have been identified in the genome of C. ochracea; therefore, the T9SS may be involved in biofilm formation by C. ochracea. Here we constructed three ortholog-deficient C. ochracea mutants lacking sprB (which encodes a gliding motility adhesin) or gldK or sprT (which encode T9SS proteins in F. johnsoniae). Gliding motility was lost in each mutant, suggesting that, in C. ochracea, the proteins encoded by sprB, gldK, and sprT are necessary for gliding motility, and SprB is transported to the cell surface by the T9SS. For the ΔgldK, ΔsprT, and ΔsprB strains, the amounts of crystal violet-associated biofilm, relative to wild-type values, were 49%, 34%, and 65%, respectively, at 48 h. Confocal laser scanning and scanning electron microscopy revealed that the biofilms formed by wild-type C. ochracea were denser and bacterial cells were closer together than in those formed by the mutant strains. Together, these results indicate that proteins exported by the T9SS are key elements of the gliding motility and biofilm formation of C. ochracea.     

Granular tau oligomers as intermediates of tau filaments

Neurofibrillary tangles (NFTs) are pathological hallmarks of several neurodegenerative disorders, including Alzheimer's disease (AD). NFTs are composed of microtubule-binding protein tau, which assembles to form paired helical filaments (PHFs) and straight filaments. Here we show by atomic force microscopy that AD brain tissue and in vitro tau form granular and fibrillar tau aggregates. CD spectral analysis and immunostaining with conformation-dependent antibodies indicated that tau may undergo conformational changes during fibril formation. Enriched granules generated filaments, suggesting that granular tau aggregates may be an intermediate form of tau fibrils. The amount of granular tau aggregates was elevated in prefrontal cortex of Braak stage I cases compared to that of Braak stage 0 cases, suggesting that granular tau aggregation precedes PHF formation. Thus, granular tau aggregates may be a relevant marker for the early diagnosis of tauopathy. Reducing the level of these aggregates may be a promising therapy for tauopathies and for promoting healthy brain aging.     

Heme synthase (ferrochelatase) catalyzes the removal of iron from heme and demetalation of metalloporphyrins

The red pigments in meat products, including cooked cured ham, arise from the reaction of myoglobin with nitric oxide generated from exogenous nitrite. Since carcinogenic nitrosoamines may be generated by the treatment of meats with nitrite, the production of nitrite-free meat products is an attractive alternative. Raw dry-cured (Parma) hams are produced by the treatment of meats with salts other than nitrite. Analysis of pigments in raw dry-cured hams reveals that the main pigment is zinc protoporphyrin, suggesting that the conversion of heme to zinc protoporphyrin occurs via an iron-removal reaction from myoglobin heme during the processing of raw hams. Purification of the iron-removal enzyme showed that it was identical to ferrochelatase. Recombinant ferrochelatase in combination with NADH-cytochrome b5 reductase catalyzed NADH-dependent iron-removal reaction from hemin and hemoproteins. Metal ions such as zinc and cobalt were also removed from the corresponding metalloporphyrins. The addition of zinc ions led to the formation of zinc protoporphyrin. In cultured cells, the conversion of zinc mesoporphyrin to mesoheme was observed to be dependent on ferrochelatase and could be markedly induced during erythroid differentiation. This is the first demonstration of a new enzyme reaction, the reverse reaction of ferrochelatase, which may contribute to a new route of the recycling of protoporphyrin and heme in cells.     

Topology of acyltransferase motifs and substrate specificity and accessibility in 1-acyl-sn-glycero-3-phosphate acyltransferase 1

1-acyl-sn-glycero-3-phosphate (AGP) acyltransferases (AGPAT) are involved in de novo biosynthesis of glycerolipids, such as phospholipids and triacylglycerol. Alignment of amino acid sequences from AGPAT, sn-glycerol-3-phosphate acyltransferase, and dihydroxyacetonephosphate acyltransferase reveals four regions with strong homology (acyltransferase motifs I-IV). The invariant amino acids within these regions may be part of a catalytically important site in this group of acyl-CoA acyltransferases. However, in human AGPAT1 a transmembrane domain is predicted to separate motif I on the cytosolic side from motifs II-III on the lumenal side, with motif IV near surface of the membrane. The topology of motifs I and III was confirmed by experiments with recombinant AGPAT1 containing potential glycosylation site near the motifs. This topology conflicts with the expectation that catalytically important sites are near one another, raising questions of whether the acyltransferase motifs really are important for AGPAT catalysis, and how substrates access motifs II-III on the lumenal side of the endoplasmic reticulum membrane. Using human AGPAT1 as a model, we have examined the catalytic roles of highly conserved residues in the four acyltransferase motifs by site-directed mutagenesis. Modifications of the sidechain structures of His104, Asp109, Phe146, Arg149, Glu178, Gly179, Thr180, Arg181 and Ile208 all affected AGPAT1 activity, indicating that the acyltransferase motifs indeed are important for AGPAT catalysis. In addition, we examined substrate accessibility to the catalytic domain of human AGPAT1 using a competition assay. Lysophosphatidic acid (LPA) with fatty acid chains shorter than 10 carbons did not access the catalytic domain, suggesting that LPA hydrophobicity is important. In contrast, short chain acyl-CoAs did access the catalytic domain but did not serve as the second substrate. These results suggest that motifs II and III are involved in LPA binding and motifs I and IV are involved in acyl-CoA binding.     

Effects of genetic backgrounds on hyperbilirubinemia in radixin-deficient mice due to different expression levels of Mrp3

ERM (ezrin/radixin/moesin) proteins are organizers of apical actin cortical layer in general. We previously reported that the knockout of radixin resulted in Rdx(-/-) mice with displacement/loss of the canalicular transporter Mrp2, giving rise to Dubin-Johnson syndrome-like conjugated hyperbilirubinemia in the mixed genetic background (C57BL/6-129/Sv) (Kikuchi, et al. (2002) Nature Genetics 31, 320-325). However, when these mice were kept under mixed genetic background for years (late mixed backgrounds; LMB), the conjugated hyperbilirubinemia gradually became inconspicuous, while evidence of liver injury increased. We examined the effect of genetic background by backcrossing LMB Rdx(-/-) mice to C57BL/6 and 129/Sv wild type mice with the result that the Rdx(-/-) congenic mice regained hyperbilirubinemia with reduced hepatocellular damage. As revealed by immunofluorescence and western blots, the localization/expression of apical transporters, Mrp2, CD26, P-gps, and Bsep were not influenced by backcrossing, though those of a basolateral transporter, Mrp3, were strikingly increased by backcrossing.     

Rho-kinase regulates endothelin-1-stimulated IL-6 synthesis via p38 MAP kinase in osteoblasts

We have previously reported that endothelin-1 (ET-1) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, through p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rho-kinase in the ET-1-stimulated IL-6 synthesis in MC3T3-E1 cells. ET-1 time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific inhibitor of Rho-kinase, significantly suppressed the IL-6 synthesis induced by ET-1 as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, reduced the ET-1-stimulated IL-6 synthesis. Y27632 as well as fasudil attenuated the ET-1-induced phosphorylation of p38 MAP kinase but not p44/p42 MAP kinase. These results strongly suggest that Rho-kinase regulates ET-1-stimulated IL-6 synthesis through p38 MAP kinase activation in osteoblasts.