Muscarinic acetylcholine receptors in rat gastric mucosa. A radioautographic study using a potent muscarinic antagonist, 3H-pirenzepine

The muscarinic cholinergic innervation of the rat gastric mucosa was investigated by localizing the muscarinic receptors using a tritiated muscarinic antagonist, pirenzepine. Radioautography was performed by freeze drying stomach tissue, which was then embedded in Epon and wet sectioned with ethylene glycol, and dry mounting on emulsion film by the wire-loop method to prevent loss of the labelled substance during fixation and the radioautographic procedure. Light and electron microscopy showed that the specific pirenzepine-binding sites were localized predominantly on parietal cells, chief cells and perivascular plexuses. Analysis of the grain distribution on parietal cells revealed that the silver grains corresponding to the pirenzepine-binding sites were mainly on the basolateral plasma membrane. On the other hand, the surface mucous or mucous neck cells had few pirenzepine-binding sites.     

The juvenile myoclonic epilepsy-related protein EFHC1 interacts with the redox-sensitive TRPM2 channel linked to cell death

The transient receptor potential M2 channel (TRPM2) is the Ca(2+)-permeable cation channel controlled by cellular redox status via β-NAD(+) and ADP-ribose (ADPR). TRPM2 activity has been reported to underlie susceptibility to cell death and biological processes such as inflammatory cell migration and insulin secretion. However, little is known about the intracellular mechanisms that regulate oxidative stress-induced cell death via TRPM2. We report here a molecular and functional interaction between the TRPM2 channel and EF-hand motif-containing protein EFHC1, whose mutation causes juvenile myoclonic epilepsy (JME) via mechanisms including neuronal apoptosis. In situ hybridization analysis demonstrates TRPM2 and EFHC1 are coexpressed in hippocampal neurons and ventricle cells, while immunoprecipitation analysis demonstrates physical interaction of the N- and C-terminal cytoplasmic regions of TRPM2 with the EFHC1 protein. Coexpression of EFHC1 significantly potentiates hydrogen peroxide (H(2)O(2))- and ADPR-induced Ca(2+) responses and cationic currents via recombinant TRPM2 in HEK293 cells. Furthermore, EFHC1 enhances TRPM2-conferred susceptibility of HEK293 cells to H(2)O(2)-induced cell death, which is reversed by JME mutations. These results reveal a positive regulatory action of EFHC1 on TRPM2 activity, suggesting that TRPM2 contributes to the expression of JME phenotypes by mediating disruptive effects of JME mutations of EFHC1 on biological processes including cell death.     

Chemotactic responses of osteoblastic MC3T3-E1 cells toward zinc chloride

Although zinc (Zn) is known to participate in bone formation, its exact role in the remodeling of this tissue has not been fully clarified. The present study was designed to investigate whether Zn has a role at the resorptive sites in vitro. We investigated the migration of osteoblastic MC3T3-E1 cells in response to Zn using a Boyden chamber assay. Exposure of MC3T3-E1 cells to Zn stimulated the migration of MC3T3-E1 cells. Checkerboard analysis revealed that the migration of MC3T3-E1 cells toward Zn was a directional (chemotaxis) rather than a random (chemokinesis) motion. Pretreatment of MC3T3-E1 cells with pertussis toxin completely blocked the chemotactic response of cells to Zn, indicating that it is mediated by G-protein-coupled receptors. Because the bone is one of the major Zn storage sites, we suggest that Zn released from bone-resorptive sites plays an important role in the recruitment of osteoblasts and bone renewal.     

The distinct role of catalase and DNA repair systems in protection against hydrogen peroxide in Escherichia coli

The katEkatG mutant of E. coli, UM1, had no assayable catalase activities in the extract and showed increased (about 20 fold) sensitivity to killing by H2O2 when compared with its parental strain CSH7. The mutant strain was able to reactivate H2O2-damaged lambda phage. On the other hand, recA and polA mutants were also highly sensitive to H2O2, but they had normal level of catalase activities. RecA derivatives of UM1 were much more sensitive to H2O2 than UM1 and recA strains. The induction of umu operon occurred in UM1 at lower (1/10-1/20) doses of H2O2 than in CSH7. From the results it is concluded that the lethal effect of H2O2 is due to DNA damage induced by it and that catalase and DNA repair systems have a distinct role in protection against H2O2 in E. coli.     

Changes in element concentration and distribution in breast-milk fractions of a healthy lactating mother

Daily changes in components of breast milk with number of days of lactation after delivery were demonstrated by determining concentrations and distributions of several elements simultaneously. Concentrations of calcium, copper, magnesium, phosphorus, sulfur, and zinc were determined simultaneously by inductively coupled argon plasma-atomic-emission spectrometry (ICP) for whole milk and milk fractions (skimmed milk and whey) collected from 2 to 196 d postpartum from a healthy lactating mother. Calcium and phosphorus concentrations increased in transitional milk. With days postpartum, the other elements decreased from the highest concentrations in colostrum milk, the modes of decrease being characteristic for each element. Distributions of copper, iron, phosphorus, sulfur, and zinc in whey were determined on a gel-filtration column by HPLC with ICP detection (HPLC-ICP method). Distributions of the five elements and absorbance peaks at 254 and 280 nm changed dramatically day by day at the beginning (colostrum milk), resulting in constant distributions after 30 d (mature milk). These results suggest the important roles of daily changing constituents in breast milk, especially in colostrum milk, in the nutrition of the newborn. Several element peaks on a gelfiltration column were identified by comparison with standard samples.     

Multiple pathways for repair of oxidative DNA damages caused by X rays and hydrogen peroxide in Escherichia coli.

The responses of Escherichia coli to X rays and hydrogen peroxide were examined in mutants which are deficient in one or more DNA repair genes. Mutant cells deficient in either exonuclease III (xthA) or endonuclease IV (nfo) had normal resistance to X rays, but an xthA-nfo double mutant showed a sensitivity increased over that of either parental strain. A DNA polymerase I mutant (polA) was more sensitive than the xthA-nfo mutant. Cells bearing mutations in all of the polA, xthA, and nfo genes were more sensitive to X rays than polA and xthA-nfo mutants. Similar repair responses were obtained by exposing these mutant cells to hydrogen peroxide, with the exception of the xthA mutant, which was hypersensitive to this agent. The DNA polymerase III mutant (polC(Ts)) was slightly more sensitive to the agents than the wild-type strain at the restrictive temperature. The sensitivity of the polC-xthA-nfo mutant to X rays and hydrogen peroxide was greater than that of polC but almost the same as that of the xthA-nfo mutant. From these results it appears that there are at least four repair pathways, the DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase III-, and exonuclease III/endonuclease IV-dependent pathways, for the repair of oxidative DNA damages in E. coli.     

Escherichia coli MutY protein has a guanine-DNA glycosylase that acts on 7,8-dihydro-8-oxoguanine:guanine mispair to prevent spontaneous G:C-->C:G transversions.

Low rates of spontaneous G:C-->C:G transversions would be achieved not only by the correction of base mismatches during DNA replication but also by the prevention and removal of oxidative base damage in DNA. Escherichia coli must have several pathways to repair such mismatches and DNA modifications. In this study, we attempted to identify mutator loci leading to G:C-->C:G transversions in E.coli. The strain CC103 carrying a specific mutation in lacZ was mutagenized by random miniTn 10 insertion mutagenesis. In this strain, only the G:C-->C:G change can revert the glutamic acid at codon 461, which is essential for sufficient beta-galactosidase activity to allow growth on lactose. Mutator strains were detected as colonies with significantly increased rates of papillae formation on glucose minimal plates containing P-Gal and X-Gal. We screened approximately 40 000 colonies and selected several mutator strains. The strain GC39 showed the highest mutation rate to Lac+. The gene responsible for the mutator phenotypes, mut39 , was mapped at around 67 min on the E.coli chromosome. The sequencing of the miniTn 10 -flanking DNA region revealed that the mut39 was identical to the mutY gene of E.coli. The plasmid carrying the mutY + gene reduced spontaneous G:C-->T:A and G:C-->C:G mutations in both mutY and mut39 strains. Purified MutY protein bound to the oligonucleotides containing 7,8-dihydro-8-oxo-guanine (8-oxoG):G and 8-oxoG:A. Furthermore, we found that the MutY protein had a DNA glycosylase activity which removes unmodified guanine from the 8-oxoG:G mispair. These results demonstrate that the MutY protein prevents the generation of G:C-->C:G transversions by removing guanine from the 8-oxoG:G mispair in E.coli.     

Evolutionary Dynamics of Nitrogen Fixation in the Legume–Rhizobia Symbiosis

The stabilization of host–symbiont mutualism against the emergence of parasitic individuals is pivotal to the evolution of cooperation. One of the most famous symbioses occurs between legumes and their colonizing rhizobia, in which rhizobia extract nutrients (or benefits) from legume plants while supplying them with nitrogen resources produced by nitrogen fixation (or costs). Natural environments, however, are widely populated by ineffective rhizobia that extract benefits without paying costs and thus proliferate more efficiently than nitrogen-fixing cooperators. How and why this mutualism becomes stabilized and evolutionarily persists has been extensively discussed. To better understand the evolutionary dynamics of this symbiosis system, we construct a simple model based on the continuous snowdrift game with multiple interacting players. We investigate the model using adaptive dynamics and numerical simulations. We find that symbiotic evolution depends on the cost–benefit balance, and that cheaters widely emerge when the cost and benefit are similar in strength. In this scenario, the persistence of the symbiotic system is compatible with the presence of cheaters. This result suggests that the symbiotic relationship is robust to the emergence of cheaters, and may explain the prevalence of cheating rhizobia in nature. In addition, various stabilizing mechanisms, such as partner fidelity feedback, partner choice, and host sanction, can reinforce the symbiotic relationship by affecting the fitness of symbionts in various ways. This result suggests that the symbiotic relationship is cooperatively stabilized by various mechanisms. In addition, mixed nodule populations are thought to encourage cheater emergence, but our model predicts that, in certain situations, cheaters can disappear from such populations. These findings provide a theoretical basis of the evolutionary dynamics of legume–rhizobia symbioses, which is extendable to other single-host, multiple-colonizer systems.     

A common variant of leucine-rich repeat-containing 16A (LRRC16A) gene is associated with gout susceptibility

Gout is a common disease resulting from hyperuricemia which causes acute arthritis. Recently, genome-wide association studies revealed an association between serum uric acid levels and a common variant of leucine-rich repeat-containing 16A (LRRC16A) gene. However, it remains to be clarified whether LRRC16A contributes to the susceptibility to gout. In this study, we investigated the relationship between rs742132 in LRRC16A and gout. A total of 545 Japanese male gout cases and 1,115 male individuals as a control group were genotyped. rs742132 A/A genotype significantly increased the risk of gout, conferring an odds ratio of 1.30 (95 % CI 1.05–1.60; p = 0.015). LRRC16A encodes a protein called capping protein ARP2/3 and myosin-I linker (CARMIL), which serves as an inhibitor of the actin capping protein (CP). CP is an essential element of the actin cytoskeleton, which binds to the barbed end of the actin filament and regulates its polymerization. In the apical membrane of proximal tubular cells in the human kidney, the urate-transporting multimolecular complex (urate transportsome) is proposed to consist of several urate transporters and scaffolding proteins, which interact with the actin cytoskeleton. Thus, if there is a CARMIL dysfunction and regulatory disability in actin polymerization, urate transportsome may be unable to operate appropriately. We have shown for the first time that CARMIL/LRRC16A was associated with gout, which could be due to urate transportsome failure.