Studies on cytogenesis in adult rat adrenal cortex: circadian and zonal variations and their modulation by adrenocorticotropic hormone

Circadian rhythms and zonal variations in the cell proliferation of adult rat adrenal cortex were studied by following the cells in the DNA-synthesizing stage (S-phase) as assessed by 5-bromo-2'-deoxyuridine incorporation into the cell-nuclei and/or by visualizing proliferating cell nuclear antigen. The S-phase cells were observed throughout the day in two regions of the adrenal cortex: (i) a region from the inner half of the zona glomerulosa to near the outer margin of the zona fasciculata, and (ii) the outer one-fourth portion of the zona fasciculata. Very little change in number was observed in the former region between day and night, while a burst of cell proliferation occurred in early morning at 3-4 a.m. in the latter region. A prominent rise in the plasma adrenocorticotropic hormone (ACTH) concentration preceded the burst of cell proliferation by about 4 h. Upon raising the plasma ACTH concentration by administration of ACTH or metyrapone, prominent cell proliferation also occurred in the same portion of the zona fasciculata 4-6 h after the provoked ACTH surge. Thus at least two sites in rat adrenal cortex are responsible for cytogenesis in this endocrine organ, and respond differentially to day/night cycles and circulating ACTH levels.     

Transformation of bile acids by Clostridium perfringens

Thirty-five strains of Clostridium perfringens were examined for their ability to transform bile acids, both in growing cultures and by washed whole cells. All of the strains oxidized the 3 alpha-hydroxy group to an oxo group, and all except three converted the same alpha-hydroxy group into a beta-configuration. The oxidative 3 alpha-dehydrogenation was barely detectable under anaerobic cultural conditions but was clearly demonstrated in an aerated system using washed whole cells, with a pH optimum between 7.0 and 9.0. The epimerizing reaction amounting to 10 to 20% conversion was observed in anaerobic cultures and also with resting cells, irrespective of oxygen supply. Both reactions were carried out with seven conventional 3 alpha-hydroxy bile acids, thus producing a series of 3-oxo and 3 beta-hydroxy derivatives that could be examined for gas-liquid chromatographic and mass spectrometric behavior. No evidence for the occurrence of 7 alpha- and 12 alpha-hydroxysteroid dehydrogenase activities among the test strains was found. A highly potent deconjugating hydrolase was elaborated by all of the strains.     

Estrogen induces rapid decrease in dendritic thorns of CA3 pyramidal neurons in adult male rat hippocampus

Modulation of hippocampal synaptic plasticity by estrogen has been attracting much attention. Thorns of thorny excrescences of CA3 hippocampal neurons are post-synaptic regions whose presynaptic partners are mossy fiber terminals. Here we demonstrated the rapid effect of estradiol on the density of thorns of thorny excrescences, by imaging Lucifer Yellow-injected CA3 neurons in adult male rat hippocampal slices. The application of 1nM estradiol induced rapid decrease in the density of thorns on pyramidal neurons within 2h. The estradiol-mediated decrease in the density of thorns was blocked by CNQX (AMPA receptor antagonist) and PD98059 (MAP kinase inhibitor), but not by MK-801 (NMDA receptor antagonist). ERalpha agonist PPT induced the same suppressive effect as that induced by estradiol on the density of thorns, but ERbeta agonist DPN did not affect the density of thorns. Note that a 1nM estradiol treatment did not affect the density of spines in the stratum radiatum and stratum oriens. A search for synaptic ERalpha was performed using purified RC-19 antibody. The localization of ERalpha (67kDa) in the CA3 mossy fiber terminals and thorns was demonstrated using immunogold electron microscopy. These results imply that estradiol drives the signaling pathway including ERalpha and MAP kinase.     

Inhibitory effects of flavonoids on the reduction of progesterone to 20alpha-hydroxyprogesterone in rat liver

The first aim of this study is to characterize the reduction of progesterone in rat liver. Progesterone was mainly reduced to 20alpha-hydroxyprogesterone in the cytosolic fraction of rat liver. The amount of 20alpha-hydroxyprogesterone formed from progesterone in the cytosolic fraction was significantly larger in the males than in the females and this enzyme reaction proceeded not only in the presence of NADPH, but also in the presence of NADH. Furthermore, we attempted to evaluate the inhibitory effects of 15 flavonoids on the NADPH-dependent reduction of progesterone to 20alpha-hydroxyprogesterone in liver cytosol of male rats. The order of the inhibitory potencies was luteolin>apigenin>quercetin>myricetin=fisetin=kaempferol. Other flavonoids exhibited lower inhibitory potencies. Energy-minimized molecular models demonstrated that a planar benzopyrone ring (A and C rings) with a coplanar phenyl ring (B ring) is a structural characteristic determining the inhibitory effects of flavonoids other than isoflavones.     

Cell surface‐associated aggregation‐promoting factor from Lactobacillus gasseri SBT2055 facilitates host colonization and competitive exclusion of Campylobacter jejuni

Campylobacter jejuni, one of the most common causes of gastroenteritis worldwide, is transmitted to humans through poultry. We previously reported that Lactobacillus gasseri SBT2055 (LG2055) reduced C. jejuni infection in human epithelial cells in vitro and inhibited pathogen colonization of chickens in vivo. This suggested that the LG2055 adhesion and/or co‐aggregation phenotype mediated by cell‐surface aggregation‐promoting factors (APFs) may be important for the competitive exclusion of C. jejuni. Here, we show that cell surface‐associated APF1 promoted LG2055 self‐aggregation and adhesion to human epithelial cells and exhibited high affinity for the extracellular matrix component fibronectin. These effects were absent in the apf1 knockout mutant, indicating the role of APF1 in LG2055‐mediated inhibition of C. jejuni in epithelial cells and chicken colonization. Similar to APF1, APF2 promoted the co‐aggregation of LG2055 and C. jejuni but did not inhibit C. jejuni infection. Our data suggest a pivotal role for APF1 in mediating the interaction of LG2055 with human intestinal cells and in inhibiting C. jejuni colonization of the gastrointestinal tract. We thus provide new insight into the health‐promoting effects of probiotics and mechanisms of competitive exclusion in poultry. Further research is needed to determine whether the probiotic strains reach the epithelial surface.     

The mammalian peroxin Pex5pL, the longer isoform of the mobile peroxisome targeting signal (PTS) type 1 transporter, translocates the Pex7p.PTS2 protein complex into peroxisomes via its initial docking site, Pex14p

In mammals, two isoforms of the peroxisome targeting signal (PTS) type 1 receptor Pex5p, i.e. Pex5pS and Pex5pL with an internal 37-amino acid insertion, have previously been identified. Expression of either type of Pex5p complements the impaired PTS1 import in Chinese hamster ovary pex5 mutants, but only Pex5pL can rescue the PTS2 import defect noted in a subgroup of pex5 mutants such as ZP105. In this work, we found that Pex5pL directly interacts with the PTS2 receptor Pex7p, carrying its cargo PTS2 protein in the cytosol. Pex5pL, but not Pex5pS, mediated the binding of PTS2 protein to Pex14p by translocating Pex7p, demonstrating that Pex5pL plays a pivotal role in peroxisomal PTS2 import. Pex5p was localized mostly in the cytosol in wild-type CHO-K1 and Pex14p-deficient mutant cells, whereas it accumulated in the peroxisomal remnants in cell mutants defective in Pex13p or the RING family peroxins such as Pex2p and Pex12p. Furthermore, overexpression of Pex14p, but not Pex10p, Pex12p, or Pex13p, caused accumulation of Pex5p in peroxisomal membranes, with concomitant interference with PTS1 and PTS2 import. Therefore, Pex5p carrying the cargoes most likely docks with the initial site (Pex14p) in a putative import machinery, subsequently translocating to other components such as Pex13p, Pex2p, Pex10p, and Pex12p.     

Metabolomics‐based systematic prediction of yeast lifespan and its application for semi‐rational screening of ageing‐related mutants

Metabolomics – the comprehensive analysis of metabolites – was recently used to classify yeast mutants with no overt phenotype using raw data as metabolic fingerprints or footprints. In this study, we demonstrate the estimation of a complicated phenotype, longevity, and semi‐rational screening for relevant mutants using metabolic profiles as strain‐specific fingerprints. The fingerprints used in our experiments are profiled data consisting of individually identified and quantified metabolites rather than raw spectrum data. We chose yeast replicative lifespan as a model phenotype. Several yeast mutants that affect lifespan were selected for analysis, and they were subjected to metabolic profiling using mass spectrometry. Fingerprinting based on the profiles revealed a correlation between lifespan and metabolic profile. Amino acids and nucleotide derivatives were the main contributors to this correlation. Furthermore, we established a multivariate model to predict lifespan from a metabolic profile. The model facilitated the identification of putative longevity mutants. This work represents a novel approach to evaluate and screen complicated and quantitative phenotype by means of metabolomics.     

Mechanical tensile properties of the aortic wall in the premature rat exposed to the microgravity environment during space flight for 16 days

Under microgravity environment, blood shifts headward and thereafter decrease in volume to adapt to the environment, which could affect cardiovascular hemodynamics and their regulatory mechanisms. Baroreceptor sensitivity is known to be reduced in newborn animals and to gradually increase with development. The baroreceptor is a stretch receptor; therefore its function is closely related to the rheological properties and fine structure of the aortic wall in which the baroreceptor lies. The mechanical and histological properties could be altered under microgravity conditions in the process of development with change in circulatory function. In the present study, we investigated the mechanical tensile characteristics and histological structure of the aortic wall in the proximal thoracic aorta of premature rats bred in the microgravity environment of the space shuttle for 16 days.     

Identification of novel drug-resistant EGFR mutant inhibitors by in silico screening using comprehensive assessments of protein structures

EGFR is a target protein for the treatment of non small cell lung cancer (NSCLC). The mutations associated with the activation of EGFR kinase activity, such as L858R and G719S, destabilize the inactive conformation of EGFR and are closely linked with the development of NSCLC. The additional T790M mutation reportedly causes drug resistance against the commercially available EGFR inhibitors, gefitinib and erlotinib. In this study, we searched for novel G719S/T790M EGFR inhibitors by a new in silico screening strategy, using two datasets. The results of in silico screening using protein-ligand docking are affected by the selection of 3D structure of the target protein. As the first strategy, we chose the 3D structures for in silico screening by test dockings using the G719S/T790M crystal structure, its molecular dynamics snapshots, and known inhibitors of the drug-resistant EGFR. In the second strategy, we selected the 3D structures by test dockings using all of the EGFR structures, regardless of the mutations, and all of the known EGFR inhibitors. Using each of the 3D structures selected by the strategies, 1000 compounds were chosen from the 71,588 compounds. Kinase assays identified 15 G719S/T790M EGFR inhibitors, including two compounds with novel scaffolds. Analyses of their structure-activity relationships revealed that interactions with the mutated Met790 residue specifically increase the inhibitory activity against G719S/T790M EGFR.