Characterization of extracellular glucoamylase from the ectomycorrhizal mushroom <Emphasis Type="Italic">Lyophyllum shimeji</Emphasis>

To investigate the function of amylases in the fruit-body formation of an ectomycorrhizal fungus, Lyophyllum shimeji, we purified the extracellular amylase in the medium of this fungus. The purified enzyme was obtained from 1.7l stationary culture filtrate, with 4.2% recovery, and showed a single protein band on SDS-PAGE. The molecular mass was about 25kDa. The enzyme was most active at around 40°C and pH 5.0 and stable over pH 4.5–6.5 for 30min at 37°C. This amylase was remarkably activated by the presence of Ca²⁺ ion (7.7 times that of the control), but Ba²⁺ and Ag⁺ completely inhibited the activity. The amylase readily hydrolyzed the -1,4 glucosidic linkage such as dextrin and amylose A (MW, 2900), converting into glucose, and hydrolyzed the -1,6 glucosidic linkage of isomaltohexaose and amylopectin. However, the enzyme did not hydrolyze the cyclic polysaccharides. On the other hand, when a low molecular mass amylose A was hydrolyzed by this amylase, -anomer glucose was produced. From these results, we concluded that the amylase from L. shimeji seems to be a glucoamylase.     

Changes in the expression of tumor necrosis factor (TNF) alpha,TNFalpha receptor (TNFR) 2, and TNFR-associated factor 2 in granulosa cells during atresia in pig ovaries

Tumor necrosis factor (TNF) alpha can induce both cell death and cell proliferation and exerts its effects by binding to either TNF receptor (TNFR) 1 or 2. When TNFalpha-bound TNFR2 interacts with TNFR-associated factor 2 (TRAF2), expression of survival/antiapoptotic genes is up-regulated. In the present study we determined the changes in localization of TNFalpha and TRAF2 and their mRNAs and the expression of TNFR2 in granulosa cells during follicular atresia in pig ovaries. In healthy follicles, intense signals for TNFalpha and TRAF2 and their mRNAs were demonstrated in the outer zone of the granulosa layer, where many proliferating cells and no apoptotic cells were observed. In atretic follicles, decreased or trace staining for TRAF2 and its mRNA and decreased expression of TNFR2 were observed in the granulosa layer, where many apoptotic cells were seen. These findings suggested that TNFalpha acts as a survival factor in granulosa cells during follicular atresia in pig ovaries.     

Dll3 pudgy mutation differentially disrupts dynamic expression of somite genes

Mutations in the notch ligand delta-like 3 have been identified in both the pudgy mouse (Dll3(pu); Kusumi et al.: Nat Genet 19:274-278, 1998) and the human disorder spondylocostal dysostosis (SCD; Bulman et al.: Nat Genet 24:438-441, 2000), and a targeted mutation has been generated (Dll3(neo); Dunwoodie et al.: Development 129:1795-1806, 2002). Vertebral and rib malformations deriving from defects in somitic patterning are key features of these disorders. In the mouse, notch pathway genes such as Lfng, Hes1, Hes7, and Hey2 display dynamic patterns of expression in paraxial mesoderm, cycling in synchrony with somite formation (Aulehla and Johnson: Dev Biol 207:49-61, 1999; Forsberg et al.: Curr Biol 8:1027-1030, 1998; Jouve et al.: Development 127:1421-1429, 2000; McGrew et al.: Curr Biol 8:979-982, 1998; Nakagawa et al.: Dev Biol 216:72-84, 1999). We report here that the Dll3(pu) mutation has different effects on the expression of cycling (Lfng and Hes7) and stage-specific genes (Hey3 and Mesp2). This suggests a more complex situation than a single oscillatory mechanism in somitogenesis and provides an explanation for the unique radiological features of the human DLL3-type of SCD.     

Molecular phylogenetic diversity of the bacterial community in the gut of the termite Coptotermes formosanus

The phylogenetic diversity of the bacterial community in the gut of the termite Coptotermes formosanus was investigated using a 16S rRNA gene clone library constructed by PCR. After screening by restriction fragment length polymorphism (RFLP) analysis, 49 out of 261 clones with unique RFLP patterns were sequenced and phylogenetically analyzed. Many of the clones (94%) were derived from Bacteroidales, Spirochaetes, and low G + C content gram-positive bacteria consisting of Clostridiales, Mycoplasmatales, Bacillales, and Lactobacillales. In addition, a few clones derived from Actinobacteria, Proteobacteria, Planctomycetes, Verrucomicrobia, and the candidate phylum "Synergistes" were also found. The most frequently identified RFLP type, BCf1-03, was assigned to the order Bacteroideales, and it constituted about 70% of the analyzed clones. The phylogenetic analysis revealed that the representative clones found in this study tended to form some clusters with the sequences cloned from the termite gut in several other studies, suggesting the existence of termite-specific bacterial lineages.     

Xis protein binding to the left arm stimulates excision of conjugative transposon Tn916

Tn916 and related conjugative transposons are clinically significant vectors for the transfer of antibiotic resistance among human pathogens, and they excise from their donor organisms using the transposon-encoded integrase ((Tn916)Int) and excisionase ((Tn916)Xis) proteins. In this study, we have investigated the role of the (Tn916)Xis protein in stimulating excisive recombination. The functional relevance of (Tn916)Xis binding sites on the arms of the transposon has been assessed in vivo using a transposon excision assay. Our results indicate that in Escherichia coli the stimulatory effect of the (Tn916)Xis protein is mediated by sequence-specific binding to either of its two binding sites on the left arm of the transposon. These sites lie in between the core and arm sites recognized by (Tn916)Int, suggesting that the (Tn916)Xis protein enhances excision in a manner similar to the excisionase protein of bacteriophage lambda, serving an architectural role in the stabilization of protein-nucleic acid structures required for strand synapsis. However, our finding that excision in E. coli is significantly enhanced by the host factor HU, but does not depend on the integration host factor or the factor for inversion stimulation, defines clear mechanistic differences between Tn916 and bacteriophage lambda recombination.     

The inhibitory effect of ouabain on the response of slowly adapting pulmonary stretch receptors to hyperinflation in the rabbit

The combined effects of ouabain (Na(+)-K(+) ATPase inhibitor) and hyperinflation (inflation volume=three tidal volumes) on slowly adapting pulmonary stretch receptors (SARs) were studied before and after administration of nifedipine (an L-type Ca(2+) channel blocker) and KB-R7943 (a reverse-mode Na(+)-Ca(2+) exchanger blocker) in anesthetized, artificially ventilated rabbits after bilateral vagotomy. Before ouabain administration, hyperinflation stimulated SAR activity. After 20 min of ouabain administration (30 microg/kg) the SARs increased discharge rates in normal inflation. Under these conditions, hyperinflation initially stimulated SAR activity but subsequently inhibited the activity at peak inflation. Additional administration of 60 microg/kg ouabain (total dose=90 microg/kg) caused a further stimulation of SAR activity, but 20 min later both normal inflation and hyperinflation resulted in a greater inhibition of the receptor activity. The hyperinflation-induced SAR inhibition in the presence of ouabain (30 microg/kg) was not significantly altered by administration of either nifedipine (2 and 4 mg/kg) or KB-R7943 (1 and 3 mg/kg). In another series of experiments, we further examined the combined effects of ouabain and hyperinflation in veratridine (a Na(+) channel opener, 40 microg/kg)-treated animals. After recovery from the veratridine effect on SAR activity, which vigorously stimulated the receptor activity, ouabain treatment (30 microg/kg) that silenced the receptor activity at peak inflation greatly inhibited hyperinflation-induced SAR stimulation. These results suggest that hyperinflation-induced SAR inhibition in the presence of ouabain may be related to a Na(+) overload, but not to a Ca(2+) influx via activation of L-type Ca(2+) channels, in the SAR endings.     

Genetic control of temperature preference in the nematode Caenorhabditis elegans

Animals modify behavioral outputs in response to environmental changes. C. elegans exhibits thermotaxis, where well-fed animals show attraction to their cultivation temperature on a thermal gradient without food. We show here that feeding-state-dependent modulation of thermotaxis is a powerful behavioral paradigm for elucidating the mechanism underlying neural plasticity, learning, and memory in higher animals. Starved experience alone could induce aversive response to cultivation temperature. Changing both cultivation temperature and feeding state simultaneously evoked transient attraction to or aversion to the previous cultivation temperature: recultivation of starved animals with food immediately induced attraction to the temperature associated with starvation, although the animals eventually exhibited thermotaxis to the new temperature associated with food. These results suggest that the change in feeding state quickly stimulates the switch between attraction and aversion for the temperature in memory and that the acquisition of new temperature memory establishes more slowly. We isolated aho (abnormal hunger orientation) mutants that are defective in starvation-induced cultivation-temperature avoidance. Some aho mutants responded normally to changes in feeding state with respect to locomotory activity, implying that the primary thermosensation followed by temperature memory formation remains normal and the modulatory aspect of thermotaxis is specifically impaired in these mutants.     

Chronic nicotine in hearts with healed ventricular myocardial infarction promotes atrial flutter that resembles typical human atrial flutter

The potential of chronic nicotine exposure for atrial fibrillation (AF) and atrial flutter (AFL) in hearts with and without chronic myocardial infarction (MI) remains poorly explored. MI was created in dogs by permanent occlusion of the left anterior descending coronary artery, and dogs were administered nicotine (5 mg.kg(-1).day(-1) sc) for 1 mo using osmotic minipumps. High-resolution epicardial (1,792 bipolar electrodes) and endocardial Halo catheters were used to map activation during induced atrial rhythms. Nicotine promoted inducible sustained AFL at a mean cycle length of 134 +/- 10 ms in all MI dogs (n = 6) requiring pacing and electrical shocks for termination. No AFL could be induced in MI dogs (n = 6), control (non-MI) dogs (n = 3) not exposed to nicotine, and dogs with no MI and exposed to nicotine (n = 3). Activation maps during AFL showed a single reentrant wavefront in the right atrium that rotated either clockwise (60%) or counterclockwise (40%) around the crista terminalis and through the isthmus. Ablation of the isthmus prevented the induction of AFL. Nicotine caused a significant (P < 0.01) but highly heterogeneous increase in atrial interstitial fibrosis (2- to 10-fold increase in left and right atria, respectively) in the MI group but only a 2-fold increase in the right atrium in the non-MI group. Nicotine also flattened (P < 0.05) the slope of the epicardial monophasic action potential duration (electrical restitution) curve of both atria in the MI but not in non-MI dogs. Two-dimensional simulation in an excitable matrix containing an isthmus and nicotine's restitutional and reduced gap junctional coupling (fibrosis) parameters replicated the experiments. Chronic nicotine in hearts with MI promotes AFL that closely resembles typical human AFL. Increased atrial interstitial fibrosis and flattened electrical restitution are important substrates for the AFL.