Digastric muscle activities in anoxic infant rats

The digastric muscle acts for both feeding (including mastication and swallowing) and respiration. In this study, we examined whether or not the muscle activity is detectable during anoxia in developing rats. Rats at 4 different ages, days 5, 10, 16, and 24, were exposed to 100% N2 under pentobarbital or ketamine-xylazine anesthesia, and the electromyograms of digastric muscles (dEMG) and the diaphragm (diaEMG) were examined simultaneously. Prior to the anoxic exposure, at all ages, the dEMG was similar to or less apparent than the diaEMG, which was detected at each inspiratory movement. In anoxia, we first observed dEMG activity, mostly sporadic (days 5 and 10) or mostly tonic (days 16 and 24), when diaEMG activity was temporarily suppressed (we termed it Phase 1). Second, synchronous phasic or tonic dEMG and phasic diaEMG were recorded temporarily before terminal apnea (we termed it Phase 2). These phenomena were also obtained in vagotomized rats (all ages) or in rats injected with the N-methyl-D-aspartate receptor antagonist MK-801 (dizocilpine (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate) (days 16 and 24). In conclusion, our results suggest that in anoxia, dEMG activity is detectable during diaEMG suppression in early anoxia, irrespective of the developmental age, the anesthetic (pentobarbital or ketamine-xylazine), vagotomy, or MK-801 injections.     

Activation of Cdc42 by trans interactions of the cell adhesion molecules nectins through c-Src and Cdc42-GEF FRG

Nectins, Ca2+ -independent immunoglobulin-like cell-cell adhesion molecules, initiate cell-cell adhesion by their trans interactions and recruit cadherins to cooperatively form adherens junctions (AJs). In addition, the trans interactions of nectins induce the activation of Cdc42 and Rac small G proteins, which increases the velocity of the formation of AJs. We examined here how nectins induce the activation of Cdc42 in MDCK epithelial cells and L fibroblasts. Nectins recruited and activated c-Src at the nectin-based cell-cell adhesion sites. FRG, a GDP/GTP exchange factor specific for Cdc42, was then recruited there, tyrosine phosphorylated by c-Src, and activated, causing an increase in the GTP-bound active form of Cdc42. Inhibition of the nectin-induced activation of c-Src suppressed the nectin-induced activation of FRG and Cdc42. Inhibition of the nectin-induced activation of FRG or depletion of FRG by RNA interference suppressed the nectin-induced activation of Cdc42. These results indicate that nectins induce the activation of Cdc42 through c-Src and FRG locally at the nectin-based cell-cell adhesion sites.     

Enhancing the staggered fluctuations of an actin filament sliding on Chara myosin

We examined both longitudinal and transversal fluctuations of displacements of an actin filament sliding upon Chara myosin molecules. Although the magnitude of transversal fluctuations remained rather independent of ATP concentration, the longitudinal ones were found to increase their magnitude as the concentration increased. In addition, the longitudinal fluctuations gradually increased as the sliding velocity of the filament increased.     

Higher-order molecular packing in amyloid-like fibrils constructed with linear arrangements of hydrophobic and hydrogen-bonding side-chains

Various mutants of the protein fragment, barnase module-1 (1-24) were investigated in order to reveal the structural principle of amyloid-like fibrils. By means of circular dichroism spectroscopy, X-ray diffraction, electron microscopy, and thioflavin T binding assay, we found that the molecules containing two beta-strands and an intervening turn structure are assembled to form a cross-beta structure. Stabilization by both the hydrophobic interactions and hydrogen bonding between the respective paired side-chains on the coupled beta-strands was essential for fibril formation. These two types of interaction can also arrange the corresponding residues in lines on both sheet surfaces of protofilaments with a cross-beta structure. This leads to the most probable fibril structure constructed with the line-matching interactions between protofilaments. Consideration of the geometrical symmetry resulted in our finding that a limited number of essential models for molecular packing in fibril structure are stable, which would rationally explain the occurrence of two or three morphologies from an identical molecular species. The ribbon-like fibrils exhibited striped texture along the axis, which was assigned to a stacked two-sheet repeat as a structural unit. The comprehensively proposed structural model, that is, the sheet-sheet interaction between left-handed cross-beta structures, results in a slightly right-handed twist of beta-sheet stacking, which reasonably elucidates the intrinsic sizes of the fibril width and its helical period along the fibril axis, as the bias in the orientation of the hydrogen-bonded beta-strand pair at the lateral edge is larger than that at the central protofilament.     

Calpain inhibitor-1 protects the rat heart from ischemia-reperfusion injury: analysis by mechanical work and energetics

We hypothesized that calpain inhibitor-1 protected left ventricular (LV) function from ischemia-reperfusion injury by inhibiting the proteolysis of alpha-fodrin. To test this hypothesis, we investigated the effect of calpain inhibitor-1 on LV mechanical work and energetics in the cross-circulated rat hearts that underwent 15-min global ischemia and 60-min reperfusion (n = 9). After ischemia-reperfusion with calpain inhibitor-1, mean end-systolic pressure at midrange LV volume and systolic pressure-volume area (PVA) at midrange LV volume (total mechanical energy per beat) were hardly changed, although they were significantly (P < 0.01) decreased after ischemia-reperfusion without calpain inhibitor-1. Mean myocardial oxygen consumption per beat (Vo(2)) intercepts (PVA-independent Vo(2); Vo(2) for the total Ca(2+) handling in excitation-contraction coupling and basal metabolism) of Vo(2)-PVA linear relations were also unchanged after ischemia-reperfusion with calpain inhibitor-1, although they were significantly (P < 0.01) decreased after ischemia-reperfusion without calpain inhibitor-1. There were no significant differences in O(2) costs of LV PVA and contractility among the hearts in control (or normal) postischemia-reperfusion and postischemia-reperfusion with calpain inhibitor-1. Western blot analysis of alpha-fodrin and the immunostaining of 150-kDa products of alpha-fodrin confirmed that calpain inhibitor-1 almost completely protected the proteolysis of alpha-fodrin. Our results indicate that calpain inhibitor-1 prevents the heart from ischemia-reperfusion injury associated with the impairment of total Ca(2+) handling by directly inhibiting the proteolysis of alpha-fodrin.     

One-step purification of metallothionein extracted from two different sources

We describe a one-step purification of hepatic metallothionein from the Amazon fish Colossoma macropomum injected with cadmium and from the copper-loaded metallothionein from the yeast Saccharomyces cerevisiae, performed by affinity chromatography through metal-chelating columns. Yeast metallothionein was purified from Cu2+-loaded resin and eluted by a continuous EDTA gradient whereas hepatic metallothionein extracted from fishes was purified by Ni2+-loaded resin and eluted by a continuous imidazol gradient. Purified metallothioneins were evaluated by SDS-PAGE and characterized by UV spectra of the apo- and Cd2+-loaded protein. This method allowed high purity and yield as well as rapid one-step extraction of both metal-loaded and apoprotein.     

Analysis of potential contamination factors in musculoskeletal tissues

Tissue Banks have become the main source for bone grafts, due to preference for homologous tissues. Notwithstanding the use of aseptic techniques for procurement of tissues and judicious selection of donors, microorganisms are frequently found in procured bones. Purpose of this study is to evaluate the factors that increase safety of procurement and minimize discard of procured tissues. Microbiological contamination was analyzed in 1271 musculoskeletal tissues removed from 138 multi-organ donors over a period extending from 2006 to 2016. Effects of various risk factors related with contamination were estimated using a logistic regression model. Microbiological contamination rate in the tissues was 17.1%; low pathogenic microorganisms were cultivated in 12.9% of the tissues, while highly pathogenic ones were cultivated in 4.2% of the tissues. Evolution of one single team was monitored during that period, verifying a fall in the general contamination level from 22.5 to 9.2%. Absence of antibiotics increased low pathogenic contamination risk. Every additional day in intensive care unit (ICU) increased the risk of highly pathogenic contamination. Time elapsed between death and the beginning of removal procedures was found to be relevant for both low pathogenic and highly pathogenic microorganisms. Among the studied factors, the following contributed for a significant increase in contamination by microorganisms in removed tissues: lack of use of prophylactic antibiotic therapy in donors, quantity of removed tissues, length of admission in ICU and the time elapsed between aortic clamping and beginning of the removal procedure.     

Hypersomnia with ADHD: a possible subtype of narcolepsy type 2

Patients with attention deficit/hyperactivity disorder (ADHD) suffer from hypersomnia; indeed, we have often encountered ADHD patients that fulfill the diagnostic criteria for narcolepsy type 2 (NA 2). Because not all patients with NA 2 carry the HLA-DQB1*06:02 allele, which is closely associated with narcolepsy type 1 (NA 1), NA 2 is believed to be heterogeneous. To reveal the contribution of ADHD in hypersomnia, we studied the characteristics of hypersomnia patients with ADHD, especially those diagnosed with NA 2. Participants were 77 of 185 consecutive outpatients who were diagnosed with NA 2 or idiopathic hypersomnia. We investigated sleep variables in (a) participants with hypersomnia with/without ADHD and (b) patients with NA 2 with/without ADHD and those with/without the DQB1*06:02 allele. The proportion of those diagnosed with NA 2 was higher in hypersomnia patients with ADHD compared to those without ADHD. None of the NA 2 patients with ADHD carried the narcolepsy-specific DQB1*06:02 allele. These patients with NA 2 with ADHD exhibited short REM latencies on the MSLT (similarly to DQB1*06:02-positive patients with NA 2 without ADHD), but less REM-related phenomena than patients with NA 2 without ADHD. Hypersomnia patients with ADHD tended to show short REM latencies, and fulfilled NA 2 diagnostic criteria in the absence of the DQB1*06:02 allele, suggesting a different etiology from NA 1. These findings support the hypotheses of noradrenergic dysregulation and delayed brain maturation that have been proposed for the pathophysiology of ADHD.