Characterization of adenylate and guanylate cyclases in rat peritoneal mast cells

Adenylate and guanylate cyclase activities were confirmed in crude homogenates from rat peritoneal mast cells. Both enzyme activities were associated with the 105, 000 X g particulate fractions, but not detected in the supernatant fractions. The optimal pH for both cyclase activities was 8.2. Mn⁺⁺ was essentially required for guanylate cylcase activity, while adenylate cyclase activity was observed in the presence of either Mg⁺⁺ or Mn⁺⁺. The apparent Km values of adenylate cyclase for Mn⁺⁺-ATP and Mg⁺⁺-ATP were 160 μM and 340 μM, respectively, whereas the value of guanylate cyclase for Mn⁺⁺-GTP was 100 μM. Adenylate cyclase was activated by 10 mM NaF. However, both adenylate and guanylate cyclase activities were neither stimulated nor inhibited by the addition of various kinds of agents which stimulate or inhibit the release of histamine from mast cells.     

Spatial Analysis of Slowly Oscillating Electric Activity in the Gut of Mice Using Low Impedance Arrayed Microelectrodes

Smooth and elaborate gut motility is based on cellular cooperation, including smooth muscle, enteric neurons and special interstitial cells acting as pacemaker cells. Therefore, spatial characterization of electric activity in tissues containing these electric excitable cells is required for a precise understanding of gut motility. Furthermore, tools to evaluate spatial electric activity in a small area would be useful for the investigation of model animals. We thus employed a microelectrode array (MEA) system to simultaneously measure a set of 8×8 field potentials in a square area of ∼1 mm². The size of each recording electrode was 50×50 µm², however the surface area was increased by fixing platinum black particles. The impedance of microelectrode was sufficiently low to apply a high-pass filter of 0.1 Hz. Mapping of spectral power, and auto-correlation and cross-correlation parameters characterized the spatial properties of spontaneous electric activity in the ileum of wild-type (WT) and W/W^v mice, the latter serving as a model of impaired network of pacemaking interstitial cells. Namely, electric activities measured varied in both size and cooperativity in W/W^v mice, despite the small area. In the ileum of WT mice, procedures suppressing the excitability of smooth muscle and neurons altered the propagation of spontaneous electric activity, but had little change in the period of oscillations. In conclusion, MEA with low impedance electrodes enables to measure slowly oscillating electric activity, and is useful to evaluate both histological and functional changes in the spatio-temporal property of gut electric activity.     

Echolocation sounds and hearing of the greater Japanese horseshoe bat (Rhinolophus ferrumequinum nippon)

Summary The relationship between the orientation sounds and hearing sensitivity in the greater Japanese horseshoe bat,Rhinolophus ferrumequinum nippon was studied.An orientation pulse consisted of a constant frequency (CF) component followed by a short downward frequency-modulated (FM) component. Sometimes, an initial upward FM component preceded the CF component. Duration of pulses was about 30 ms and the CF of resting pulses (RF) averaged 65.5 kHz. The best frequency (BF) at the lowest threshold in audiograms as measured by the pinna reflex averaged 66.1 kHz. Audiograms showed remarkable sharp cut-offs on both sides near the BF. The frequency difference between the BF and the RF was about 0.6 kHz, and the RF was always below the BF. The values of RF and BF were characteristically different from those of the European subspecies,Rhinolophus ferrumequinum ferrumequinum.Abbreviations BF best frequency - CF constant frequency - FM frequency modulated - RF resting frequency     

Estimation of transport distance of fine particulate organic matter in relation to channel morphology in tailwaters of the Lake Biwa and reservoir dams

Knowledge on transport and deposition of fine particulate organic matter (FPOM) from reservoir dams is increasingly required for habitat management and restoration of dam tailwater ecosystems. Variations in the transport distance of FPOM, however, have never been studied well, particularly in relation to channel morphology, due to channel size restrictions of artificial tracers such as corn pollen when applied to larger river channels. This study aims to show the relations between FPOM retention efficiency and channel morphology in dam tailwaters using lentic plankters as tracers. We estimated the mean transport distance, S _p, by calculating downstream reduction ratios of lentic tracer plankters and calculated the deposition velocity, v _dep. Suspended FPOM samples were collected in tailwaters of two river channels below reservoir dams and two artificial canals below Lake Biwa in the Yodo River system. The longest S _p (19.2 km) and the shortest one (2.2 km) were recorded in the deep canal and shallow canal, respectively, showing a positive correlation with channel hydraulic radius. The values of v _dep were 4.7–6.4 times higher in river channels than in artificial canals. These results indicate that increasing complexity of bed morphology can minimize S _p, whereas bed degradation and armored bed materials may lead to increased S _p. Advantages of lentic plankters as tracers for estimating distance ranges of reservoir dam impact on river ecosystems are also discussed.     

Production, crystallization, and preliminary X-ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1-47) of troponin I.

Troponin is a ternary protein complex consisting of subunits TnC. TnI, and TnT, and plays a key role in calcium regulation of the skeletal and cardiac muscle contraction. In the present study, a partial complex (CI47) was prepared from Escherichia coli-expressed rabbit skeletal muscle TnC and fragment 1-47 of TnI, which is obtained by chemical cleavage of an E. coli-expressed mutant of rabbit skeletal muscle TnI. Within the ternary troponin complex, CI47 is thought to form a core that is resistant to proteolytic digestion, and the interaction within CI47 likely maintains the integrity of the troponin complex. Complex CI47 was crystallized in the presence of sodium citrate. The addition of trehalose improved the diffraction pattern of the crystals substantially. The crystal lattice belongs to the space group P3(1)(2)21, with unit cell dimensions a = b = 48.2 A, c = 162 A. The asymmetric unit presumably contains one CI47 complex. Soaking with p-chloromercuribenzenesulfonate (PCMBS) resulted in loss of isomorphism, but enhanced the quality of the crystals. The crystals diffracted up to 2.3 A resolution, with completeness of 91% and R(merge) = 6.4%. The crystals of PCMBS-derivative should be suitable for X-ray studies using the multiple-wavelength anomalous diffraction technique. This is the first step for elucidating the structure of the full troponin complex.     

Prophages integrating into prophages: A mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified attB sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different attB sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species.     

Mechanism of How Salt-Gradient-Induced Charges Affect the Translocation of DNA Molecules through a Nanopore

Experiments using nanopores demonstrated that a salt gradient enhances the capture rate of DNA and reduces its translocation speed. These two effects can help to enable electrical DNA sequencing with nanopores. Here, we provide a quantitative theoretical evaluation that shows the positive net charges, which accumulate around the pore entrance due to the salt gradient, are responsible for the two observed effects: they reinforce the electric capture field, resulting in promoted molecule capture rate; and they induce cationic electroosmotic flow through the nanopore, thus significantly retarding the motion of the anionic DNA through the nanopore. Our multiphysical simulation results show that, during the polymer trapping stage, the former effect plays the major role, thus resulting in promoted DNA capture rate, while during the nanopore-penetrating stage the latter effect dominates and consequently reduces the DNA translocation speed significantly. Quantitative agreement with experimental results has been reached by further taking nanopore wall surface charges into account.