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.     

DNA strand breaks in mammalian tissues induced by methylarsenics

DNA damage induced by administration of dimethylarsinic acid (DMAA) to rats and mice was investigated. At 12 h after administration of DMAA, DNA single-strand breaks were induced markedly in lung. The majority of dimethylarsine, one of the main metabolites, in the expired air was excreted within 6–18 h after administration of DMAA to rats. In vitro experiments using nuclei isolated from lung of mice indicated that DNA strand breaks were caused by dimethylarsine. Furthermore, the strand breaks after exposure to dimethylarsine were reduced in the presence of catalase and/or superoxide dismutase. These results strongly suggest that the strand breaks are induced not by dimethylarsine itself but by active oxygen, e.g., O ₂ ^? and ·OH, produced both by dimethylarsine and molecular oxygen. When DNA was exposed to dimethylarsine, thiobarbituric acid (TBA)-reactive intermediates andcis-thymine glycol were produced. Dimethylarsine appears to induce DNA damage by the mechanism similar to the damage produced by ionizing radiation.     

Endothelin-3 is a novel neuropeptide: isolation and sequence determination of endothelin-1 and endothelin-3 in porcine brain

The molecular forms of endothelin (ET) related peptides were investigated in porcine brain by using high performance liquid chromatography coupled with three specific radioimmunoassays. ET-1 and its oxidized form were isolated and sequenced as in the case of porcine spinal cord. A very small amount of big ET-1 (1-39) and its C-terminal fragment (big ET-1 (22-39] were also detected. Furthermore, immunoreactive (ir)-ET-3 was isolated and sequenced; its partial primary structure was identical to that of human (rat) ET-3. The concentrations of ir-ET-1 and ir-ET-3 in porcine brain were 140 fmol/g tissue and 5 fmol/g tissue, respectively. These results indicate that besides ET-1, ET-3 is a novel neuropeptide in the central nervous system.     

Presence of endothelin-1 in porcine spinal cord: isolation and sequence determination

We investigated the molecular forms of endothelin (ET) related peptides in porcine spinal cord by high performance liquid chromatography coupled with radioimmunoassays using three antisera raised against ET-1 and C-terminal fragments of ET-1 and big ET-1. ET-1 and its oxidized form were isolated as major immunoreactive peptides and sequenced. Furthermore, immunoreactivities like ET-3 and big ET-1(22-39) (contents: less than 8% and less than 1% of ET-1, respectively) were detected based on their chromatographic retention times and characteristics of immunoreactivity to the antisera. Big ET-1 was only scarcely detected. Immunohistochemical study showed the presence of ET-1-like immunoreactivity in motoneurons, dorsal horn neurons and dot- and fiber-like structures in the dorsal horn of lumbar spinal cord. These results indicate that ET-1 is present not only in endothelial cells but also in spinal cord, and that big ET-1 is converted into ET-1 in spinal cord by specific processing between Trp21-Val22. The data also indicate that ET-1 may act as a neuropeptide in the central nervous system.     

The growth inhibitor of African green monkey (BSC-1) cells is transforming growth factors beta 1 and beta 2

The growth inhibitory activity in conditioned medium of African green monkey kidney epithelial (BSC-1) cells that has been shown to arise, at least in part, from transforming growth factor beta 2 (TGF-beta 2) [Hanks, S. K., Armour, R., Baldwin, J. H., Maldonado, F., Spiess, J., & Holley, R. W. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 79-82] was tested for growth inhibitory activity prior to and following acidification. Similar to TGF-beta 1 from human platelets, the inhibitory activity from BSC-1 cells demonstrated an 8-10-fold stimulation following acidification, showing that the activity was secreted from the cells in latent form. Conditioned medium from BSC-1 cells was collected, acidified, and fractionated by procedures that separate TGF-beta 1 and -2. Biological activity was assayed by using the BSC-1 cell proliferation assay. Two active proteins with properties similar to known TGF-beta 1 and TGF-beta 2 were identified. Identity was confirmed by using immunological and amino acid sequencing techniques. These results were consistent with Northern blot analysis of total BSC-1 RNA, using cDNA probes for TGF-beta 1 and TGF-beta 2, which demonstrated strong signals for both mRNAs. Metabolic labeling in conjunction with two-dimensional gel electrophoresis revealed that the cells secrete approximately 10% TGF-beta 1 and 90% TGF-beta 2.     

Hormone-Autonomous Suspension Culture from Leaf Explants of Sugar Beets in Liquid Medium

Hormone autonomous callus was institutioniated reproduciblyon MS agar medium with 0.25 mg/liter of BA as the sole planthormone (AI medium) from young leaf explants of sugar beets.When leaf explants were inoculated into AI medium and culturedon a reciprocal shaker, single cells began to be released fromthe cut surfaces of the leaf pieces after 6 days, followed byactive release. When the single cells which had been releasedwere transferred to fresh liquid MS medium without any planthormones, they could divide and grow autonomously, giving riseto hormone-autonomous suspension cultures. The effects of BAon induction of hormone-autonomous cells are discussed. (Received March 12, 1987; Accepted October 13, 1987)     

Dose and time-dependent mesoderm induction and outgrowth formation by activin A in Xenopus laevis.

We examined the quality of mesoderm induced by the action of activin A on the Xenopus presumptive ectoderm when various concentrations and treatment times were employed. The minimum concentration of activin A to induce mesodermal tissues was inversely proportional to its treatment time. The explants differentiated into different types of mesodermal tissues, from ventral-type to dorsal-type depending on the concentration of activin A and its treatment time. To confirm whether activin A has a role in establishing axial organization, activin A was injected into the blastocoel of late blastulae. About 70% of the injected embryos formed secondary tail-shaped outgrowths in which muscle and neural tube differentiated. The amount of activin A to form secondary outgrowths was 0.5-2.5 pg, roughly consistent with the amount estimated from in vitro experiments. As we have detected almost the same amount of activin homologue in the early embryos (Asashima et al., 1991a), we speculate that activin A may be the natural mesodermal inducer, and that it is responsible for establishing axial organization in the Xenopus embryo.     

The production of anorexigenic substances by intestinal flora

The role of intestinal flora in the production of anorexigenic substance was investigated. Proteus mirabilis (P. mirabilis) and Escherichia coli (E. coli) were found to produce an anorexigenic substance, while Enterococcus faecalis (E. faecalis, type 1 and 2) and Staphylococcus intermedius (S. intermedius) did not. The anorexigenic substance was purified and was detected as, a single though broad band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of the final form of the purified substance was 120 units/mg carbohydrate. The substance contained no protein residue and appeared to be a lipopolysaccharide. The evidence that intestinal flora produces an anorexigenic substance leads to an interesting assumption that the intestinal flora may be responsible for regulating food intake.     

Effects of taurine on the electrical and mechanical activities of embryonic chick heart

The effect of taurine (2-aminoethanesulphonic acid) on myocardial slow action potentials (APs) and accompanying contractions was examined in isolated perfused chick hearts and reaggregated cultured cells. Isoproterenol (ISO), histamine (HIS), or tetraethylammonium (TEA) induced slow APs and contractions in hearts whose fast Na+ channels had been inactivated by elevated K+. Taurine (10 mM) not only failed to induce slow APs, but actually decreased ISO (10(-8) M), HIS (10(-4) M), or TEA (10 mM) induced slow APs and contractions transiently (about 30s-2 min after the addition of taurine). The properties of the slow APs recovered to control levels by 7-13 min after the addition of the taurine; at this time, there was an increase in developed tension of the contraction accompanying the slow APs. These results suggest that the positive inotropic action of taurine is not mediated through an increase in the slow inward Ca2+ current. However, the transient depression of Ca2+-dependent slow APs by taurine probably explains the transient negative inotropic effect of taurine.     

Simulation analysis of excitation conduction in the heart: Propagation of excitation in different tissues

The normal excitation and conduction in the heart are maintained by the coordination between the dynamics of ionic conductance of each cell and the electrical coupling between cells. To examine functional roles of these two factors, we proposed a spatially-discrete model of conduction of excitation in which the individual cells were assumed isopotential. This approximation was reasoned by comparing the apparent space constant with the measured junctional resistance between myocardial cells. We used the four reconstruction models previously reported for five kinds of myocardial cells. Coupling coefficients between adjacent cells were determined quantitatively from the apparent space constants. We first investigated to what extent the pacemaker activity of the sinoatrial node depends on the number and the coupling coefficient of its cells, by using a one-dimensional model system composed of the sinoatrial node cells and the atrial cells. Extensive computer simulation revealed the following two conditions for the pacemaker activity of the sinoatrial node. The number of the sinoatrial node cells and their coupling coefficients must be large enough to provide the atrium with the sufficient electric current flow. The number of the sinoatrial node cells must be large so that the period of the compound system is close to the intrinsic period of the sinoatrial node cell. In this simulation the same sinoatrial node cells produced action potentials of different shapes depending on where they were located in the sinoatrial node. Therefore it seems premature to classify the myocardial cells only from their waveforms obtained by electrical recordings in the compound tissue. Second, we investigated the very slow conduction in the atrioventricular node compared to, for example, the ventricle. This was assumed to be due to the inherent property of the membrane dynamics of the atrioventricular node cell, or to the small value of the coupling coefficient (weak intercellular coupling), or to the electrical load imposed on the atrioventricular node by the Purkinje fibers, because the relatively small atrioventricular node must provide the Purkinje fibers with sufficient electric current flow. Relative contributions of these three factors to the slow conduction were evaluated using the model system composed of only the atrioventricular cells or that composed of the atrioventricular and Purkinje cells. We found that the weak coupling has the strongest effect. In the model system composed of the atrioventricular cells, the propagation failure was not observed even for very small values of the coupling coefficient.(ABSTRACT TRUNCATED AT 400 WORDS)