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.     

Purification and characterization of human liver branched-chain alpha-keto acid dehydrogenase complex

Human liver BCKADH complex was purified. On SDS-polyacrylamide gel electrophoresis, the purified enzyme complex gave three major bands having molecular weights of 51,000, 46,000, and 36,000, and one minor band with a molecular weight of 55,000. The minor band corresponded in molecular weight to lipoamide oxidoreductase which was purified separately. The purified BCKADH represented only approximately 20% of the maximum activity when assayed without addition of exogenous lipoamide oxidoreductase, indicating that lipoamide oxidoreductase component was readily dissociable from the complex. The BCKADH effectively oxidized all of KIV, KIC, and KMV, yielding apparent Km values in the range of 14-17 microM for those alpha-keto acids. Vmax values obtained were 0.86, 0.61, and 0.51 mumole NADH produced/min/mg of protein for KIV, KIC, and KMV, respectively, in the presence of excess amount of lipoamide oxidoreductase. This ratio of Vmax values was practically identical to those of specific activities obtained with respective branched-chain alpha-keto acids at each purification step. The enzyme complex also oxidized pyruvate and alpha-ketoglutarate to a lesser extent. Kinetic experiments gave Km values of 0.98 and 2.9 mM for pyruvate and alpha-ketoglutarate, respectively, with Vmax of 0.43 and 0.08 mumole NADH produced/min/mg of protein. NAD and CoASH were absolutely required for the reaction. Km values for NAD and CoASH were estimated to be 47 and 25 microM, respectively.     

Chest wall and respiratory system mechanics in cats: effects of flow and volume

The effects of inspiratory flow rate and inflation volume on the resistive properties of the chest wall were investigated in six anesthetized paralyzed cats by use of the technique of rapid airway occlusion during constant flow inflation. This allowed measurement of the intrinsic resistance (Rw,min) and overall dynamic inspiratory impedance (Rw,max), which includes the additional pressure losses due to time constant inequalities within the chest wall tissues and/or stress adaptation. These results, together with our previous data pertaining to the lung (Kochi et al., J. Appl. Physiol. 64: 441-450, 1988), allowed us to determine Rmin and Rmax of the total respiratory system (rs). We observed that 1) Rw,max and Rrs,max exhibited marked frequency dependence; 2) Rw,min was independent of flow (V) and inspired volume (delta V), whereas Rrs,min increased linearly with V and decreased with increasing delta V; 3) Rw,max decreased with increasing V, whereas Rrs,max exhibited a minimum value at a flow rate substantially higher than the resting range of V; 4) both Rw,max and Rrs,max increased with increasing delta V. We conclude that during resting breathing, flow resistance of the chest wall and total respiratory system, as conventionally measured, includes a significant component reflecting time constant inequalities and/or stress adaptation phenomena.     

Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver

Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.     

Effect of Protease Inhibitors on Early Events of Apoptosis

Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization ofin situDNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence ofin situDNA strand breaks. DNA stability was estimated by the measure of its sensitivityin situto denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of ≥50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNAin situ,and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to ≥50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.     

Strain differences to effects of aging on concentrations of amino acids in cerebrospinal fluid between Sprague Dawley rat and Wistar Kyoto rat.

Age-related alterations and differences of weights and those of amino acid concentrations in the cerebrospinal fluid (CSF) were evaluated between Sprague Dawley (SD) rats and Wistar Kyoto (WKY) rats from eight to twenty weeks of age. The weights of SD rats were heavier than WKY rats at all ages. The age-related alterations of the CSF concentration of many amino acids within each strain were significant but showed no significant trend with age. Between the strains, the concentration differences of excitatory and inhibitory amino acids were not frequent although the concentrations of arginine, alanine and threonine were significantly higher in SD rats than in WKY rats. These results suggest that the different CSF concentrations of amino acids may relate to characteristics of rat strains.     

Differential Localization of the γ3 and γ12 Subunits of G Proteins in the Mammalian Brain

Abstract: The localization of two forms of the γ subunit of G proteins, γ₃ and γ₁₂, was examined in the mammalian brain. Concentrations of these two γ subunits increased markedly, as did those of glial fibrillary acidic protein, during postnatal development in the rat cerebral cortex. In aged human brains, by contrast, the concentration of γ₃ tended to decrease with age, whereas that of γ₁₂ in the temporal cortex increased slightly. An immunohistochemical study of human brains revealed that γ₃ was abundant in the neuropil, whereas γ₁₂ was localized in glial cells. In the hippocampal formation of aged human brains, levels of γ₁₂-positive cells, as well as levels of glial fibrillary acidic protein- and vimentin-positive astrocytes, increased, in particular in the CA1 subfield and the prosubiculum, in which there was a decrease in the number of pyramidal cells. The appearance of γ₁₂-positive cells associated with the loss of pyramidal cells was also observed in the hippocampus of rats that had been treated with kainic acid. These results indicate that γ₁₂ is strongly expressed in reactive astrocytes. In a study of cultured neural cells, we found that γ₁₂ was predominant in glioma cells, such as C6 and GA-1 cells, in contrast with the specific localization of γ₃ in PC12 pheochromocytoma cells, which are neuron-like cells. Taken together, the results indicate that γ₃ and γ₁₂ are selectively expressed in neuronal and glial cells, respectively, and that concentrations of γ₃ and γ₁₂ in the brain are related to the numbers and/or extent of maturation of these cells.     

Regulation of hormonal induction of chick liver cytosol-specific phosphoenolpyruvate carboxykinase

Administration of glucagon, epinephrine, or dibutyryl cAMP to chicks induced cytosol-specific phosphoenolpyruvate carboxykinase in liver. In vitro translation assay with poly(A)+RNA indicated that this induction was due to the increase in phosphoenolpyruvate carboxykinase-coding mRNA synthesis which resulted from an increased level of hepatic cAMP. Either hydrocortisone or alpha-adrenergic agonist was ineffective for the induction by itself, but showed a significant effect when administered together with one of the inducing agents given above. In particular, hydrocortisone enhanced the synthesis of phosphoenolpyruvate carboxykinase-specific mRNA without changing the profile of the time courses of the induction and of hepatic cAMP level. Those observations suggest that the fundamental machinery required for induction of cytosol-specific phosphoenolpyruvate carboxykinase in liver is shared in common between rat and chick, and that the absence of appreciable induction of cytosol-specific hepatic phosphoenolpyruvate carboxykinase in starved chicks is due to neither lack nor impairment of the hormone-mediated induction mechanism, but is due to the difference in usage of the genetic information between the two animal species.     

Regulation by induction of branched-chain 2-oxo acid dehydrogenase complex in clofibrate-fed rat liver.

The activity of branched-chain 2-oxo acid dehydrogenase complex increased 3.0-fold in liver of rats fed on 0.1%(w/w) clofibrate. Immunotitration experiments with antibodies against the constituent enzymes of the complex revealed that this increase resulted mainly from the increased amounts of only two(a decarboxylase and a lipoate acyltransferase) of three components of the complex and that the other component(dihydrolipoamide dehydrogenase) remained unchanged in its content, irrespective of clofibrate administration. The increases of both enzyme components were associated with increases in their mRNA levels which were estimated by in vitro translation with poly(A)+ RNA.