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.     

Intracellular localization of alkaline phosphatase in freshly isolated foetal rat hepatocytes

Summary The cytochemical localization of alkaline phosphatase activity in foetal rat hepatocytes was examined in relation to the pattern of cell to cell attachment during cell isolation and culture. In foetal hepatocytesin vivo, alkaline phosphatase was exclusively localized on the bile canalicular membrane. In freshly isolated foetal hepatocytes, however, the activity was present in the endoplasmic reticulum, nuclear envelope, Golgi apparatus, tubulo-vesicular organelles, and over the entire plasma membrane. In monolayer cells cultured for one or two days, the activity was localized on the reconstituted bile canalicular membrane, plasma membrane sites adjacent to neighbouring cells and on the bottom surface of the monolayer, but was detected in none of the intracellular organelles. Biochemical alkaline phosphatase activity did not change during isolation of the cells. These results suggest that, in foetal hepatocytes, loss of cell—cell contact may induce a temporal disturbance, or dedifferentiation, in their membrane system.     

Efficient synthesis and secretion of a thermophilic alpha-amylase by protein-producing Bacillus brevis 47 carrying the Bacillus stearothermophilus amylase gene.

Bacillus subtilis and Bacillus brevis 47-5, carrying the Bacillus stearothermophilus alpha-amylase gene on pUB110 (pBAM101), synthesized the same alpha-amylase as the donor strain as determined by the enzyme's thermal stability and NH2-terminal amino acid sequence. Regardless of the host, the 34-amino acid signal peptide of the enzyme was processed at exactly the same site between two alanine residues. B. brevis 47-5(pBAM101) secreted the enzyme most efficiently of the hosts examined, 100, 15, and 5 times more than B. stearothermophilus, Escherichia coli HB101(pH1301), and B. subtilis 1A289(pBAM101), respectively. The efficient secretion of the enzyme in B. brevis 47-5(pBAM101) was suggested to be due to the unique properties of the cell wall of this organism.     

A single gene directs synthesis of a precursor protein with beta- and alpha-amylase activities in Bacillus polymyxa.

The Bacillus polymyxa amylase gene comprises 3,588 nucleotides. The mature amylase comprises 1,161 amino acids with a molecular weight of 127,314. The gene appeared to be divided into two portions by the direct-repeat sequence located at almost the middle of the gene. The 5' region upstream of the direct-repeat sequence was shown to be responsible for the synthesis of beta-amylase. The 3' region downstream of the direct-repeat sequence contained four sequences homologous with those in other alpha-amylases, such as Taka-amylase A. The 48-kilodalton (kDa) amylase isolated from B. polymyxa was proven to have alpha-amylase activity. The amino acid sequences of the peptides generated from the 48-kDa amylase showed complete agreement with the predicted amino acid sequence of the C-terminal portion. The B. polymyxa amylase gene was therefore concluded to contain in-phase beta- and alpha-amylase-coding sequences in the 5' and 3' regions, respectively. A precursor protein, a 130-kDa amylase, directed by a plasmid, pYN520, carrying the entire amylase gene, had both beta- and alpha-amylase activities. This represents the first report of a single protein precursor in procaryotes that gives rise to two enzymes.     

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.     

Kinetics and collagenolytic role of eosinophils in chronic gastric ulcer in the rat

 An association between eosinophils and tissue damage has been observed in numerous disorders. However, few reports have addressed the role of infiltrating eosinophils in gastric ulcer healing. The aim of this study was to investigate the kinetics and role of eosinophils infiltrating experimental chronic gastric ulcers in the rat. We developed a monoclonal antibody against human matrix metalloproteinase 1 (MMP1) purified from conditioned culture medium of human skin fibroblasts. Acetic acid-induced gastric ulcers were resected from rats on days 1, 3, 5, 10, 20, 40, and 180 after the days of induction (day 0). Tissue specimens were immunostained with this antibody and examined with an electron microscope. Few eosinophils were observed in the granulation tissue until day 20. By days 40 and 180, MMP1-positive eosinophils had increased in the granulation tissue of open ulcers. Azan staining revealed dispersed collagen fibers around infiltrating eosinophils. In contrast, scars demonstrated few eosinophils in fibrous tissue on days 40 and 180. Eosinophils which express MMP1 infiltrate granulation tissue at the chronic stage of gastric ulceration. The results suggest that eosinophils may play a role in tissue remodeling and deterioration of ulceration. Accepted: 18 March 1997     

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.     

Increased production of alpha-amylase by Bacillus amyloliquefaciens in the presence of glycine

The production of alpha-amylase by Bacillus amyloliquefaciens increased by a factor of 300 when glycine was added to a chemically defined simple medium at the early-logarithmic phase of growth. Glycine was not metabolized to a significant extent under the conditions used, but it considerably prevented the lowering of the pH of the culture.