Formation of biologically active protein from the subunits of islets-activating protein (IAP), a new protein isolated from Bordetella pertussis.

Based on the finding reported in the preceding paper (Kanbayashi, et al.: J. Biochem) that subunits of islets-activating protein (IAP), a new protein purified from the culture media of Bordetella pertussis, were inactive as such, but regained the original biological activities when recombined, the conditions required for recovery of the biological activities were studied. Essentially the same biological activities as the native IAP were recovered when the smallest subunit, F-3, was incubated with one of the other subunits, F-1 and F-2, at a pH of around 7, at temperatures below 30 degrees C and for longer than 12 h. During the incubation, association products were formed which were isolated by gel filtration as homogenous proteins that consisted of two subunits probably in a molar ratio of 1 : 1. The native IAP (consisting of two IAP subunits including F-3) were equipotent in enhancing insulin secretory responses, in inhibiting epinephrine-induced hyperglycemia, in inducing leukocytosis and in increasing histamine sensitivity in experimental animals.     

Fetal mouse lung in circumfusion system cultures

Summary Fetal mouse lungs were cultivated, using the dual-rotary circumfusion system for tissue culture, and their histotypic development was surveyed for 75 days by phase-contrast and electron microscopy. Alveoli, terminal bronchioles and alveolar macrophages were photographed periodically with still and time-lapse phase-contrast microscopy. Their histotypic appearance was confirmed by electron micrographs of the 1- and 2 1/2-month-old specimens. These revealed typical alveoli surrounded by a basal lamina and composed of types I and II pneumocytes containing various lamellar-body forms within the type II cells, the alveolar lumen, and the alveolar macrophages. There was a shift from almost all type II cells in the 1-month-old alveoli to the presence of frequent type I cells as constituents of the alveoli in the 2 1/2-month-old cultures. The terminal bronchioles were tubules consisting of ciliated cells with Clara cells interspersed between them. The ciliated cells contained as many as 30 cilia or basal bodies per section and numerous microvilli. They were attached to each other and to the Clara cells by junctional complexes and accessory desmosomes which were generally in the apical ends of the cells. The Clara cells typically had glycogen granules interspersed between lamellae of the endoplasmic reticulum, contained numerous well dispersed mitochondria, occasional lysosome-like granules and crystalloid bodies which appeared to be tubular. Some Clara cells presented a moderately dense secretory granule in the center of the whorl of the endoplasmic reticulum. This work supported by Grant HL19684 from the National Heart and Lung Institute, National Institutes of Health. Pregnant Strong A mice were kindly supplied by Dr. Henry Browning of the Department of Anatomy.     

Ca2(+)-independent secretory mechanism of thyrotropin-releasing hormone (TRH) involves protein kinase C in rat pituitary cells

Thyrotropin-releasing hormone (TRH) stimulates biphasic prolactin (PRL) secretion from rat pituitary GH3 cells. The pretreatment of cells with EGTA (100 microM) plus arachidonic acid (15 microM), a condition which decreased TRH-responsive intracellular Ca2+ pools, eliminated the activity of TRH on burst PRL secretion (2 min) but did not alter that on sustained PRL secretion (30 min). However, the treatment of cells with EGTA, arachidonic acid and H-7 (300 microM), a potent inhibitor of protein kinase C (PKC), almost completely suppressed the activity of TRH for sustained PRL secretion. In cells down-modulated for PKC, TRH abolished this Ca2(+)-independent sustained PRL secretion. These results suggest that TRH acts through a separate, Ca2(+)-independent secretory mechanism, besides by modulating the Ca2(+)-dependent mechanism and that PKC is involved in this Ca2(+)-independent secretory pathway.     

Effect of short — chain fatty acids on electrical activity of the small intestinal mucosa of rat

Intravenous administration of short-chain fatty acid (SCFA), such as propionate, butyrate, valerate and caproate, caused a transient increase in transmural potential difference (p.d.) across the small intestine of rat $\text{in vivo}$. There was a sigmoid relationship between the change in the p.d. and the logarithm of the dose of SCFA. The median effective dose of propionate, n-butyrate, n-valerate and n-caproate, which was calculated from the each dose-response curve obtained from the terminal ileum, 1.31, 1.43, 0.83 and 0.81 μmole, respectively. Repeated administrations of the same dose of propionate evoked progressively smaller response. The dose-response curve of propionate was shifted to the left by neostigmine and to the right by atropine, suggesting that the action of SCFA may be mediated by acetylcholine, which was released from a nerve ending.     

Kinetics of photoinactivation and photooxidation of mitomycin C in the presence of riboflavin

The kinetic relation between the photoinactivation and photooxidation of mitomycin C in the presence of riboflavin was investigated. The photoinactivation was tested for lambda-phage induction in Escherichia coli K-12 (lambda) cells and colony formation of E. coli Bs-1 cells. Mitomycin C lost its phage-inducing and antibiotic activities when the antibiotic was irradiated in vitro with visible light in the presence of riboflavin. The loss of phage-inducing activity followed a Stern-Volmer type equation with respect to the dose of irradiation, and the inactivation constant was evaluated to be 0.96X10(-4)m2/J. The initial rate of decay of mitomycin C by photooxidation in the presence of riboflavin obeyed first order kinetics, and its cross section was estimated to be 2.51X10(-6)m/J independent of the intensity of incident light. The cross section for photooxidation was found to be proportional to the inactivation constant. These results suggest that the photoinactivation of mitomycin C is caused by its photooxidation. In order to rationalize this conclusion, a mechanism of photooxidation was proposed and reactions in vivo of the photoproduct were discussed in relation to the inactivation.     

N-acetylchitosan gel: A polyhydrate of chitin

N-Acetylchitosan gel, a polyhydrate of chitin, and partially O-acetylated N-acetylchitosan gel were produced by a facile acetylation of chitosan with acetic anhydride in 10% acetic acid and in aqueous acetic acid/methanol at room temperature. Under the same conditions, a series of N-acylchitosan gels was produced in reaction with the other carboxylic anhydrides. The gels thus produced were colorless, transparent and rigid, and stable on heating. The gels were insoluble in cold and boiling water, formic acid, aqueous acids, and the other solvents examined. Significant changes in specific rotation occurred in the acylation of chitosan and its aggregation.     

Inhibitory effect of norepinephrine on immunoreactive corticotropin-releasing factor release from the rat hypothalamus in vitro

Effects of catecholamines on immunoreactive corticotropin-releasing factor (I-CRF) release from the rat hypothalamus were examined using a rat hypothalamic perifusion system and a rat CRF RIA in vitro. Norepinephrine had a potent inhibitory effect on I-CRF release in a dose-dependent manner at 0.1 nM-1 microM concentrations, but dopamine did not. This inhibitory effect of norepinephrine was completely blocked by propranolol, but only partially blocked by phentolamine. Isoproterenol also had a potent inhibitory effect at 0.01-100 nM concentrations, and a high dose of phenylephrine (10 nM) inhibited I-CRF release. Clonidine did not influence I-CRF release. These results suggest that norepinephrine inhibits I-CRF release mainly through the beta-adrenergic receptor and partially through the alpha 1-receptor.     

Tissue culture on a chip: Developmental biology applications of self‐organized capillary networks in microfluidic devices

Organ culture systems are used to elucidate the mechanisms of pattern formation in developmental biology. Various organ culture techniques have been used, but the lack of microcirculation in such cultures impedes the long‐term maintenance of larger tissues. Recent advances in microfluidic devices now enable us to utilize self‐organized perfusable capillary networks in organ cultures. In this review, we will overview past approaches to organ culture and current technical advances in microfluidic devices, and discuss possible applications of microfluidics towards the study of developmental biology.     

Detection and identification of Escherichia coli,Shigella, and Salmonella by microarrays using the gyrB gene

Commonly, 16S ribosome RNA (16S rRNA) sequence analysis has been used for identifying enteric bacteria. However, it may not always be applicable for distinguishing closely related bacteria. Therefore, we selected gyrB genes that encode the subunit B protein of DNA gyrase (a topoisomerase type II protein) as target genes. The molecular evolution rate of gyrB genes is higher than that of 16S rRNA, and gyrB genes are distributed universally among bacterial species. Microarray technology includes the methods of arraying cDNA or oligonucleotides on substrates such as glass slides while acquiring a lot of information simultaneously. Thus, it is possible to identify the enteric bacteria easily using microarray technology. We devised a simple method of rapidly identifying bacterial species through the combined use of gyrB genes and microarrays. Closely related bacteria were not identified at the species level using 16S rRNA sequence analysis, whereas they were identified at the species level based on the reaction patterns of oligonucleotides on our microarrays using gyrB genes.     

Genome-wide screening of genes required for swarming motility in Escherichia coli K-12

Escherichia coli K-12 has the ability to migrate on semisolid media by means of swarming motility. A systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection) was used to identify the genes involved in the swarming motility of this bacterium. Of the 3,985 nonessential gene mutants, 294 were found to exhibit a strongly repressed-swarming phenotype. Further, 216 of the 294 mutants displayed no significant defects in swimming motility; therefore, the 216 genes were considered to be specifically associated with the swarming phenotype. The swarming-associated genes were classified into various functional categories, indicating that swarming is a specialized form of motility that requires a wide variety of cellular activities. These genes include genes for tricarboxylic acid cycle and glucose metabolism, iron acquisition, chaperones and protein-folding catalysts, signal transduction, and biosynthesis of cell surface components, such as lipopolysaccharide, the enterobacterial common antigen, and type 1 fimbriae. Lipopolysaccharide and the enterobacterial common antigen may be important surface-acting components that contribute to the reduction of surface tension, thereby facilitating the swarm migration in the E. coli K-12 strain.