Biochemical switching device realizing McCulloch-Pitts type equation

We analyzed cyclic enzyme systems, one of the best candidates for biochemical switching devices, especially focusing on their control mode against external perturbations. Since these systems have the reliability of ON-OFF types of operation (McCulloch-Pitts' neuronic equation), we shall present here the mechanical difference between these systems and electronic switching circuit, especially on the mnemonic mechanism of biochemical switching devices.     

Effect of C-reactive protein on peritoneal macrophages. II. Human C-reactive protein activates peritoneal macrophages of guinea pigs to release superoxide anion in vitro

The effect of human C-reactive protein (CRP) on macrophage function was studied in an assay of superoxide anion (O2-) production. Peritoneal exudate macrophages (PEM) of guinea pigs exposed in vitro to various doses of CRP for 72 hr resulted in the development of O2- production dose-dependently, measured by increases in superoxide dismutase-inhibitable nitro blue tetrazolium reduction. The O2--producing activity of PEM cultured without CRP, used as a control, decreased markedly in proportion to incubation time. The O2- production by PEM exposed to CRP for 18 hr when control PEM were still high in O2- production, was decreased by larger doses of CRP, while PEM cultured without CRP for 72 hr, when O2- production by control PEM was very low, followed by incubation with CRP for another 18 hr, produced O2- CRP-dose-dependently as in the case of that observed after 72-hr incubation with CRP. These results indicate that CRP is capable of activating macrophages and acts on macrophage function as a modulator. CRP possesses migration inhibitory factor (MIF)-like activity (as reported in the preceding paper) and also macrophage-activating factor (MAF)-like activity, indicating that CRP may play a functional role at the site of inflammation and tissue damage by accumulating and activating macrophages.     

Filtering rates on natural bacteria by Daphnia longispina and Eodiaptomus japonicus in Lake Biwa

Filtering rates on [³H]thymidine-labelled natural unattachedbacteria and that on [¹⁴C]bicarbonate-labelled natural planktonwhich pass through the 25 µm-mesh-size screen were measuredfor Daphnia longispina and Eodiaptomus japonicus in Lake Biwa.Errors associated with the radioisotope technique, i.e the lossof labels after feeding trials and the self-absorption of thebeta emittance of ³H, were checked and corrected for the calculationof the filtering rates. It was suggested that Daphnia collectsbacteria efficiently, although the efficiency is somewhat variabledepending on food particle composition (i.e. presence and absenceof larger particles) and feeding condition (i.e. animal densityand physical disturbance). By contrast, copepodites of Eodiaptomuswere suggested to be less efficient bacteria feeders. Food resourceexploitation strategies of these two co-existing zooplanktersare discussed.     

Inhibition of bovine adrenocortical mitochondrial cytochrome P-450(11)beta-mediated reactions by imidazole derivatives and mineralocorticoid analogs

The effects of several imidazole antimycotic agents, an imidazole and several mineralocorticoid analogs on the cytochrome P-450(11)beta-catalyzed 11 beta-hydroxylation of 11-deoxycorticosterone and aldosterone synthesis were examined. Ketoconazole, clotrimazole, miconazole and etomidate were found to be potent inhibitors of the reactions, causing 50% inhibition of the 11 beta-hydroxylase activity at concentrations between 10(-8) and 10(-7) M. The potency of etomidate as to the inhibition of aldosterone- and 18-hydroxycorticosterone-production was found to be almost equal to that in the case of 11 beta-hydroxylation. Spironolactone and other newly synthesized mineralocorticoid analogs were also found to inhibit the cytochrome P-450(11)beta-mediated reactions. The ID50 values of these drugs for inhibition of the 11 beta-hydroxylase activity were almost equal to those in the case of the aldosterone- and 18-hydroxycorticosterone-biosynthetic activities. The results of kinetical studies indicated that one of the mineralocorticoid analogs, Compound 23-0586, acts as a competitive inhibitor for the cytochrome P-450(11)beta-mediated reactions.     

Secretion of soluble functional insulin receptors by transfected NIH3T3 cells

In order to determine whether the human insulin receptor ectodomain can be expressed as a functional protein, the coding regions for the transmembrane and cytoplasmic domain of a full-length human insulin receptor cDNA were deleted by site-directed mutagenesis, and the resultant construct was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH3T3 cells, a cell line secreting an insulin binding protein was isolated. The insulin binding alpha subunit had an Mr of 138,000 and a beta subunit of Mr 48,000 (compared to 147,000 and 105,000 for the full-length human insulin receptor expressed in NIH3T3 cells). This difference in size of the alpha subunit was due to a difference in glycosylation as N-glycanase digestion reduced the apparent size of the alpha subunits of secreted and normal membrane-bound receptors to identical values. The secreted receptor formed disulfide-linked heterotetrameric structures with an Mr of 280,000. It was synthesized as an Mr 160,000 precursor which was cleaved into mature subunits with a t1/2 of 3 h. Increasing expression of the cDNA by induction with sodium butyrate lead to the appearance of an Mr 180,000 protein in the medium as well as the mature alpha and beta subunits. A Scatchard plot of insulin binding to the secreted receptor was curvilinear with a Kd of 7 X 10(-10) M for the high affinity sites and 10(-7) M for the low affinity site (compared to Kd values of 1.1 X 10(-9) M and 10(-7) M, respectively, for human insulin receptors expressed in these cells.     

Inhibition of RNA synthesis in embryo of Xenopus laevis by protease inhibitor

We have studied the role of proteases during the development of Xenopus laevis embryos with the aid of protease inhibitors. The activity of proteases was found to be only minimal in the unfertilized egg and during the initiation of development, but activity began to increase at the morula stage. When the activity of proteases was inhibited by antipain, an inhibitor of endopeptidase activity, RNA synthesis in the embryo was inhibited. To examine the relationship between the inhibitory effect of antipain on protease activity and its effect on RNA synthesis, antipain was reduced with NaBH4 to inactivate its protease inhibitory activity. The reduced antipain did not inhibit RNA synthesis in the embryo. Antipain effectively inhibited synthesis of both rRNA and poly(A)+RNA but not 4S RNA. We therefore suggest that protease activity plays an important role in the initiation and/or continuation of RNA synthesis.     

Glyoxylate Cycle Enzymes in Peroxisomes Isolated from Petals of Pumpkin (Cucurbita sp.) during Senescence

The activities of the two unique enzymes of the glyoxylate cycle,isocitrate lyase (EC 4.1.3.1 [EC] ) and malate synthase (EC 4.1.3.2 [EC] ),were undetectable in petals of pumpkin (Cucurbita sp. AmakuriNankin) until the end of blooming, but they appeared duringsenescence. The activity of catalase (EC 1.11.1.6 [EC] ) increased,glycolate oxidase (EC 1.1.3.1 [EC] ) activity did not change, whilehydroxypyruvate reductase (EC 1.1.1.81 [EC] ) activity peaked at fullblooming stage and declined thereafter. After fractionationof cellular organelles on a sucrose density gradient, we detectedisocitrate lyase and malate synthase activities in peroxisomalfractions only from petals at the senescing stage. Northernblot analysis revealed that malate synthase mRNA increased duringpetal senescence. Citrate synthase (EC 4.1.3.7 [EC] ) and malate dehydrogenase(EC 1.1.1.37 [EC] ) activities were also present, while aconitase(EC 4.2.1.3 [EC] ) was not detectable in peroxisomal fractions. Moreoverthe presence of 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35 [EC] )and urate oxidase (EC 1.7.3.3 [EC] ) in the peroxisomal fractionsfrom senescing petals indicates that peroxisomes could be involvedboth in the ß-oxidation pathway and in the purinecatabolism during petal senescence. (Received May 25, 1991; Accepted September 25, 1991)     

Innervation of the C cells of chicken ultimobranchial glands studied by immunohistochemistry, fluorescence microscopy, and electron microscopy

Innervation of the ultimobranchial glands in the chicken was investigated by immunohistochemistry, fluorescence microscopy and electron microscopy. The nerve fibers distributed in ultimobranchial glands were clearly visualized by immunoperoxidase staining with antiserum to neurofilament triplet proteins (200K-, 150K- and 68K-dalton) extracted from chicken peripheral nerves. The ultimobranchial glands received numerous nerve fibers originating from both the recurrent laryngeal nerves and direct vagal branches. The left and right sides of the ultimobranchial region were asymmetrical. The left ultimobranchial gland had intimate contact with the vagus nerve trunk, especially with the distal vagal ganglion, but was somewhat separated from the recurrent nerve. The right gland touched the recurrent nerve, the medial edge being frequently penetrated by the nerve, but the gland was separated from the vagal trunk. The left gland was innervated mainly by the branches from the distal vagal ganglion, whereas the right gland received mostly the branches from the recurrent nerve. The carotid body was located cranially near to the ultimobranchial gland. Large nerve bundles in the ultimobranchial gland ran toward and entered into the carotid body. By fluorescence microscopy, nerve fibers in ultimobranchial glands were observed associated with blood vessels. Only a few fluorescent nerve fibers were present in close proximity to C cell groups; the C cells of ultimobranchial glands may receive very few adrenergic sympathetic fibers. By electron microscopy, numerous axons ensheathed with Schwann cell cytoplasm were in close contact with the surfaces of C cells. In addition, naked axons regarded as axon terminals or "en passant" synapses came into direct contact with C cells. The morphology of these axon terminals and synaptic endings suggest that ultimobranchial C cells of chickens are supplied mainly with cholinergic efferent type fibers. In the region where large nerve bundles and complex ramifications of nerve fibers were present, Schwann cell perikarya investing the axons were closely juxtaposed with C cells; long cytoplasmic processes of Schwann cells encompassed large portions of the cell surface. All of these features suggest that C-cell activity, i.e., secretion of hormones and catecholamines, may be regulated by nerve stimuli.