Improved production of L-lysine by disruption of stationary phase-specific rmf gene in Escherichia coli

Growth and rate, at which fermentation products are formed in cells, generally decreases during the stationary phase as a result of changes in gene expression. We focused on the rmf gene, which encodes the ribosome modulation factor protein, as a target for strain modification in order to improve the rate of L-lysine production in Escherichia coli. Increased expression of the rmf gene during the stationary phase was confirmed under various cultivation conditions using DNA macroarray analysis. Mutants with disrupted rmf were then generated from an L-lysine-producing E. coli strain. The rates of L-lysine accumulation and production were significantly increased in disruptants that were cultivated with excess phosphate. By contrast, a higher biomass was generated in disruptants that were grown under limited phosphate conditions. These results demonstrate that disruption of the rmf gene significantly affects L-lysine production and growth in E. coli.     

An electron microscopic study on the interstitial cells of the gizzard in the love-bird (Uroloncha domestica)

Summary A type of cells morphologically resembling fibroblasts or Schwann cells and identical with the interstitial cells as firstly described byCajal was studied electronmicroscopically.Examinations of serial sections reveal that cell membranes of these cells make close appositions (nexus) to those of all surrounding smooth muscle cells. The surfaces of these cells are also provided with nerve endings of certain axons derived from plexus myentericus.On the basis of these findings, the possible nature and function of interstitial cells are discussed.This work was supported in part by NIH Grant NB-06052.     

Cholesterol esterase accelerates intestinal cholesterol absorption

Lipid bilayers of dimyristoyl phosphatidylcholine (DMPC) containing opioid peptide dynorphin A(1-17) are found to be spontaneously aligned to the applied magnetic field near at the phase transition temperature between the gel and liquid crystalline states (T(m)=24 degrees C), as examined by 31P NMR spectroscopy. The specific interaction between the peptide and lipid bilayer leading to this property was also examined by optical microscopy, light scattering, and potassium ion-selective electrode, together with a comparative study on dynorphin A(1-13). A substantial change in the light scattering intensity was noted for DMPC containing dynorphin A(1-17) near at T(m) but not for the system containing A(1-13). Besides, reversible change in morphology of bilayer, from small lipid particles to large vesicles, was observed by optical microscope at T(m). These results indicate that lysis and fusion of the lipid bilayers are induced by the presence of dynorphin A(1-17). It turned out that the bilayers are spontaneously aligned to the magnetic field above T(m) in parallel with the bilayer surface, because a single 31P NMR signal appeared at the perpendicular position of the 31P chemical shift tensor. In contrast, no such magnetic ordering was noted for DMPC bilayers containing dynorphin A(1-13). It was proved that DMPC bilayer in the presence of dynorphin A(1-17) forms vesicles above T(m), because leakage of potassium ion from the lipid bilayers was observed by potassium ion-selective electrode after adding Triton X-100. It is concluded that DMPC bilayer consists of elongated vesicles with the long axis parallel to the magnetic field, together with the data of microscopic observation of cylindrical shape of the vesicles. Further, the long axis is found to be at least five times longer than the short axis of the elongated vesicles in view of simulated 31P NMR lineshape.     

Contribution of K(ir)2 potassium channels to ATP-induced cell death in brain capillary endothelial cells and reconstructed HEK293 cell model

Cellular turnover of brain capillary endothelial cells (BCECs) by the balance of cell proliferation and death is essential for maintaining the homeostasis of the blood-brain barrier. Stimulation of metabotropic ATP receptors (P2Y) transiently increased intracellular Ca2(+) concentration ([Ca2(+)](i)) in t-BBEC 117, a cell line derived from bovine BCECs. The [Ca2(+)](i) rise induced membrane hyperpolarization via the activation of apamin-sensitive small-conductance Ca2(+)-activated K(+) channels (SK2) and enhanced cell proliferation in t-BBEC 117. Here, we found anomalous membrane hyperpolarization lasting for over 10 min in response to ATP in ～15% of t-BBEC 117, in which inward rectifier K(+) channel (K(ir)2.1) was extensively expressed. Once anomalous hyperpolarization was triggered by ATP, it was removed by Ba2(+) but not by apamin. Prolonged exposure to ATPγS increased the relative population of t-BBEC 117, in which the expression of K(ir)2.1 mRNAs was significantly higher and Ba2(+)-sensitive anomalous hyperpolarization was observed. The cultivation of t-BBEC 117 in serum-free medium also increased this population and reduced the cell number. The reduction of cell number was enhanced by the addition of ATPγS and the enhancement was antagonized by Ba2(+). In the human embryonic kidney 293 cell model, where SK2 and K(ir)2.1 were heterologously coexpressed, [Ca2(+)](i) rise by P2Y stimulation triggered anomalous hyperpolarization and cell death. In conclusion, P2Y stimulation in BCECs enhances cell proliferation by SK2 activation in the majority of cells but also triggers cell death in a certain population showing a substantial expression of K(ir)2.1. This dual action of P2Y stimulation may effectively facilitate BCEC turnover.     

Campest-5-en-3-one, an oxidized derivative of campesterol, activates PPARalpha, promotes energy consumption and reduces visceral fat deposition in rats

Dietary campest-5-en-3-one (campestenone), an oxidized derivative of campesterol, significantly reduced visceral fat weight and the concentration of triacylglycerol in serum and liver of rats. Dietary campestenone dramatically increased the activities and the mRNA expressions of mitochondrial and peroxisomal enzymes involved in beta-oxidation in the liver. Campestenone activated human peroxisome proliferator-activated receptor (PPAR) alpha as determined using the novel GAL4 ligand-binding domain chimera assay system with coactivator coexpression. In contrast, dietary campestenone reduced the activities and the mRNA expressions of enzymes involved in fatty acid synthesis, except for the malic enzyme. Dietary campestenone decreased the sterol regulatory element binding protein-1 (SREBP-1) mRNA level. Energy expenditure was significantly higher in the feeding of campestenone in rats. Dietary campestenone reduced hepatic cholesterol concentration and increased fecal excretion of neutral steroids originated from cholesterol. Lymphatic absorption of cholesterol was reduced by the coadministration of campestenone in rats cannulated in the thoracic duct. These observations suggest a possibility that campestenone has an ability to prevent coronary heart disease by improving obesity and abnormality of lipid metabolism.     

Quantification of galectin-7 and its localization in adult mouse tissues

We developed a method to quantify galectin-7 extracted from adult mouse tissues by Western blot analysis. More than 0.5 ng of galectin-7 per mg of tissue was detectable by this method. The amounts of galectin-7 in tissues were determined as follows: skin, 62 +/- 3 ng/mg; esophagus, 23 +/- 8 ng/mg; stomach, 18 +/- 6 ng/mg; anus, 13 +/- 1 ng/mg; and tongue, 12 +/- 2 ng/mg. This indicates that galectin-7 production coincides with the degree of stratification of the epithelia. Interestingly, we also detected significant amounts of galectin-7, 5.9 plus minus 1.4 and 2.7 +/- 0.6 ng/mg, in the trachea and ovaries, respectively. Moreover, we found that galectin-7 is localized in the pseudostratified epithelium of the trachea and stromal epithelium of the ovaries by immunohistochemistry. Thus, galectin-7 protein might be produced primarily in stratified epithelia, but also in some wet epithelia, and plays a unique role in cell-mucus contact, or the growth of ovarian follicles.     

Pigment Epithelium-derived Factor (PEDF) Ameliorates Advanced Glycation End Product (AGE)-induced Hepatic Insulin Resistance In Vitro by Suppressing Rac-1 Activation

Advanced glycation end products (AGEs) could be implicated in insulin resistance. However, the molecular mechanisms underlying this are not fully understood. Since pigment epithelium-derived factor (PEDF) blocks the AGE-signaling pathways, we examined here whether and how PEDF improves insulin resistance in AGE-exposed hepatoma cells, Hep3B cells. Proteins were extracted from Hep3B cells, immunoprecipitated with or without insulin receptor substrate-1 (IRS-1) antibodies, and subjected to Western blot analysis. Glycogen synthesis was measured using [ (14)C]-d-glucose. AGE induced Rac-1 activation and increased phosphorylation of IRS-1 at serine-307 residues, JNK, c-JUN, and IkappaB kinase in association with decreased IkappaB levels in Hep3B cells. PEDF or overexpression of dominant negative Rac-1 blocked these effects of AGE on Hep3B cells. Further, AGEs decreased tyrosine phosphorylation of IRS-1, and subsequently reduced the association of p85 subunit of phosphatidylinositol 3-kinase with IRS-1 and glycogen synthesis in insulin-exposed Hep3B cells, all of which were inhibited by PEDF. Our present study suggests that PEDF could improve the AGE-elicited insulin resistance in Hep3B cells by inhibiting JNK- and IkappaB kinase-dependent serine phosphorylation of IRS-1 via suppression of Rac-1 activation. PEDF may play a protective role against hepatic insulin resistance in diabetes.     

Initial lesions in the mouse brain induced by intraperitoneal injection of hypertonic saline

A dose of 30 ml/kg of 8.5% solution of sodium chloride was intraperitoneally injected into ddY mice and time course observations were conducted. Colloidal carbon was infused 6 hr after injection and distribution of carbon in the brain was observed. A transient increase in hematocrit value, serum osmotic pressure and ascites was observed 1 hr arter injection. Distribution of colloidal carbon in the brain showed less penetration of the basal ganglia. Histopathological examination of the brain showed degeneration of pyramidal cells in hippocampus 6 hr after injection. Electron microscopical examination revealed intracytoplasmic microvacuoles, swollen mitochondria and dilated cisternae in rough-surfaced endoplasmic reticulum of pyramidal cells. It was suggested that serum viscosity was increased by rapid transport of water from blood into the peritoneal cavity or urine, due to the high osmotic pressure of hypertonic saline injected, resulting in a decrease in blood flow and ischemic changes in the brain.