Induction of angiogenesis in chick yolk-sac membrane by polyamines and its inhibition by tissue inhibitors of metalloproteinases (TIMP and TIMP-2).

Treatment of yolk-sac membranes of 4-day-old chick embryos with spermine or spermidine resulted in angiogenesis in the membranes. The angiogenic activity of spermine was stronger than that of spermidine. Putrescine, polylysine and histamine did not induce angiogenesis in the membranes. Administration of putrescine, spermidine and spermine increased their respective levels in yolk-sac membranes, but no interconversion of these amines was observed. The increases in spermidine and spermine levels in yolk-sac membranes preceded induction of angiogenesis. The angiogenesis induced by spermine was inhibited by tissue inhibitors of metalloproteinases, that is, TIMP and TIMP-2. These findings suggest that spermine and spermidine are angiogenesis factors in yolk-sac membranes of chick embryos and that matrix metalloproteinases represented by collagenase are involved in their action.     

Cell-division inhibitor produced by a killer strain of cellular slime mold Polysphondylium pallidum

The killer strain CK-8 of cellular slime mold Polysphondylium pallidum produces a cell-division inhibitor, in addition to a killer factor. This inhibitor represses cell division of many strains and species of cellular slime molds, except CK-8 itself and its complementary mating-type strain. It is sensitive to both heat and trypsin, and capable of binding to Con A. Its apparent molecular mass is more than 100 kDa. Repression of cell division by this inhibitor is reversed by trypsin treatment of the cells.     

An improved culture medium for mouse blastocysts

Summary Eagle's basal medium, modified to contain essential amino acids at the concentrations optimal for mouse blastocyst hatching, attachment, and outgrowth, supported in vitro development of the mouse blastocyst better than other tissue culture media tested. This medium was improved for growth and differentiation of the inner cell mass by doubling the concentration of amino acids and glucose and by adding uridine (10⁻⁵ M) and β-mercaptoethanol (10⁻⁵ M). In this improved medium nearly all blastocysts grown from the two-cell stage hatched and formed trophoblast outgrowths, and 62% developed into two-layer egg cylinders. This work was supported by the U.S. Department of Energy.     

Expression of an mNSC1 in mammalian cells.

We have cloned a cDNA inducing a cation-permeable current (mNSC1) from pancreatic beta-cells, which shows niflumate-sensitive current in Xenopus oocytes. To elucidate the expression in mammalian cells, mNSC1 was expressed in CHO cells. The reversal potential by mNSC1 was shifted toward positive which was significantly reversed by flufenamic acid. Single-channel analysis showed a characteristic of a Ca-activated nonselective cation channel. Therefore, we may conclude that mNSC1 expresses a fenamates-sensitive cation channel, inducing membrane depolarization in a mammalian cell.     

Transformation of cucumber (Cucumis sativus L.) plants using Agrobacterium tumefaciens and regeneration from hypocotyl explants

Summary Transgenic cucumber (Cucumis sativus L.) plants were successfully obtained from hypocotyl explants inoculated with Agrobacterium tumefaciens, which harbored a binary vector plasmid with NOS-nptII, CaMV 35S-I-gus and CaMV 35S-hph genes. Acetosyringone enhanced the efficiency of transformation at the cut surface cells of hypocotyl explants during five days of co-cultivation. Transformed cells were more effectively selected using 20–30 mg/l hygromycin B than using 50–100 mg/l kanamycin. Shoot regeneration occurred within 4–6 wks, and 12 of 21 regenerated plantlets displayed strong GUS expression in the very young leaves. All of 8 GUS-positive R0 plants examined showed single or a few positive bands by Southern blot analysis. The expression of the CaMV 35S-I-gus gene was observed in various tissues and organs of R0 and R1 transgenic cucumber plants.     

Py-GC/MS analysis of sediments from Lake Biwa,Japan: characterization and sources of humic acids

Humic substances extracted and purified from bottom sediments of northern Lake Biwa, Japan, in November 2012 and 2013 were characterized using elemental analysis, Fourier-transform infrared spectroscopy, hydrogen-1 nuclear magnetic resonance (¹H NMR) analysis, and pyrolysis gas chromatography/mass spectrometry (Py-GC/MS). The infrared (IR) bands in the spectra of humic acids showed the presence of amide linkages and polysaccharides. Results of ¹H NMR analysis showed that the humic acids contained approximately twice the number of aliphatic protons as those in the Japanese soil standards used for comparison. Results of the Py-GC/MS analysis, which evaluates pyrolysis temperature dependency of the amount of pyrolysis products, showed that the generation of pyrolysis products in humic acids also differed from that in Japanese soil standards but was similar to that of phytoplankton in Lake Biwa. This analysis method is the first to provide extensive information about the chemical structure of humic substances; conventional Py-GC/MS provides limited information for a single temperature. Data suggest that humic acids in lake sediments are related to chemical characteristics of phytoplankton. Results shed new light on the origins of humic substances in deep-water-lake sediments and provide insights into material recycling in such sediments.     

Simultaneous determination of citalopram and its metabolites by high-performance liquid chromatography with column switching and fluorescence detection by direct plasma injection

High-performance liquid chromatography with a successive column-switching technique was developed for simultaneous determination of citalopram and its four metabolites in plasma. Plasma samples were injected directly, and the target compounds were purified and concentrated with an inexpensive commercial octadecyl guard column. Then, the six-port valve was switched, and the compounds retained in the column were eluted by the back-flush method using 20 mM phosphate buffer (pH 4.6)-acetonitrile (70:30, v/v) containing 0.1% diethylamine and separated with an ODS column. The compounds were assayed with a fluorescence detector at an excitation wavelength of 249 nm and an emission wavelength of 302 nm. At least 30 plasma samples could be treated with an octadecyl guard column. The limits of quantitation of this method were 2.0 ng/ml for citalopram, desmethylcitalopram, didesmethylcitalopram, citalopram propionic acid and citalopram N-oxide. This method was applied to a pharmacokinetic study in dogs and a toxicokinetic study in rats.