The present study was designed to test whether there are diurnal changes in the firing rate of sympathetic nerves to brown adipose tissue and whether these diurnal rhythms influenced the response to insulin injected into the suprachiasmatic nu-
For chemical characterization of glycosphingolipids, it is necessary to determine the chemical compositions of three constituents, i.e., sugars, fatty acids, and sphingoids. A new rapid analytical method is described using a one-pot reaction in a household microwave oven, producing sugars, fatty acids, and especially sphingoids free of byproducts, from a single aliquot of a biological sample. Glycosphingolipids were hydrolyzed by microwave exposure with 0.1 M NaOH/CH 3 OH for 2 min followed by 1 M HCl/ CH 3 OH for 45 s. The alkaline methanolysis step produced intermediate lysoglycosphingolipids virtually free of byproducts such as the O -methyl ethers usually seen. The fatty acid methyl esters were extracted with n-hexane, and other reaction products were dried, taken up in aqueous alkaline methanol, and shaken with chloroform. Sphingoids partitioned into the organic phase under these conditions, whereas the sugar portion that partitioned into the aqueous phase was re-N -acetylated and remethanolyzed for 30 s by microwave exposure.Analysis of the profiles of glycosphingolipid constituents obtained using the microwave oven method showed that they were quantitatively and qualitatively comparable to those obtained by time-consuming conventional methods, which require reaction for several hours. Analysis of the three constituents, including analysis by gas chromatography, may be obtained within 1 day using the method described here.
Monitoring the environmental factors during shake-flask culture of microorganisms can help to optimise the initial steps of bioprocess development. Herein, we developed a circulation direct monitoring and sampling system (CDMSS) that can monitor the behaviour of CO2 and O2 in the gas–liquid phases and obtain a sample without interrupting the shaking of the culture in Erlenmeyer flasks capped with breathable culture plugs. Shake-flask culturing of Escherichia coli using this set-up indicated that a high concentration of CO2 accumulated not only in the headspace (maximum ~100 mg/L) but also in the culture broth (maximum ~85 mg/L) during the logarithmic phase (4.5–9.0 h). By packing a CO2 absorbent in the gas circulation unit of CDMSS, a specialised shake-flask culture was developed to remove CO2 from the headspace. It was posited that removing CO2 from the headspace would suppress increases in the dissolved CO2 concentration in the culture broth (maximum ~15 mg/L). Furthermore, the logarithmic growth phase (4.5–12.0 h) was extended, the U.O.D.580 and pH value increased, and acetic acid concentration was reduced, compared with the control. To our knowledge, this is the first report of a method aimed at improving the growth of E. coli cells without changing the composition of the medium, temperature, and shaking conditions.
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