Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment

Gold particles coated with -glucuronidase (GUS) mRNA with a 5 cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.     

Novel function of guanidine hydrochloride in reverse micellar extraction of lysozyme from chicken egg white

The efficiency of guanidine hydrochloride (GuHCl) addition in the suppression of gel formation and the extraction of lysozyme during reverse micellar extraction from chicken egg white was investigated. A low concentration of GuHCl in the feed permitted the successful extraction of lysozyme in its native form without gel formation, which is perceived as a novel function of GuHCl. The highest recovery and specific activity of lysozyme were obtained at a GuHCl concentration of 0.06 M in 25 mM AOT reverse micellar extraction from 20-fold-diluted natural chicken egg white. Lysozyme and ovalbumin CD spectra in the corresponding GuHCl aqueous solutions revealed no changes in the higher order structures of the proteins. Furthermore, the specific activity of lysozyme in the feed was well preserved in the GuHCl system. (c) 1995 John Wiley & Sons, Inc.     

Vitamin D receptor contains multiple dimerization interfaces that are functionally different.

The vitamin D receptor mediates the signal of 1 alpha, 25-dihydroxyvitamin D3 by binding to vitamin D responsive elements in DNA as a homodimer or as a heterodimer composed of one vitamin D receptor subunit and one retinoid X receptor subunit. We have mapped the dimerization interfaces of the vitamin D receptor that is involved in homo- or heterodimer formation in the absence of DNA. While deletion of the first zinc finger region of vitamin D receptor diminished homodimerization activity, it did not affect heterodimerization. In contrast, a deletion just beyond the zinc finger region affected heterodimerization with retinoid X receptor, but not homodimerization. The zinc finger region alone could form a homodimer with full-length vitamin D receptor, but not a heterodimer with retinoid X receptor. The carboxy-terminal region was also necessary for heterodimer formation. This region showed only a weak dimerization activity in the absence of ligand, but this was dramatically increased in the presence of ligand for both homo- and heterodimerization. These results suggest that the vitamin D receptor has at least three dimerization interfaces whose functions are apparently distinguishable. These are located in the first zinc finger region, the region just beyond this zinc finger and in the carboxy-terminal region.     

Products of phosphatidylglycerol turnover in two Bacillus strains with and without lipoteichoic acid in the cells

In order to understand the phosphatidylglycerol turnover mechanism, especially the differential turnover of diacylated and unacylated glycerol moieties of the lipid, products of phosphatidylglycerol metabolism were surveyed in vivo in Bacillus subtilis W23 and an alkalophile, Bacillus sp. strain A007. When cells of B. subtilis W23 labeled with radioactive glycerol were chased, lipoteichoic acid accumulated 90% of the radioactivity lost from the unacylated glycerol moiety of phosphatidylglycerol. Also, lipids other that phosphatidylglycerol, except diacylglycerol, and glycerol and glycerophosphate incorporated much less radioactivity. The [32P]phosphoryl group was also transferred from phosphatidylglycerol to lipoteichoic acid almost quantitatively in B. subtilis W23. A unique metabolism of phosphatidylglycerol was found in Bacillus sp. strain A007 which lacked phosphoglycolipid and lipoteichoic acid, that is, the turnover of phosphatidylglycerol of this organism was less extensive compared with that of B. subtilis W23, and both glycerol moieties of the lipid were metabolized at an identical rate. These results suggested that the major reaction involved in the turnover of phosphatidylglycerol was the transfer of glycerophosphate residue to lipoteichoic acid in a bacterium which possessed lipoteichoic acid and that several minor reactions also were involved in phosphatidylglycerol turnover.     

Inhibition of cholesterol absorption and synthesis in rats by sesamin

The effects of sesamin, a lignan from sesame oil, on various aspects of cholesterol metabolism were examined in rats maintained on various dietary regimens. When given at a dietary level of 0.5% for 4 weeks, sesamin reduced the concentration of serum and liver cholesterol significantly irrespective of the presence or absence of cholesterol in the diet, except for one experiment in which the purified diet free of cholesterol was given. On feeding sesamin, there was a decrease in lymphatic absorption of cholesterol accompanying an increase in fecal excretion of neutral, but not acidic, steroids, particularly when the cholesterol-enriched diet was given. Sesamin inhibited micellar solubility of cholesterol, but not bile acids, whereas it neither bound taurocholate nor affected the absorption of fatty acids. Only a marginal proportion (ca. 0.15%) of sesamin administered intragastrically was recovered in the lymph. There was a significant reduction in the activity of liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase after feeding sesamin, although the activity of hepatic cholesterol 7 alpha-hydroxylase, drug metabolizing enzymes, and alcohol dehydrogenase remained uninfluenced. Although the weight and phospholipid concentration of the liver increased unequivocally on feeding sesamin, the histological examination by microscopy showed no abnormality, and the activity of serum GOT and GPT remained unchanged. Since sesamin lowered both serum and liver cholesterol levels by inhibiting absorption and synthesis of cholesterol simultaneously, it deserves further study as a possible hypocholesterolemic agent of natural origin.     

A novel dihydrodiol dehydrogenase in bovine liver cytosol: purification and characterization of multiple forms of dihydrodiol dehydrogenase.

Three enzymes (DD1, DD2, and DD3) having dihydrodiol dehydrogenase activity were purified to homogeneity from bovine cytosol. DD1 and DD2 were identified as 3 alpha-hydroxysteroid dehydrogenase and high-Km aldehyde reductase, respectively, as judged from their molecular weights, substrate specificities and inhibitor sensitivities. DD3 was a unique enzyme which could specifically catalyze the dehydrogenation of trans-benzenedihydrodiol and trans-naphthalenedihydrodiol without any activity toward the other tested alcohols, aldehydes, ketones, and quinones. The Km value of DD3 (0.18 mM) for benzenedihydrodiol was lower than those of other dihydrodiol dehydrogenases so far reported. DD3 immunologically crossreacted with DD1, but showed no crossreactivity with DD2. Additionally, DD3 was inhibited in a competitive manner, with a low Ki value of 1 microM, by androsterone, which was a good substrate for DD1. It was assumed that DD3 is a novel enzyme which is specific to dihydrodiols, exhibiting similarity to DD1 in immunological and structural properties.     

Archaea contain a novel diether phosphoglycolipid with a polar head group identical to the conserved core of eucaryal glycosyl phosphatidylinositol.

The structure of a major ether polar lipid of the methanogenic archaeon Methanosarcina barkeri was identified as glucosaminyl archaetidylinositol. This lipid had archaeol (2,3-di-O-phytanyl-sn-glycerol) as a core lipid portion, and the polar head group consisted of 1 mol each of phosphate, myo-inositol and D-GlcN. The polar head group was identified by means of chemical degradations, phosphatidylinositol-specific phospholipase C treatment, permethylation analysis, and fast atom bombardment-mass spectrometry as glucosaminylinositol phosphate, which was linked to the glycerol backbone via a phosphodiester bond. The stereochemical configuration of the phospho-myo-inositol residue of glucosaminyl archaetidylinositol was determined to be 1-D-myo-inositol 1-phosphate by measuring optical rotation of phospho-myo-inositol prepared by nitrous acid deamination and alkaline hydrolysis from the lipid. 1H NMR of the intact lipid showed that GlcN was linked to C-6 position of myo-inositol as an alpha-anomer. It is, finally, concluded that the complete structure of this lipid is 2,3-di-O-phytanyl-sn-glycero-1-phospho- 1'[6'-O-(2"-amino-2"-deoxy-alpha-D-glucopyranosyl)]-1'-D-myo-inositol. This lipid has a hybrid nature of an archaeal feature in alkyl glycerol diether core portion and an eucaryal feature in the polar head group identical to the conserved core structure (GlcNp(alpha 1-6)-myo-inositol 1-phosphate) of glycosylated phosphatidylinositol which serves as a membrane protein anchor in eucaryal cells.