Transgene-mediated and elicitor-induced perturbation of metabolic channeling at the entry point into the phenylpropanoid pathway

3H-l-Phenylalanine is incorporated into a range of phenylpropanoid compounds when fed to tobacco cell cultures. A significant proportion of (3)H-trans-cinnamic acid formed from (3)H-l-phenylalanine did not equilibrate with exogenous trans-cinnamic acid and therefore may be rapidly channeled through the cinnamate 4-hydroxylase (C4H) reaction to 4-coumaric acid. Such compartmentalization of trans-cinnamic acid was not observed after elicitation or in cell cultures constitutively expressing a bean phenylalanine ammonia-lyase (PAL) transgene. Channeling between PAL and C4H was confirmed in vitro in isolated microsomes from tobacco stems or cell suspension cultures. This channeling was strongly reduced in microsomes from stems or cell cultures of transgenic PAL-overexpressing plants or after elicitation of wild-type cell cultures. Protein gel blot analysis showed that tobacco PAL1 and bean PAL were localized in both soluble and microsomal fractions, whereas tobacco PAL2 was found only in the soluble fraction. We propose that metabolic channeling of trans-cinnamic acid requires the close association of specific forms of PAL with C4H on microsomal membranes.     

Effect of variety and location on growth and yield components of Roselle,HIbiscus sabdariffa L. and its infestation with the spiny bollworm Earias insulana (BOISD.)

Three roselle, Hibiscus sabdariffa L. varieties (Sudani, Masri and White) were cultivated at three different locations to recognize the transportation ability of roselle cultivation from the narrow old valley land to broad new land in Egypt. Qena as origin in situ old land, El-Kanater as ex situ old land and Nubaria as ex situ new land were the considered locations. Six growth quantitative characters and bolls infestation by spiny bollworm, Earias insulana were evaluated. Growth characters of roselle plants were affected significantly by either variety or location. Qena region was more suitable for roselle plant growth as judged with plant height, number of branches, number of fruits and sepals dry weight, followed by Nubaria followed by El-Kanater. Whereas, plants grown at Nubaria produced more fresh sepals weight than Qena or El-Kanater grown plants. As for Sudani, Nubaria exhibited the tallest plants, with the highest number of fruits and the heaviest fresh sepals as compared with the corresponding plants in Qena or El-Kanater. Values of broad sense heritability were highest for all characters in Qena. While the number of fruits per plant had the highest heritability in all locations. Dry sepals yield had highly significant correlation with all studied characters except percentage of water loss in Qena and Nubaria. Path coefficient analysis confirmed that fresh sepals yield had the highest direct and indirect effects on dried sepals yield. Chemical constituents responsible to sepal quality tended to produce significant variations due to the changes in varieties or locations. The highest levels of anthocyanins and sugars were achieved by Sudani variety, but the highest levels of free amino acids and total soluble solids were recorded for Masri variety. Moreover, Nubaria region was the most favourable for the accumulation of more anthocyanins in the sepals of all varieties followed by Qena. Plants grown at Qena produced sepals with the highest levels of sugars, free amino acids, organic acids and total soluble solids, followed by Nubaria followed by El-Kanater plants. Infestation with spiny bollworm Earias insulana was increased from Sudani up to Masri up to White varieties. Plants grown at Nubaria had the lowest number of attacks by bolls in all varieties, followed by those at El-Kanater followed by Qena plants. Spiny bollworm infestation was positively correlated with the number of branches and dry sepals weight, but negatively correlated with sepal moisture loss and anthocyanin contents. These findings clearly indicated that the Nubaria region was considered as a promising reclaimed area suitable for roselle cultivation, especially for Sudani, the most economic variety.     

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.      

Genetic analysis of chromosomal resistance to trimethoprim derived from clinical isolates of Escherichia coli

Chromosomal genes conferring resistance to trimethoprim were transferred from three independently isolated thy+ clinical strains of Escherichia coli to Escherichia coli K12 by using P1 transduction. Trimethoprim-resistant transductants were obtained less frequently than transductants of other chromosomal markers, suggesting that there were problems related to the expression of the trimethoprim resistance genes in E. coli K12. Mapping studies revealed that one of the resistance determinants was located at a similar position on the chromosome (1 min) to the fol-type mutations previously described in E. coli K12. The two remaining resistance determinants mapped at separate positions between 2.5 and 3 min on the chromosome. The presence of one of these determinants reduced the efficiency with which either donor or recipient cells carrying it could participate in conjugation mediated by the sex factor F and also resulted in phenotypic interaction with the azi gene. The mechanisms of trimethoprim resistance in the three clinical E. coli isolates studied were more complex and diverse than was expected from previous studies of E. coli K12 mutants.     

Enhancement of carbon tetrachloride-induced liver injury by a single dose of ethanol: proton magnetic resonance imaging (MRI) studies in vivo

Magnetic resonance imaging (MRI) and localized magnetic resonance spectroscopy (MRS) were used to study the effects of a single dose of ethanol, given 18 h prior to experiments, on CC14-induced acute hepatotoxicity in rats in situ. Localized edema in the centrilobular region of the liver, following exposure to ethanol and CCl4, was detected by 1H-MRI techniques. The edema was characterized by a volume selective spectroscopy (VOSY) method, which measured an increase in water concentration from ethanol and CCl4-treated rat livers, in comparison to control livers. Electron microscopy (EM) of the high intensity regions of the ethanol/CCl4 treated liver sections revealed dramatic subcellular changes such as fragmentation of the granular endoplasmic reticulum (ER), formation of large vacuoles and lipid droplets in the cytoplasmic matrix and extensive swelling of the mitochondria as well as disruption of the cristae. Pretreatment with alpha-phenyl tert-butyl nitrone (PBN), a free radical spin trap, prior to halocarbon exposure, was found to reduce the CC14-mediated high intensity region in the liver images. Electron microscopy of the PBN pretreated CCl4 exposed rat liver sections revealed only minor observable differences in subcellular organization, such as some swelling of the mitochondria, when compared to controls. In addition, these data suggest that ethanol may potentiate CCl4 hepatotoxicity by increased formation of free radical intermediates. Inhibition of the CCl4-induced edematous response in rat liver by PBN demonstrates that free radical intermediates, arising from the metabolism of CCl4, are possibly the causal factor in the initiation of the edema.     

Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.     

Circle reopening in the Tetrahymena ribozyme resembles site-specific hydrolysis at the 3' splice site.

The Tetrahymena intron, after splicing from its flanking exons, can mediate its own circularization. This is followed by site-specific hydrolysis of the phosphodiester bond formed during the circularization reaction. The structural components involved in recognition of this bond for hydrolysis have not been established. We have made base substitutions to the P9.0 pairing and at the 3'-terminal guanosine residue (G414) of the intron to investigate their effects on circle formation and reopening. We have found that disruption of either P9.0 pairing or binding of the terminal nucleotide result in the formation of a large circle, C-413:5E23 from precursor RNA molecules that have undergone hydrolysis at the 3' splice site. This circle is formed at the phosphodiester bond of the 5'-terminal guanosine residue of the upstream exon via nucleophilic attack by the 3'-terminal nucleotide of the intron. The large circle is novel since it can reopen eight bases downstream from the original circularization junction at a site resembling the normal 3' splice site, restoring a guanosine to the 3' terminus and re-establishing P9.0 pairing. The new 3' terminus of the intron is capable of recircularization at any of the three normal wild-type sites. We conclude that both P9.0 and the 3'-terminal guanosine residue are required for the selection of the phosphodiester bond hydrolysed during circle reopening.     

Oriented thick and thin filaments in amoeba proteus

Actin and myosin filaments as a foundation of contractile systems are well established from ameba to man (3). Wolpert et al. (19) isolated by differential centrifugation from Amoeba proteus a motile fraction composed of filaments which moved upon the addition of ATP. Actin filaments are found in amebas (1, 12, 13) which react with vertebrate heavy meromyosin (HMM), forming arrowhead complexes as vertebrate actin (3, 9), and are prominent within the ectoplasmic tube where some of them are attached to the plasmalemma (1, 12). Thick and thin filaments possessing the morphological characteristics of myosin and actin have been obtained from isolated ameba cytoplasm (18, 19). In addition, there are filaments exhibiting ATPase activity in amebas which react with actin (12, 16, 17). However, giant ameba (Chaos-proteus) shapes are difficult to preserve, and the excellent contributions referred to above are limited by visible distortions occurring in the amebas (rounding up, pseudopods disappearing, and cellular organelles swelling) upon fixation. Achievement of normal ameboid shape in recent glycerination work (15) led us to attempt other electron microscope fixation techniques, resulting in a surprising preservation of A. proteus with a unique orientation of thick and thin filaments in the ectoplasmic region.     

Topography of the rhodopsin molecule. Identification of the domain phosphorylated.

Studies on the light-stimulated phosphorylation of rod outer segments by [gamma-32P]ATP showed that although nearly 1 mol of [32P]phosphate was incorporated/mol of total opsin, only a small fraction of the molecules were phosphorylated, and these contained at least 2-3 mol of phosphate/mol. Rod outer segments containing the phosphorylated opsin were incubated with 11-cis-retinal to generate phosphorylated rhodopsin and then digested with papain to produce a cleaved complex comprising three fragments, heavy (H), medium (M) and light (L). It was shown that L-fragment of apparent mol.wt. 6000 contained all the phosphorylation sites. This suggests that one specific domain of rhodopsin is susceptible to multiple phosphorylation.