THE ECOLOGICAL IMPACT OF ALLELOPATHY IN AILANTHUS ALTISSIMA (SIMAROUBACEAE)

Compounds inhibitory to the growth of neighboring plant species were found in significant concentrations in the leaves and stems of young Ailanthus altissima ramets. The surrounding soil also contained appreciable concentrations of similarly acting toxins. Individuals of neighboring plant species have either incorporated active portions of inhibitory compounds or responded to Ailanthus by producing growth-inhibiting substances. Under greenhouse conditions, individuals of neighboring plant species previously unexposed to Ailanthus in the field were found to be more susceptible to the Ailanthus toxins than individuals previously exposed. Moreover, seeds produced by unexposed populations were also more susceptible to Ailanthus toxins than seeds produced by previously exposed populations. These differences demonstrated that the allelochemicals of Ailanthus altissima exhibited a measurable impact upon neighboring plant species. Since the progeny of these populations displayed a differential response to Ailanthus toxin, this phenotypic difference between the two populations may have a heritable basis.     

Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation.

Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His6) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219-246 of the complete Glg2p sequence) was isolated that contained 4-25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono- and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogen-related enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis.     

Identification of the Serratia endonuclease dimer: structural basis and implications for catalysis.

The Serratia endonuclease is an extracellularly secreted enzyme capable of cleaving both single- and double-stranded forms of DNA and RNA. It is the first member of a large class of related and usually dimeric endonucleases for which a structure is known. Using X-ray crystallography, the structure of monomer of this enzyme was reported by us previously (Miller MD et al., 1994, Nature Struct Biol 1:461-468). We now confirm the dimeric nature of this enzyme through light-scattering experiments and identify the physiologic dimer interface through crystal packing analysis. This dimerization occurs through an isologous twofold interaction localized to the carboxy-terminal subdomain of the enzyme. The dimer is a prolate ellipsoid with dimensions 30 A x 35 A x 90 A. The dimer interface is flat and contains four salt links, several hydrogen bonds, and nonpolar interactions. Buried water is prominent in this interface and it includes an unusual "cubic" water cluster. The position of the two active sites in the dimer suggests that they can act independently in their cleavage of DNA, but have a geometrical advantage in attacking substrate relative to the monomer.     

The elongation of amylose and amylopectin chains in isolated starch granules

The aim of this work was to investigate the conditions required for amylose synthesis in starch granules. Although the major granule-bound isoform of starch synthase - GBSSI - catalyses the synthesis of amylose in vivo, ¹⁴C from ADP[¹⁴C]glucose was incorporated primarily into a specific subset of amylopectin chains when supplied to starch granules isolated from pea (Pisum sativum L.) embryos and potato (Solanum tuberosum L.) tubers. Incubation of granules with soluble extracts of these organs revealed that the extracts contained compounds that increased the incorporation of ¹⁴C into amylose. These compounds were rendered inactive by treatment of the extracts with α-glucosidase, suggesting that they were malto-oligosaccharides. Consistent with this idea, provision of pure malto-oligosaccharides to isolated granules resulted in a dramatic shift in the pattern of incorporation of ¹⁴C, from amylopectin chains to amylose molecules. Comparison of the pattern of incorporation in granules from wild-type peas and lam mutant peas which lack GBSSI showed that this effect of malto-oligosaccharides was specifically on GBSSI. The significance of these results for understanding of the synthesis of amylose and amylopectin in storage organs is discussed.     

Characterization of tobacco plants expressing a bacterial salicylate hydroxylase gene

Transgenic tobacco plants that express the bacterial nahG gene encoding salicylate hydroxylase have been shown to accumulate very little salicylic acid and to be defective in their ability to induce systemic acquired resistance (SAR). In recent experiments using transgenic NahG tobacco and Arabidopsis plants, we have also demonstrated that salicylic acid plays a central role in both disease susceptibility and genetic resistance. In this paper, we further characterize tobacco plants that express the salicylate hydroxylase enzyme. We show that tobacco mosaic virus (TMV) inoculation of NahG tobacco leaves induces the accumulation of the nahG mRNA in the pathogen infected leaves, presumably due to enhanced stabilization of the bacterial mRNA. SAR-associated genes are expressed in the TMV-infected leaves, but this is localized to the area surrounding necrotic lesions. Localized acquired resistance (LAR) is not induced in the TMV-inoculated NahG plants suggesting that LAR, like SAR, is dependent on SA accumulation. When SA is applied to nahG-expressing leave's SAR gene expression does not result. We have confirmed earlier reports that the salicylate hydroxylase enzyme has a narrow substrate specificity and we find that catechol, the breakdown product of salicylic acid, neither induces acquired resistance nor prevents the SA-dependent induction of the SAR genes.     

Membrane potential and input resistance are ambiguous measures of sealing of transected cable-like structures.

For many years, membrane potential (Vm) and input resistance have been used to characterize the electrophysiological nature of a seal (barrier) that forms at the cut end of a transected axon or other extended cytoplasmic structure. Data from a mathematical and an analog model of a transected axon and other theoretical considerations show that steady-state values of Vm and input resistance measured from any cable-like structure provide a very equivocal assessment of the electrical barrier (seal) at the cut end. Extracellular assessments of injury currents almost certainly provide a better electrophysiological measure of the status of plasma membrane sealing because measurements of these currents do not depend on the cable properties of extended cytoplasmic processes after transection.     

The effects of calystegines isolated from edible fruits and vegetables on mammalian liver glycosidases

The polyhydroxylated nortropane alkaloids called calyste-ginesoccur in many plants of the Convolvulaceae, Solanaceae, andMoraceae families. Certain of these alkaloids exhibit potentinhibitory activities against glycosidases and the recentlydemonstrated occurrence of calystegines in the leaves, skins,and sprouts of potatoes (Solatium tuberosum), and in the leavesof the eggplant (S.melongena), has raised concerns regardingthe safety of these vegetables in the human diet. We have surveyedthe occurrence of calystegines in edible fruits and vegetablesof the families Convolvulaceae, Solanaceae, and Moraceae byGC-MS. Calystegines A3, B1, B2, and C1 were detected in allthe edible fruits and vegetables tested; sweet and chili peppers,potatoes, eggplants, tomatoes, Physalis fruits, sweet potatoes,and mulberries. Calystegines B1 and C1 were potent competitiveinhibitors of the bovine, human, and rat β-glucosidaseactivities, with K₁ values of 150, 10, and 1.9 µM, respectivelyfor B1 and 15,1.5, and 1 µM, respectively, for C1. CalystegineB2 was a strong competitive inhibitor of the -galactosidaseactivity in all the livers. Human β-xylosidase was inhibitedby all four nortropanes, with calystegine C1 having a K₁ of0.13 µM. Calystegines A3 and B2 selectively inhibitedthe rat liver β-glucosidase activity. The potent inhibitionof mammalian β-glucosidase and -galactosidase activitiesin vitro raises the possibility of toxicity in humans consuminglarge amounts of plants that contain these compounds. edible plants calystegines glycosidase inhibitors bovine, human, and rat liver     

Isolation of genomic and cDNA clones encoding bovine poly(A) binding protein II.

cDNA clones for bovine poly(A) binding protein II (PAB II) were isolated. Their sequence predicts a protein of 32.8 kDa, revising earlier estimates of molecular mass. The protein contains one putative RNA-binding domain of the RNP type, an acidic N-terminal and a basic C-terminal domain. Analyses of authentic PAB II were in good agreement with all predictions from the cDNA sequence except that a number of arginine residues appeared to be post-translationally modified. Poly(A) binding protein II expressed in Escherichia coli was active in poly(A) binding and reconstitution of processive polyadenylation, including poly(A) tail length control. The cDNA clones showed a number of potential PAB II binding sites in the 3' untranslated sequence. Bovine poly(A)+RNA contained two mRNAs hybridizing to a PAB II-specific probe. Analysis of a genomic clone revealed six introns in the coding sequence. The revised molecular mass led to a demonstration of PAB II oligomer formation and a reinterpretation of earlier data concerning the protein's binding to poly(A).