Elimination of ergoline alkaloids following treatment of<Emphasis Type="Italic"> Ipomoea asarifolia</Emphasis> (Convolvulaceae) with fungicides

Ergoline alkaloids are constituents of Clavicipitaceous fungi living on Poaceae plants. Ergoline alkaloids as well as volatile oil are also present in Ipomoea asarifolia Roem. & Schult (Convolvulaceae). Treatment of this plant with two fungicides (Folicur, Pronto Plus) eliminates the ergoline alkaloids but not the volatile oil. Elimination of ergoline alkaloids occurs concomitantly with loss of fungal hyphae associated with secretory glands on the upper leaf surface of the Ipomoea plant. Our observations suggest that accumulation of ergoline alkaloids in the Convolvulaceae may depend on the presence of a plant-associated fungus.Dedicated to Wolfgang Steglich, München, on the occasion of his 70th birthday     

Transfer and Expression of an Artificial Storage Protein (ASP1) Gene in Cassava (Manihot Esculenta Crantz)

In order to increase the nutritional quality of cassava storage roots, which contain up to 85% starch of their dry weight, but are deficient in protein, a synthetic ASP1 gene encoding a storage protein rich in essential amino acids (80%) was introduced into embryogenic suspensions of cassava via Agrobacterium-mediated gene transfer. Transgenic plants were regenerated from suspension lines derived from hygromycin-resistant friable embryogenic callus lines. Molecular analysis showed the stable integration of asp1 in cassava genome and its expression at RNA level in transformed suspension lines. PCR and Southern analyses proved the transgenic nature of the regenerated plant lines. The expression of asp1 at RNA level was demonstrated by RT-PCR. The ASP1 tetramer could be detected in leaves as well as in primary roots of cultured transgenic plants by western blots. These results indicate that the nutritional improvement of cassava storage roots may be achieved by constitutive expression of asp1 in transgenic plants.     

Preliminary approach to find synthetic peptides from nodavirus capsid potentially protective against sea bass viral encephalopathy and retinopathy

Four synthetic peptides of 15 amino acids (aa), corresponding to sequences of the nodavirus DIEV RNA(2) protein, were chosen to test their potential immunogenicity in sea bass. Two of these included the N or C terminal regions (N-ter or C-ter) and the sequences of the others contained a potential external site (aa 127-140: Lp1 and as 266-279: Lp2). Two heat inactivated strains of nodavirus (HI Sb1 and HI Sb2), were used as positive controls and the carrier (KLH) as a negative control. ELISAs were performed to quantify serum antibodies specific to nodavirus, to peptides, and to the carrier in order to monitor their immunogenicity. All the fish reacted to the peptides C-Ter, Lp1 and Lp2 but only 55% of animals injected with N-ter produced specific antibodies. The proportion of fish that produced antibodies that cross reacted with nodavirus was very different with regard to the antigen injected: HI Sb1=88%; HI Sb2=85%; N-ter=38%; C-ter=27%. Protection against nodavirus was investigated by challenging the fish with a virulent viral suspension. The results showed that heat-inactivated Nodavirus protect fish and the N-ter peptide is a potential protective peptide. This initial approach showed that although vaccinating fish with peptides is possible, the tools and strategies of the research used in this field still need to be adapted to fish.     

Identification and characterization of peptides that bind human ErbB-2 selected from a bacteriophage display library

The ErbB-2 receptor, a member of the tyrosine kinase type 1 family of receptors, has been implicated in many human malignancies. The overexpression of ErbB-2 in cancer cells as well as its extracellular accessibility makes it an attractive target for the development of tumor-specific agents. In this study, random peptide bacteriophage display technology was employed to identify peptides that bound the extracellular domain of human ErbB-2. The peptide KCCYSL, most frequently occurring in the affinity-selected phage population, was chemically synthesized and characterized for its binding activities to ErbB-2. The synthetic peptide exhibited high specificity for ErbB-2 and an equilibrium dissociation constant of 30 M. Peptide binding to ErbB-2 positive human breast and prostate carcinoma cells was visualized in direct cell binding assays. In conclusion, the peptide KCCYSL has the potential to be developed into a cancer imaging or therapeutic agent targeting malignant cells overexpressing the ErbB-2 receptor.     

Self-association of helical peptides in a lipid environment

The self-association of two model transmembrane helical peptides, differing in their surface topography, was compared in mixed micelles containing 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate (CHAPS) and dimyristoylphosphatidylcholine (DMPC). One peptide, Ac-KKL₂₄KK-amide (L₂₄), has large, rotationally mobile leucine side chains and a relatively rough surface. The other peptide, Ac-KKLLLLLLAALLALLAALLALLLLLLKK-amide (L₁₈A₆), has a patch of small alanines on one side of the helix that forms a smooth surface. The aggregation state of the peptides was sampled by chemical cross-linking with bis-sulfosuccinimidyl suberate (B53). A monomer-aggregate association constant was obtained from the cross-linking results in the range of 2 × 10⁵ M^–1 to 3 × 10⁵ M^–1 for both peptides. Kinetics of formation of cross-linked dimers indicated that the ratio of dimerization constants for L₁₈A₆ to L₂₄ was between 10 and 20. This suggests that the alanine patch contributes about 1.5 Kcal/mol more stabilization free energy to dimer formation of L₁₈A₆ compared to L₂₄.     

NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS

GUS continues to be the reporter of choice for many gene fusion applications, due to the unparalleled sensitivity of the encoded enzyme and the ease with which it can be quantified in cell-free extracts and visualized histochemically in cells and tissues. A compatible and functionally equivalent reporter gene would facilitate dual promoter studies and internal standardization of expression analyses in the same plant. A search for a candidate enzyme activity not found in plants, which might form the basis of a novel GUS-compatible reporter system, led us to investigate nanH, a Clostridium perfringens gene which encodes the so-called 'small' cytoplasmic sialidase. Expression of the native, AT-rich nanH gene in transgenic plants did not, however, result in detectable sialidase activity. For this reason, a codon-optimized derivative, NAN, was synthesized which possesses a GC content similar to that found in highly expressed plant genes. NAN enzyme activity was expressed at high levels in both stably and transiently transformed cells, possessed kinetic and stability properties similar to those of GUS, and showed optimal activity in GUS buffer. Moreover, NAN and GUS activity could be visualized simultaneously in polyacrylamide gels using the corresponding methylumbelliferone-based substrates, and in whole seedlings and tissue sections using the histochemical substrates 5-bromo-4-chloro-3-indolyl alpha-d-N-acetylneuraminic acid (X-NeuNAc) and 5-bromo-6-chloro-3-indolyl beta-d-glucuronide (X-GlucM), respectively.     

Cannabinoid receptor-G protein interactions: G(alphai1)-bound structures of IC3 and a mutant with altered G protein specificity

The structure of the C-terminal region of the third cytoplasmic loop (IC3) of the cannabinoid receptor one (CB1) bound to G(alphai1) has been determined using transferred nuclear Overhauser effects (NOEs). The wild-type IC3 sequence is helical when associated with G(alphai1). In contrast, a peptide containing the amino-acid inversion, Ala(341)-Leu(342) adopts a single turn. These findings correlate with the attenuated G(i) association of CB1 with the Ala(341)-Leu(342) mutation previously observed in vivo and the diminished stimulation of G(alphai1) GTPase activity by the corresponding peptide demonstrated in vitro here. These results, the first to report the structure of a GPCR domain while associated with G protein, imply the C-terminus of CB1 IC3, a region with high-sequence conservation among G-protein coupled receptors, must be helical for efficient coupling and activation of the G(i) protein.     

Sequencing of production-scale synthetic oligonucleotides by enriching for coupling failures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A technique for sequencing oligonucleotides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is described. The series of coupling failure species are extracted from the dimethoxytrityl-on, full-length oligonucleotide in crude synthetic material using C18 stationary-phase cartridges. These concentrated failure species can be easily detected by MALDI-TOF, which determines the mass difference between spectral ions to identify a particular base. The solid-phase extraction step greatly enhances ion signals and mass resolution, and sequencing information is generally obtained from the 5' end up to the first three to four nucleotides at the 3' end. Complete sequence can be generated in conjunction with snake venom phosphodiesterase digestion of purified material. This method eliminates difficulties associated with other mass spectrometric sequencing techniques involving oligonucleotide length; structure; and sugar, base, and backbone modifications. Examples of sequencing a 17-mer composed primarily of 2'-O-methylribonucleotides and a single nonnucleosidic linker and a mixed sugar backbone 51-mer with 2'-O-methylribonucleotides and a homopolymer tail are reported in this study.     

The multisubunit acetyl-CoA carboxylase is strongly associated with the chloroplast envelope through non-ionic interactions to the carboxyltransferase subunits

The committed step for de novo fatty acid biosynthesis is the carboxylation of acetyl-CoA catalyzed by acetyl-CoA carboxylase (ACCase). Plastidial ACCase from most plants is a multisubunit complex composed of multiple copies of four different polypeptides, biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), and carboxyltransferase (alpha-CT and beta-CT). Immunoblot analyses revealed these four proteins were mostly (69% of total) associated with a 17,000 g insoluble fraction from lysed pea chloroplasts. Under the same conditions only 8% of ribulose-1,5-bisphosphate carboxylase was associated with this insoluble fraction. BCCP and biotin carboxylase BC subunits freely dissociated from 17 kg insoluble fractions under high ionic strength conditions, whereas alpha-CT and beta-CT subunits remained tightly associated. Both CT subunits were highly enriched in envelope versus stroma and thylakoid preparations whereas BC and BCCP subunits were predominantly stromal-localized due to partial dissociation. Rapid solubilization of intact chloroplasts with Triton X-100 followed by centrifugation at 30 kg resulted in a pellet that was up to 8-fold enriched in ACCase activity and 21-fold enriched in BC activity. Triton-insoluble 30 kg pellets were reduced in lipid and chlorophyll content but enriched in chloroplast DNA due to the isolation of nucleoid particles. However, ACCase was not directly associated with nucleoids since enzymatic digestion of DNA or RNA had no effect on the association with Triton-insoluble matter. The amount of Triton-insoluble ACCase was similar in chloroplasts isolated from dark- or light-adapted leaves suggesting transitory starch granules were also not involved in this association. It is proposed that ACCase is associated with envelope membranes through interactions with an unidentified integral membrane protein.