Bacitracin significantly reduces degradation of peptides in plant cell cultures

Production of useful peptides using recombinant plant cells is often limited by the stability of the newly translated peptides in the plant expression system. As an initial step toward producing peptides in transformed plant cell cultures, we examined the stability of exogenously added arginine vasopressin (AVP) peptide in a suspension culture of Nicotiana plumbaginifolia cells. The peptide was lost rapidly from the plant cell culture, but the rate of loss was slowed significantly by the addition of the peptide, bacitracin, at a concentration of 150 mug/mL. At higher concentrations, bacitracin substantially inhibited the growth of the plant cells in culture. (c) 1997 John Wiley & Sons, Inc.     

Rapid semi-on-line monitoring of Fmoc solid-phase peptide synthesis by matrix-assisted laser desorption/ionization mass spectrometry

A simple yet highly effective application of matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS) for the rapid monitoring of Fmoc solid-phase peptide synthesisis described. A few beads of the resin are removed at any desired step during synthesis, thefully protected peptide is cleaved from the resin and an MS spectrum of the analytes presentis produced. Some standard side-chain protecting groups may be cleaved off during samplepreparation for MS analysis; however, these cleavages are readily identified. Using thisapproach, incomplete amino acid acylations are readily detected in approximately the sametime as by traditional tests such as ninhydrin. The semi-on-line method also lends itself toready optimization of synthesis protocols and to the examination of resin-bound peptide sidereactions which may not be detectable by chemical means.     

Insertion of leader peptidase into the thylakoid membrane during synthesis in a chloroplast translation system

The mechanisms of targeting and insertion of chloroplast-encoded thylakoid membrane proteins are poorly understood. In this study, we have used a translation system isolated from chloroplasts to begin to investigate these mechanisms. The bacterial membrane protein leader peptidase (Lep) was used as a model protein because its targeting and insertion mechanisms are well understood for Escherichia coli and for the endoplasmic reticulum. Lep could thus provide insight into the functional homologies between the different membrane systems. Lep was efficiently expressed in the chloroplast translation system, and the protein could be inserted into thylakoid membranes with the same topology as in E. coli cytoplasmic membranes, following the positive-inside rule. Insertion of Lep into the thylakoid membrane was stimulated by the trans-thylakoid proton gradient and was strongly inhibited by azide, suggesting a requirement for SecA activity. Insertion most likely occurred in a cotranslational manner, because insertion could only be observed if thylakoid membranes were present during translation reactions but not when thylakoid membranes were added after translation reactions were terminated. To halt the elongation process at different stages, we translated truncated Lep mRNAs without a stop codon, resulting in the formation of stable ribosome nascent chain complexes. These complexes showed a strong, salt-resistant affinity for the thylakoid membrane, implying a functional interaction of the ribosome with the membrane and supporting a cotranslational insertion mechanism for Lep. Our study supports a functional homology for the insertion of Lep into the thylakoid membrane and the E. coli cytoplasmic membrane.     

Sequence variability of three alleles of the self-incompatibility gene of Nicotiana alata.

Three alleles of the self-incompatibility gene of Nicotiana alata have been cloned and sequenced. A comparison of the sequences shows a surprisingly low level of homology (56%) and the presence of defined regions of homology and variability. The homologous regions include the N-terminal sequence, most of the cysteine residues and glycosylation sites, as well as other blocks throughout the sequence. We interpret these conserved regions as "framework" and nonconserved regions as "hypervariable," following the terminology used to describe analogous regions in the IgG supergene family. The low level of overall homology forms the basis of a general method for isolating S-allele-specific cDNAs. Allele-specific antibodies can be generated using synthetic peptides corresponding to one of the variable regions. When applied to sections of the pistil, these antibodies label the intercellular matrix in the stigma and transmitting tissue of the style and the cell walls in the epidermis of the placenta. HindIII digestion of genomic DNA generates a characteristic pattern of S-gene fragments for each genotype. These restriction fragment length polymorphisms can be used to assign S-genotype to progeny arising from breeding experiments.     

Distinct epitopes recognized by I-Ad-restricted T-cell clones within antigenic site E on influenza virus hemagglutinin.

A total of 14 I-Ad-restricted helper T-cell clones specific for the hemagglutinin (HA) molecule of influenza virus were isolated from spleens of BALB/c or (BALB/c X C57BL/10)F1 mice immunized with the H3 subtype influenza virus A/Memphis/71 (Mem 71) and from lymph nodes of BALB/c mice primed with purified HA. The specificity of these T-cell clones was assessed in proliferation assays by reactivity with naturally occurring strains of viruses that arose by antigenic drift and contain known amino acid sequence changes in HA and with a panel of monoclonal antibody (MAb)-selected mutants of Mem 71 with single amino acid substitutions in HA. The HA genes of those mutant viruses that failed to stimulate one or more of the T-cell clones were sequenced. The clones could be allocated to at least four groups, each group having a distinct pattern of reactivity with the panel of natural field strains. The epitopes recognized by the four groups of clones were found, by reactivity with MAb-selected mutants, to be in very close proximity to one another and probably overlapping. All of the distinct epitopes recognized by the T-cell clones were adversely affected by a single amino acid substitution, either at residue 60 or at residue 63 in the HA1 polypeptide chain, within the region known from antibody-binding studies as site E. Some, but not all, of the epitopes may be influenced by the addition of a carbohydrate side chain to the HA of a particular MAb-selected mutant and certain field strains containing an Asp----Asn substitution at residue 63. Site E is therefore a major site of H-2d helper T-cell recognition on the H3 HA.     

Improved gold chloride staining method for anatomical analysis of sensory nerve endings in the shoulder capsule and labrum as examples of loose and dense fibrous tissues

Consistency in gold chloride staining is essential for anatomical analysis of sensory nerve endings. The gold chloride stain for this purpose has been modified by many investigators, but often yields inconsistent staining, which makes it difficult to differentiate structures and to determine nerve ending distribution in large tissue samples. We introduce additional steps and major changes to the modified Gairns’ protocol. We controlled the temperature and mixing rate during tissue staining to achieve consistent staining and complete solution penetration. We subjected samples to sucrose dehydration to improve cutting efficiency. We then exposed samples to a solution containing lemon juice, formic acid and paraformaldehyde to produce optimal tissue transparency with minimal tissue deformity. We extended the time for gold chloride impregnation 1.5 fold. Gold chloride was reduced in the labrum using 25% formic acid in water for 18 h and in the capsule using 25% formic acid in citrate phosphate buffer for 2 h. Citrate binds gold nanoparticles, which minimizes aggregation in the tissue. We stored samples in fresh ultrapure water at 4° C to slow reduction and to maintain color contrast in the tissue. Tissue samples were embedded in Tissue Tek and sectioned at 80 and 100 μm instead of using glycerin and teasing the tissue apart as in Gairns’ modified gold chloride method. We attached sections directly to gelatin subbed slides after sectioning with a cryostat. The slides then were processed and coverslipped with Permount. Staining consistency was demonstrated throughout the tissue sections and neural structures were clearly identifiable.