Ca lectin gene insertion has the structural features of a transposable element

The single gene Le1, coding for soybean seed lectin, was compared to le1, a naturally occurring mutant allele containing a 3.4 kb insertion within its coding region. Le1 is devoid of introns and produces a 1.0 kb mRNA. It codes for a signal sequence of 32 amino acids and a mature protein of 253 amino acids. With the exception of six single-base substitutions, the coding and flanking sequences in le1 are identical with those in the uninterrupted gene. The insertion termini are imperfect inverted repeats flanked by a 3 bp duplication of lectin target DNA. Inverted repeats within the lectin gene are located symmetrically with respect to the insertion site and are homologous to a region of the insertion termini. These molecular traits conform with the structural aspects of transposable elements in other organisms and imply some degree of site specificity.     

Biophysical studies of signal peptides: Implications for signal sequence functions and the involvement of lipid in protein export

This review discusses efforts to understand the mode of action of signal sequences by biophysical study of synthetic peptides corresponding to these protein localization signals. On the basis of reports from several laboratories, it is now clear that signal peptides may adopt a variety of conformations, depending on their local environment. In membrane-mimetic systems like detergent micelles or lipid vesicles, they have a high tendency to form helices. Ability to take up a helical conformation appears to be required at some point in the function of a signal sequence, since some peptides corresponding to export-defective signal sequences display reduced helical potential. By contrast, functional signal sequences share a high capacity to adopt helices. High affinity for organized lipid assemblies, like monolayers or vesicles, is also a property of functional signal sequences. This correlation suggests a role for direct interaction of signal sequences with the lipids of the cytoplasmic membranein vivo. Supporting this role are studies of the influence of signal peptides on lipid structure, which reveal an ability of these peptides to pertub lipid packing and to alter the phase state of the lipids. Insertion of the signal sequencein vivo could substantially reduce the barrier for translocation of the mature chain. Lastly, synthetic signal peptides have been added to native membranes and found to inhibit translocation of precursor proteins. This approach bridges the biophysical and the biochemical aspects of protein export and promises to shed light on the functional correlates of the properties and interactions observed in model systems.     

Amritosides A, B, C and D: clerodane furano diterpene glucosides from Tinospora cordifolia

Four new clerodane furano diterpene glucosides (amritosides A, B, C and D) were isolated as their acetates from Tinospora cordifolia stems. The structures of these compounds were established on the basis of spectroscopic studies.     

Development of gene transfer technology for black tiger shrimp, Penaeus monodon

An effective foreign gene transfer method for shrimp would have several potential uses in the shrimp culture industry, such as in preventing infectious diseases. We evaluated two gene transfer methods and used black tiger shrimp, Penaeus monodon, as a model target species. For a promoter, we used the 1,592-bp promoter region of the EF-1alpha gene, a house-keeping gene, of kuruma shrimp Marsupenaeus japonicus. The promoter region was linked to either the gene for green fluorescence protein (GFP) or the gene for chloramphenicol acetyl transferase (CAT). The fusion genes were designated pJEF-GFP and pJEF-CAT, respectively. The pJEF-GFP gene was introduced into fertilized eggs of black tiger shrimp by microinjection and particle gun bombardment. The survival rate of the microinjected eggs was 17.6%, and 1.0% of the treated embryos were found to be GFP-positive. However, the GFP-positive embryos were damaged and embryogenesis did not progress. The survival rate of the particle-bombarded eggs was 60.6%, and 0.42% of the treated embryos were found to be GFP-positive. Ubiquitous GFP expression was observed from 8 hr post-fertilization and these embryos developed and hatched normally. The pJEF-CAT gene was introduced into fertilized eggs of black tiger shrimp using the optimized conditions of the particle gun bombardment. CAT activity was observed from 1 to 7 days post-fertilization, with the highest activities being observed at 5 and 7 days post-hatching.     

An in-gel digestion procedure that facilitates the identification of highly hydrophobic proteins by electrospray ionization-mass spectrometry analysis

A procedure is described for in-gel tryptic digestion of proteins that allows the direct analysis of eluted peptides in electrospray ionization (ESI) mass spectrometers without the need of a postdigestion desalting step. It is based on the following principles: (a) a thorough desalting of the protein in-gel before digestion that takes advantage of the excellent properties of acrylamide polymers for size exclusion separations, (b) exploiting the activity of trypsin in water, in the absence of inorganic buffers, and (c) a procedure for peptide extraction using solvents of proven efficacy with highly hydrophobic peptides. Quality of spectra and sequence coverage are equivalent to those obtained after digestion in ammonium bicarbonate for hydrophilic proteins detected with Coomassie blue, mass spectrometry-compatible silver or imidazole-zinc but are significantly superior for highly hydrophobic proteins, such as membrane proteins with several transmembrane domains. ATPase subunit 9 (GRAVY 1.446) is a membrane protein channel, lipid-binding protein for which both the conventional in-gel digestion protocol and in solution digestion failed. It was identified with very high sequence coverage. Sample handling after digestion is notably simplified as peptides are directly loaded into the ESI source without postdigestion processing, increasing the chances for the identification of hydrophobic peptides.     

Heterologous recombinant expression of non-originator NISTmAb

The successful development and regulatory approval of originator and biosimilar therapeutic proteins requires a systems approach to upstream and downstream processing as well as product characterization and quality control. Innovation in process design and control, product characterization strategies, and data integration represent an ecosystem whose concerted advancement may reduce time-to-market and further improve comparability and biosimilarity programs. The biopharmaceutical community has made great strides to this end, yet there currently exists no pre-competitive monoclonal antibody (mAb) expression platform for open innovation. Here, we describe the development and initial expression of an intended copy of the NISTmAb using three non-originator murine cell lines. It was found that, without optimization and in culture flasks, all three cell lines produce approximately 100 mg mAb per liter of culture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, nuclear magnetic resonance spectroscopy, intact mass spectrometry, and surface plasmon resonance were used to demonstrate that the products of all three cell lines embody quality attributes with a sufficient degree of sameness to the NISTmAb Reference Material 8671 to warrant further bioreactor studies, process improvements and optimization. The implications of the work with regard to pre-competitive innovation to support process design and feedback control, comparability and biosimilarity assessments, and process analytical technologies are discussed.     

Competition and Allometry in Kochia scoparia

Comparisons between crowded and uncrowded Kochia scoparia individualsdemonstrate pronounced effects of competition on plant allometryas well as on the distributions of different aspects of size.Non-destructive measurements of height and stem diameter and,for a subset of the populations, the number and length of leavesand branches, were taken at three times, and the plants wereharvested after the third measurement. The sequential measurementsafforded the opportunity to obtain information of the effectsof competition on allometric growth trajectories of individuals,as well as on static inter-individual allometric relationships. The distributions of most size measures appeared to be normalfor the uncrowded population. Crowded populations developeda negatively-skewed height distribution and a high-inequalitymass distribution, whereas the diameter distributions remainednormal. Plants grown without neighbours showed simple allometricrelationships between height, diameter and weight. For isolatedplants, the 'static' allometric relationship between plantsof different sizes and the allometric growth trajectory of individualswere similar. Crowded populations showed complex allometry;the static inter-individual relationships between height, diameterand weight were curvilinear (on log-log scale). There were largedifferences in the allometric growth slopes of uncrowded vs.crowded plants. Allometric relationships between stem diameterand plant mass, and between total length of leaves and totallength of branches, did not seem to be altered by competition. The data suggest that height was the most important aspect ofsize influencing future growth of individuals in the crowdedpopulation. Only plants above a certain height were able tocontinue to grow from the second to third measurement in thecrowded population. This supports the hypothesis that asymmetriccompetition for light is the cause of the allometric changesand of the increase in size variability due to competition.Copyright1994, 1999 Academic Press Allometric growth, allometry, competition, growth, Kochia     

Species‐specific physiological response of dinoflagellates to quantified small‐scale turbulence1

Turbulence has been shown to alter different aspects of the physiology of some dinoflagellates. The response appears to be species‐specific and dependent on the experimental design and setup used to generate small‐scale turbulence. We examined the variability of the response of three dinoflagellate species to the turbulence, following the same experimental design used by Berdalet (1992) on Akashiwo sanguinea (Hirasaka) Ge. Hansen et Moestrup (=Gymnodinium nelsonii G. W. Martin). In all experiments, turbulence was generated by an orbital shaker at 100 rpm, which corresponded on bulk average, to dissipation rates (ε, quantified using an acoustic Doppler velocimeter) of ≈2 cm² · s^?3. Turbulence did not appreciably affect Gymnodinium sp., a small dinoflagellate. However, Alexandrium minutum Halim and Prorocentrum triestinum J. Schiller exhibited a reduced net growth rate (33% and 28%, respectively) when shaken during the exponential growth phase. Compared to the still cultures, the shaken treatments of A. minutum and P. triestinum increased the mean cell volume (up to 1.4‐ and 2.5‐fold, respectively) and the mean DNA content (up to 1.8‐ and 5.3‐fold, respectively). Cultures affected by turbulence recovered their normal cell properties when returned to still conditions. The swimming speed of the cells exposed to agitation was half that of the unshaken ones. Overall, the response of A. minutum and P. triestinum was similar, but with lower intensity, to that observed previously on A. sanguinea. We found no clear trends related to taxonomy or morphology.