Association of carbonic anhydrase with a Calvin cycle enzyme complex in Nicotiana tabacum

Chloroplast-localized carbonic anhydrase (CA; EC 4.2.1.1), an enzyme which catalyzes the reversible hydration of CO₂, appears to be associated with other enzymes of the Calvin cycle in a large multienzyme complex. Gel-filtration fast protein liquid chromatography (FPLC) of soluble proteins obtained by osmotic lysis of tobacco (Nicotiana tabacum L. cv. Carlson) chloroplasts results in the co-elution of a protein complex of greater than 600 kDa which includes CA, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoribulokinase (PRK), and ribose-5-phosphate isomerase. Anion-exchange FPLC of chloroplast extracts indicates that there is an association of CA with other proteins that modifies its elution profile in a NaCl gradient, and that Rubisco co-elutes with the fractions containing CA. Following a protocol described by Süss et al. (1993, Proc Natl Acad Sci USA 90: 5514–5518), limited protease treatment of chloroplast extracts was used to show that the association of PRK with other chloroplast proteins appears to protect a number of lysine and arginine residues which may be involved in specific protein-protein interactions. A similar treatment of CA indicates some protection of these residues when CA is associated with other chloroplast polypeptides but the level of protection is not as profound as that exhibited by PRK. In concert with previously published immunolocalization studies, these data indicate that CA may be associated with Rubisco at the stromal periphery of a Calvin cycle enzyme complex in which PRK is more centrally located and associated with thylakoid membranes. Received: 2 June 1997 / Accepted: 28 June 1997     

Integration of Biological Control and Native Seeding to Restore Invaded Plant Communities

Disturbed natural areas frequently experience invasion by introduced plant species that can reduce native biodiversity. Biological control can suppress these introduced species, but without restoration another introduced species can invade. Integration of biological control with concurrent revegetation can both aid in weed reduction via interspecific plant competition and establish a restored native plant community. This 3‐year study investigated an integrated approach to controlling the introduced annual Mile‐a‐minute weed (Persicaria perfoliata [L.] H. Gross [Polygonaceae]) using the biocontrol weevil Rhinoncomimus latipes Korotyaev (Coleoptera: Curculionidae) and restoration planting using a native seed mix. A fully factorial design tested weevils and seeding, separately and together, using insecticide to eliminate weevils. The weevils together with the native seed mix reduced P. perfoliata percent cover in 2009 and 2010, and peak seed cluster production in 2010, compared to the insecticide ? no seed control treatment. Persicaria perfoliata final dry biomass was reduced by 75% in 2010 and by 57% in 2011 in the weevils plus seed treatment compared to the control, with weevils having the greatest effect in 2010 and the seed treatment having the greatest impact in 2011. Results suggest an additive effect of biocontrol and seeding in suppressing P. perfoliata. Seeded treatments also developed the highest native plant species richness and diversity, comprised of spontaneous recolonization in addition to species from the seed mix. Results support the use of integrated management of this invasive weed, with suppression through biological control and native revegetation together helping prevent reinvasion while restoring native plant biodiversity.     

Isolated in an ocean of grass: low levels of gene flow between termite subpopulations

Habitat fragmentation is one of the most important causes of biodiversity loss, but many species are distributed in naturally patchy habitats. Such species are often organized in highly dynamic metapopulations or in patchy populations with high gene flow between subpopulations. Yet, there are also species that exist in stable patchy habitats with small subpopulations and presumably low dispersal rates. Here, we present population genetic data for the ‘magnetic’ termite Amitermes meridionalis, which show that short distances between subpopulations do not hinder exceptionally strong genetic differentiation (F_ST: 0.339; R_ST: 0.636). Despite the strong genetic differentiation between subpopulations, we did not find evidence for genetic impoverishment. We propose that loss of genetic diversity might be counteracted by a long colony life with low colony turnover. Indeed, we found evidence for the inheritance of colonies by so‐called ‘replacement reproductives’. Inhabiting a mound for several generations might result in loss of gene diversity within a colony but maintenance of gene diversity at the subpopulation level.     

High‐throughput respiration screening of single mitochondrial substrates using permeabilized CHO cells highlights control of mitochondria metabolism

Respiration analysis using isolated mitochondria and electrochemical oxygen sensing has contributed significantly to the knowledge about mitochondrial metabolism, which is involved in energy generation but also in ageing and numerous diseases. Here, we present a high‐throughput respiration screening for functional in situ mitochondrial studies in permeabilized Chinese hamster ovary cells. The determination of oxygen uptake rates allowed a quantitative comparison between different conditions and a distinction of substrates into three groups providing an insight into tricarboxylic acid (TCA) cycle regulation. The mitochondrial metabolization of citrate, isocitrate, glutamine, and glutamate was highly stimulated by ADP supply. In contrast, the metabolization of α‐ketoglutarate, succinate, fumarate, and malate was little controlled by the energy and redox state. Metabolization of pyruvate was very strictly regulated by several independent mechanisms: phosphorylation, feedback inhibition, but also by the availability of CoA. A moderate stimulation of pyruvate metabolization was accomplished by feeding both pyruvate and aspartate simultaneously. The presented high‐throughput respiration screening provides comprehensive information about the effect of single or mixed substrates on mitochondrial metabolic activities, including transport and TCA cycle regulation, and metabolic bottlenecks. This supports the design of efficient mammalian producer strains or feeding strategies, but also the investigation of pathological and toxicological effects related to mitochondrial metabolism.     

Extensive differential protein phosphorylation as intraerythrocytic Plasmodium falciparum schizonts develop into extracellular invasive merozoites

Pathology of the most lethal form of malaria is caused by Plasmodium falciparum asexual blood stages and initiated by merozoite invasion of erythrocytes. We present a phosphoproteome analysis of extracellular merozoites revealing 1765 unique phosphorylation sites including 785 sites not previously detected in schizonts. All MS data have been deposited in the ProteomeXchange with identifier PXD001684 ( http://proteomecentral.proteomexchange.org/dataset/PXD001684 ). The observed differential phosphorylation between extra and intraerythrocytic life‐cycle stages was confirmed using both phospho‐site and phospho‐motif specific antibodies and is consistent with the core motif [K/R]xx[pS/pT] being highly represented in merozoite phosphoproteins. Comparative bioinformatic analyses highlighted protein sets and pathways with established roles in invasion. Within the merozoite phosphoprotein interaction network a subnetwork of 119 proteins with potential roles in cellular movement and invasion was identified and suggested that it is coregulated by a further small subnetwork of protein kinase A (PKA), two calcium‐dependent protein kinases (CDPKs), a phosphatidyl inositol kinase (PI3K), and a GCN2‐like elF2‐kinase with a predicted role in translational arrest and associated changes in the ubquitinome. To test this notion experimentally, we examined the overall ubiquitination level in intracellular schizonts versus extracellular merozoites and found it highly upregulated in merozoites. We propose that alterations in the phosphoproteome and ubiquitinome reflect a starvation‐induced translational arrest as intracellular schizonts transform into extracellular merozoites.     

Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle

Background

30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested—mostly from the milk—of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4).

Results

With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody’s activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.

Conclusion

Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.     

Carbon accumulation and vertical accretion in a restored versus historic salt marsh in southern Puget Sound,Washington, United States

Few comparisons exist between vertical accretion (VA) and carbon accumulation rates (CARs) in restored versus historic (i.e. reference) marshes. Here, we compare these processes in a formerly diked, sparsely vegetated, restored salt marsh (Six Gill Slough, SG), whose surface is subsided relative to the tidal frame, to an adjacent, relatively pristine, historic salt marsh (Animal Slough, AS). Six sediment cores were collected at both AS and SG approximately 6 years after restoration. Cores were analyzed for bulk density (BD), % loss of ignition, % organic carbon, and ²¹⁰Pb. We found that sharp changes in BD in surface layers of SG cores were highly reliable markers for the onset of restoration. The mean VA since restoration at SG (0.79 [SD = 0.29] cm/year) was approximately twice that of AS (0.41 [SD = 0.16] cm/year). In comparison, the VA at AS over 50 years was 0.30 (SD = 0.09) cm/year. VA consisted almost entirely of inorganic sediment at SG whereas at AS it was approximately 55%. Mean CARs at SG were somewhat greater than at AS, but the difference was not significant due to high variability (SG: 81–210 g C m^?2 year^?1; AS: 115–168 g C m^?2 year^?1). The mean CAR at AS over the past 50 years was 118 (SD = 23) g C m^?2 year^?1. This study demonstrates that a sparsely vegetated, restored salt marsh can quickly begin to accumulate carbon and that historic and restored marshes can have similar CARs despite highly divergent formation processes.