Expression, Purification, and Inhibitory Properties of Human Proteinase Inhibitor 8

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861]. In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM. The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM. A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.     

Resource distribution influences mating system in the bobuck (<Emphasis Type="Italic">Trichosurus cunninghami</Emphasis>: Marsupialia)

Mammalian mating systems are thought to be shaped by the spatial distribution and abundance of key resources, which in turn influence the spacing behaviour of individuals. In particular, female home range size is predicted to reflect the availability of key resources. We documented the availability and distribution of food and shelter resources for two neighbouring populations of bobucks, or mountain brushtail possums, Trichosurus cunninghami, that were characterised by different mating systems: our “forest population” was socially monogamous, whereas the “roadside population” was polygynous. Both silver wattle, Acacia dealbata, the main food resource for bobucks, and den-trees, which provided shelter, occurred at significantly higher density at the roadside site. The pattern of distribution of these two resources also differed between the sites. Both food and den-trees were scattered evenly throughout the roadside habitat. In contrast, den-trees were located predominantly at one end of the forest site, while silver wattle trees were located at the other. There was no significant difference in the amount of silver wattle, or in the number of den-trees, located within the home ranges of individual females at the two sites. However, forest females had home ranges, on average, almost three times the size of those of roadside females. At the roadside site, the size of female home ranges varied inversely with the density of silver wattle, indicating that these females ranged over as large an area as necessary to gain access to sufficient silver wattle trees. There was no such relationship among forest females. These populations provide a clear example of resource distribution determining female home range size. This influenced the number of female home ranges a male’s home range overlapped with, which in turn determined the mating system. Such clear links between resource availability and mating system have not previously been established in a marsupial.     

Impacts of the invasive fungus Austropuccinia psidii (myrtle rust) on three Australian Myrtaceae species of coastal swamp woodland

Exotic fungal pathogens can substantially affect individuals and populations of susceptible native plant species, potentially resulting in changes in community structure and composition. Austropuccinia psidii (myrtle rust) is a pathogenic fungus native to South America that affects species in the plant family Myrtaceae. The pathogen was introduced accidentally to Australia and first detected in NSW in April 2010. Ecological impacts have been poorly studied in the native range of A. psidii and even less in its Australian introduced range. In order to assess the potential impact of A. psidii on coastal swamp woodland, two glasshouse experiments were conducted using three co‐occurring species: Melaleuca quinquenervia, Leptospermum laevigatum and Baeckea linifolia. Plants of each species were grown individually (Experiment 1) and in mixed species assemblages (Experiment 2), with half inoculated with A. psidii and the other half remaining as controls. Infection level was assessed and impact on seedling survival and growth recorded. In both experiments L. laevigatum and M. quinquenervia seedlings were heavily infected and showed high degrees of susceptibility with negative effects on growth (height, biomass and number of leaves). In contrast, no B. linifolia seedling presented visible symptoms of disease, although seedlings showed reduced growth. Melaleuca quinquenervia seedlings had greater infection levels and suffered greater growth reductions than L. laevigatum in both experiments. However, there was no significant difference in the relative abundance of the three species in the mixed‐species experiment. This study provides a better understanding of the potential impacts of A. psidii in this vegetation community and has significant implications for the conservation and management of Australian Myrtaceae‐dominated plant communities generally.     

Development of a transformation system in the swainsonine producing, slow growing endophytic fungus, Undifilum oxytropis

Undifilum oxytropis (Phylum: Ascomycota; Family: Pleosporaceae) is a slow growing endophytic fungus that produces a toxic alkaloid, swainsonine. This endophyte resides in locoweeds, which are perennial flowering legumes. Consumption of this fungus by grazing animals induces a neurological disorder called locoism. The alkaloid swainsonine, an α-mannosidase inhibitor, is responsible for the field toxicity related to locoism. Little is known about the biosynthetic pathway of swainsonine in endophytic fungi. Genetic manipulation of endophytic fungi is important to better understand biochemical pathways involved in alkaloid synthesis, but no transformation system has been available for studying such enzymes in Undifilum. In this study we report the development of protoplast and transformation system for U. oxytropis. Fungal mycelia required for generating protoplasts were grown in liquid culture, then harvested and processed with various enzymes. Protoplasts were transformed with a fungal specific vector driving the expression of Enhanced Green Florescent Protein (EGFP). The quality of transformed protoplasts and transformation efficiency were monitored during the process. In all cases, resistance to antibiotic hygromycin B was maintained. Such manipulation will open avenues for future research to decipher fungal metabolic pathways.     

Multiple forms of Go alpha mRNA: analysis of the 3'-untranslated regions

Go, a guanine nucleotide binding protein found predominantly in neural tissues, interacts in vitro with rhodopsin, muscarinic, and other receptors and has been implicated in the regulation of ion channels. Despite the virtual identity of reported cDNA sequences for the alpha subunit of Go (Go alpha), multiple molecular weight forms of mRNA have been identified in tissues from all species examined. To investigate the molecular basis for the size heterogeneity of Go alpha mRNAs, four cDNA clones were isolated from the same retinal lambda gt10 cDNA library that was used earlier to isolate lambda GO9, a clone encompassing the complete coding region of Go alpha. These clones were identified as Go alpha clones based on nucleotide sequence identity with lambda GO9 in the coding region; they diverge, however, from lambda GO9 in the 3'-untranslated region 28 nucleotides past the stop codon. An oligonucleotide probe complementary to a portion of the 3'-untranslated region of lambda GO9 that differs from the newly isolated clones hybridized with 3.0- and 4.0-kb mRNAs present in bovine brain and retina whereas a similar probe for the unique region of the new clones hybridized with a 4.0-kb mRNA in both tissues and with a 2.0-kb mRNA found predominantly in retina. A similar hybridization pattern was observed when brain poly(A+) RNA from other species was hybridized with the different 3'-untranslated region probes. It appears that differences in the 3'-untranslated regions could, in part, be the basis for the observed heterogeneity in Go alpha mRNAs.     

Similar molecules spatially correlate with lipofuscin and N-retinylidene-N-retinylethanolamine in the mouse but not in the human retinal pigment epithelium

The accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD) in humans. The exact composition of lipofuscin is not known but its best characterized component is N-retinylidene-N-retinylethanolamine (A2E), a byproduct of the retinoid visual cycle. Utilizing our recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI–IMS)-based technique to determine the spatial distribution of A2E, this study compares the relationships of lipofuscin fluorescence and A2E in the murine and human RPE on representative normal tissue. To identify molecules with similar spatial patterns, the images of A2E and lipofuscin were correlated with all the individual images in the MALDI–IMS dataset. In the murine RPE, there was a remarkable correlation between A2E and lipofuscin. In the human RPE, however, minimal correlation was detected. These results were reflected in the marked distinctions between the molecules that spatially correlated with the images of lipofuscin and A2E in the human RPE. While the distribution of murine lipofuscin showed highest similarities with some of the known A2E-adducts, the composition of human lipofuscin was significantly different. These results indicate that A2E metabolism may be altered in the human compared to the murine RPE.     

Evolutionary temperature compensation of carbon fixation in marine phytoplankton

The efficiency of carbon sequestration by the biological pump could decline in the coming decades because respiration tends to increase more with temperature than photosynthesis. Despite these differences in the short‐term temperature sensitivities of photosynthesis and respiration, it remains unknown whether the long‐term impacts of global warming on metabolic rates of phytoplankton can be modulated by evolutionary adaptation. We found that respiration was consistently more temperature dependent than photosynthesis across 18 diverse marine phytoplankton, resulting in universal declines in the rate of carbon fixation with short‐term increases in temperature. Long‐term experimental evolution under high temperature reversed the short‐term stimulation of metabolic rates, resulting in increased rates of carbon fixation. Our findings suggest that thermal adaptation may therefore have an ameliorating impact on the efficiency of phytoplankton as primary mediators of the biological carbon pump.     

SAUR Inhibition of PP2C-D Phosphatases Activates Plasma Membrane H+-ATPases to Promote Cell Expansion in Arabidopsis

The plant hormone auxin promotes cell expansion. Forty years ago, the acid growth theory was proposed, whereby auxin promotes proton efflux to acidify the apoplast and facilitate the uptake of solutes and water to drive plant cell expansion. However, the underlying molecular and genetic bases of this process remain unclear. We have previously shown that the SAUR19-24 subfamily of auxin-induced SMALL AUXIN UP-RNA (SAUR) genes promotes cell expansion. Here, we demonstrate that SAUR proteins provide a mechanistic link between auxin and plasma membrane H⁺-ATPases (PM H⁺-ATPases) in Arabidopsis thaliana. Plants overexpressing stabilized SAUR19 fusion proteins exhibit increased PM H⁺-ATPase activity, and the increased growth phenotypes conferred by SAUR19 overexpression are dependent upon normal PM H⁺-ATPase function. We find that SAUR19 stimulates PM H⁺-ATPase activity by promoting phosphorylation of the C-terminal autoinhibitory domain. Additionally, we identify a regulatory mechanism by which SAUR19 modulates PM H⁺-ATPase phosphorylation status. SAUR19 as well as additional SAUR proteins interact with the PP2C-D subfamily of type 2C protein phosphatases. We demonstrate that these phosphatases are inhibited upon SAUR binding, act antagonistically to SAURs in vivo, can physically interact with PM H⁺-ATPases, and negatively regulate PM H⁺-ATPase activity. Our findings provide a molecular framework for elucidating auxin-mediated control of plant cell expansion.     

Non-random Escape Pathways from a Broadly Neutralizing Human Monoclonal Antibody Map to a Highly Conserved Region on the Hepatitis C Virus E2 Glycoprotein Encompassing Amino Acids 412–423

A challenge for hepatitis C virus (HCV) vaccine development is to define epitopes that are able to elicit protective antibodies against this highly diverse virus. The E2 glycoprotein region located at residues 412–423 is conserved and antibodies to 412–423 have broadly neutralizing activities. However, an adaptive mutation, N417S, is associated with a glycan shift in a variant that cannot be neutralized by a murine but by human monoclonal antibodies (HMAbs) against 412–423. To determine whether HCV escapes from these antibodies, we analyzed variants that emerged when cell culture infectious HCV virions (HCVcc) were passaged under increasing concentrations of a specific HMAb, HC33.1. Multiple nonrandom escape pathways were identified. Two pathways occurred in the context of an N-glycan shift mutation at N417T. At low antibody concentrations, substitutions of two residues outside of the epitope, N434D and K610R, led to variants having improved in vitro viral fitness and reduced sensitivity to HC33.1 binding and neutralization. At moderate concentrations, a S419N mutation occurred within 412–423 in escape variants that have greatly reduced sensitivity to HC33.1 but compromised viral fitness. Importantly, the variants generated from these pathways differed in their stability. N434D and K610R-associated variants were stable and became dominant as the virions were passaged. The S419N mutation reverted back to N419S when immune pressure was reduced by removing HC33.1. At high antibody concentrations, a mutation at L413I was observed in variants that were resistant to HC33.1 neutralization. Collectively, the combination of multiple escape pathways enabled the virus to persist under a wide range of antibody concentrations. Moreover, these findings pose a different challenge to vaccine development beyond the identification of highly conserved epitopes. It will be necessary for a vaccine to induce high potency antibodies that prevent the formation of escape variants, which can co-exist with lower potency or levels of neutralizing activities.