Catalytic implications of the higher plant ADP-glucose pyrophosphorylase large subunit

ADP-glucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis, is composed of a pair of catalytic small subunits (SSs) and a pair of catalytically disabled large subunits (LSs). The N-terminal region of the LS has been known to be essential for the allosteric regulatory properties of the heterotetrameric enzyme. To gain further insight on the role of this region and the LS itself in enzyme function, the six proline residues found in the N-terminal region of the potato tuber AGPase were subjected to scanning mutagenesis. The wildtype and various mutant heterotetramers were expressed using our newly developed host-vector system, purified, and their kinetic parameters assessed. While P(17)L, P(26)L, and P(55)L mutations only moderately affected the kinetic properties, P(52)L and P(66)L gave rise to significant and contrasting changes in allosteric properties: P(66)L enzyme displayed up-regulatory properties toward 3-PGA while the P(52)L enzyme had down-regulatory properties. Unlike the other mutants, however, various mutations at P(44) led to only moderate changes in regulatory properties, but had severely impaired catalytic rates, apparent substrate affinities, and responsiveness to metabolic effectors, indicating Pro-44 or the LS is essential for optimal catalysis and activation of the AGPase heterotetramer. The catalytic importance of the LS is further supported by photoaffinity labeling studies, which revealed that the LS binds ATP at the same efficiency as the SS. These results indicate that the LS, although considered having no catalytic activity, may mimic many of the catalytic events undertaken by the SS and, thereby, influences net catalysis of the heterotetrameric enzyme.     

The promoter of the wheat puroindoline-a gene (<Emphasis Type="Italic">PinA</Emphasis>) exhibits a more complex pattern of activity than that of the <Emphasis Type="Italic">PinB</Emphasis> gene and is induced by wounding and pathogen attack in rice

Puroindolines form the molecular basis of wheat grain hardness. However, little is known about puroindoline gene regulation. We previously reported that the Triticum aestivum puroindoline-b gene (PinB) promoter directs β-glucuronidase gene (uidA) seed-specific expression in transgenic rice. In this study, we isolated a puroindoline-a gene (PinA), analyzed PinA promoter activity by 5′ deletions and compared PinA and PinB promoters in transgenic rice. Seeds of PinA-1214 and PinB-1063 transgenic plants strongly expressed uidA in endosperm, in the aleurone layer and in epidermis cells in a developmentally regulated manner. The GUS activity was also observed in PinA-1214 embryos. Whereas the PinB promoter is seed specific, the PinA promoter also directed, but to a lower level, uidA expression in roots of seedlings and in the vascular tissues of palea and pollen grains of dehiscent anthers during flower development. In addition, the PinA promoter was induced by wounding and by Magnaporthe grisea. By deletion analysis, we showed that the “390-bp” PinA promoter drives the same expression pattern as the “1214-bp” promoter. Moreover, the “214-bp” PinA promoter drives uidA expression solely in pollen grains of dehiscent anthers. The presence of putative cis-regulatory elements that may be related to PinA expression is discussed from an evolutionary point of view. By electrophoretic mobility shift assay, we showed that putative cis-elements (WUN-box, TCA motifs and as-1-like binding sites) whose presence in the PinA promoter may be related to wounding and/or the pathogen response form complexes with nuclear extracts isolated from wounded wheat leaves.     

The <Emphasis Type="Italic">Alphavirus</Emphasis> 6K Protein Activates Endogenous Ionic Conductances when Expressed in <Emphasis Type="Italic">Xenopus</Emphasis> Oocytes

The Alphavirus Sindbis 6K protein is involved in several functions. It contributes to the processing and membrane insertion of E1 and PE2 viral envelope glycoproteins and to virus budding. It also permeabilizes Escherichia coli and mammalian cells. These viroporin-like properties have been proposed to help virus budding by modifying membrane permeabilities. We expressed Sindbis virus 6K cRNA in Xenopus oocytes to further characterize the effect of 6K on membrane conductances and permeabilization. Although no intrinsic channel properties were seen, cell shrinkage was observed within 24 h. Voltage-clamp experiments showed that 6K upregulated endogenous currents: a hyperpolarization-activated inward current (I _in) and a calcium-dependent chloride current (I _Cl). 6K was located at both the plasma and the endoplasmic reticulum membranes. The plasma membrane current upregulation likely results from disruption of the calcium homeostasis of the cell at the endoplasmic reticulum level. Indeed, 6K cRNA expression induced reticular calcium store depletion and capacitative calcium entry activation. By experimental modifications of the incubation medium, we showed that downstream of these events cell shrinkage resulted from a 6K -induced KCl efflux (I _Cl upregulation leads to chloride efflux, which itself electrically drives potassium efflux), which was responsible for an osmotic water efflux. Our data confirm that 6K specifically triggers a sequential cascade of events that leads to cytoplasmic calcium elevation and cell permeabilization, which likely play a role in the Sindbis virus life cycle.     

Modeling regulation mechanisms in the immune system

We develop a mathematical framework for modeling regulatory mechanisms in the immune system. The model describes dynamics of key components of the immune network within two compartments: lymph node and tissue. We demonstrate using numerical simulations that our system can eliminate virus-infected cells, which are characterized by a tendency to increase without control (in absence of an immune response), while tolerating normal cells, which are characterized by a tendency to approach a stable equilibrium population. We experiment with different combinations of T cell reactivities that lead to effective systems and conclude that slightly self-reactive T cells can exist within the immune system and are controlled by regulatory cells. We observe that CD8+ T cell dynamics has two phases. In the first phase, CD8+ cells remain sequestered within the lymph node during a period of proliferation. In the second phase, the CD8+ population emigrates to the tissue and destroys its target population. We also conclude that a self-tolerant system must have a mechanism of central tolerance to ensure that self-reactive T cells are not too self-reactive. Furthermore, the effectiveness of a system depends on a balance between the reactivities of the effector and regulatory T cell populations, where the effectors are slightly more reactive than the regulatory cells.     

Modeling the Western Arctic Caribou Herd During a Positive Growth Phase: Potential Effects of Wolves and Radiocollars

ABSTRACT Population modeling exercises can lead to both expected and unexpected results useful for wildlife research and management, even though inferences must often be qualitative, given underlying assumptions. Our main objective was to use empirical data on wolf (Canis lupus) kill rates and growth of the Western Arctic caribou (Rangifer tarandus) herd (WAH) of Alaska, USA, to assess the potential for predator regulation. We used available data and published literature to construct a deterministic density-dependent population model fitted to trends of the WAH from 1976 through 2003. By increasing wolf densities in the baseline model, we failed to reject the hypothesis that wolves at a density of 6.5 wolves per 1,000 km² could regulate a caribou herd at a density of 0.4 caribou per km². In addition, our model may be conservative by underestimating the regulatory potential of wolves. We suggest that this relatively simple predator-preysystem shows signs of a predation—food 2-state model. Elasticities from matrix models may be deceiving. Although herd growth is most sensitive to changes in adult female survival, survival of younger cohorts may be more easily influenced by natural conditions or management action. Management of the WAH near maximum sustained yield may not be attainable if desired, but modeling exercises such as this elucidate options. In conducting this research, we also discovered by Monte Carlo simulation that survival and productivity data from radiocollared females and calves were negatively biased and failed to predict herd growth. Thus, researchers should consider potential effects of neck collars on vital rates of female tundra caribou and concomitant offspring when using sample data to model population dynamics or test hypotheses.