Wiring diagrams of MAPK regulation by MEKK1, 2, and 3.

Mitogen-activated protein kinase (MAPK) pathways are activated by a plethora of stimuli. The literature is filled with papers describing the activation of different MAPKs by almost any stimulus or insult imaginable to cells. In this review, we use signal transduction wiring diagrams to illustrate putative upstream regulators for the MAPK kinase kinases, MEKK1, 2, and 3. Targeted gene disruption of MEKK1, 2, or 3 defined phenotypes for each MEKK associated with loss of specific MAPK regulation. Genetic analysis of MEKK function clearly defines specific components of the wiring diagram that require MEKK1, 2, or 3 for physiological responses. We propose that signal transduction network wiring diagrams are valuable tools for hypothesis building and filtering physiologically relevant phenotypic responses from less connected protein relations in the regulation of MAPK pathways.     

Allozyme Variation Patterns and Multiple Hybridization Origins: Clonal Variation Among Four Sibling Parthenogenetic Caucasian Rock Lizards

Allozyme electrophoresis of four sibling parthenogenetic Caucasian rock lizards Darevskia unisexualis, D.uzzelli, D.sapphirina, and D.bendimahiensis found seven clones and five variable loci. The data supported the hypothesis that D.raddei and D.valentini are the parental species of all four parthenogens. Variation patterns in Darevskia were summarized. Species that originated from a single F₁ typically consisted of one widespread clone with a few rare clones. Species with multiple origins displayed variation only slightly higher than species with a single origin. This is contrary to other genera of parthenogenetic lizards, in which cases massive clonal variations were observed. This revised version was published online in August 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">Physcomitrella patens</Emphasis> arabinogalactan proteins contain abundant terminal 3-<Emphasis Type="Italic">O</Emphasis>-methyl-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosyl residues not found in angiosperms

A biochemical investigation of arabinogalactan proteins (AGPs) in Physcomitrella patens was undertaken with particular emphasis on the glycan chains. Following homogenization and differential centrifugation of moss gametophytes, AGPs were obtained by Yariv phenylglycoside-induced precipitation from the soluble, microsomal membrane, and cell wall fractions. Crossed-electrophoresis indicated that each of these three AGP fractions was a mixture of several AGPs. The soluble AGP fraction was selected for further separation by anion-exchange and gel-permeation chromatography. The latter indicated molecular masses of ∼100 and 224 kDa for the two major soluble AGP subfractions. The AGPs in both of these subfractions contained the abundant (1,3,6)-linked galactopyranosyl residues, terminal arabinofuranosyl residues, and (1,4)-linked glucuronopyranosyl residues that are typical of many angiosperm AGPs. Unexpectedly, however, the moss AGP glycan chains contained about 15 mol% terminal 3-O-methyl-l-rhamnosyl residues, which have not been found in angiosperm AGPs. This unusual and relatively nonpolar sugar, also called l-acofriose, is likely to have considerable effects on the overall polarity of Physcomitrella AGPs. A review of the literature indicates that the capacity to synthesize polymers containing 3-O-methyl-l-rhamnosyl residues is present in a variety of bacteria, algae and lower land plants but became less common through evolution to the extent that this sugar has been found in only a few species of angiosperms where it occurs as a single residue on steroidal glycosides.     

Acorn mast drives long-term dynamics of rodent and songbird populations

Resource pulses can have cascading effects on the dynamics of multiple trophic levels. Acorn mast is a pulsed resource in oak-dominated forests that has significant direct effects on acorn predators and indirect effects on their predators, prey, and pathogens. We evaluated changes in acorn mast, rodent abundance, raptor abundance, and reproductive success of a ground-nesting songbird over a 24-year period (1980–2004) in the southern Appalachian Mountains in an effort to determine the relationships among the four trophic levels. In particular, we examined the following: acorn mast from red oaks (Quercus rubra) and white oaks (Q. alba), abundance of white-footed mice (Peromyscus leucopus) and deer mice (P. maniculatus), population estimates of seven raptor species from three feeding guilds, and nest failure and number of juveniles of dark-eyed juncos (Junco hyemalis). Finally, we recorded seasonal temperature and precipitation to determine the effects of weather on each trophic level. We found that weather patterns had delayed effects of up to 3 years on these trophic interactions. Variation in acorn mast, the keystone resource in this community, was explained by weather conditions as far back as 2 years before the mast event. Acorn mast, in turn, was a strongly positive predictor of rodent abundance the following year, whereas spring and summer temperature and raptor abundance negatively affected rodent abundance. Dark-eyed junco nests were more likely to fail in years in which there were more rodents and raptors. Nest failure rate was a strong predictor of the number of juvenile juncos caught at the end of the summer. Our results improve our understanding of the complex ecological interactions in oak-dominated forests by illustrating the importance of abiotic and biotic factors at different trophic levels. Ethan D. Clotfelter and Amy B. Pedersen contributed equally to the writing of this paper.     

Membrane adhesion in photosynthetic bacterial membranes. Light harvesting complex I (LHI) appears to be the main adhesion factor

We have reconstituted pigment-protein complexes isolated from Rhodopseudomonas palustris photosynthetic membranes into phospholipid liposomes. The various complexes were tested for their ability to promote adhesion of the liposome membrane in the presence and absence of Mg²⁺ ions. Samples containing a reaction center (RC)/light-harvesting I (LHI) complex appeared to stack in a manner resembling control thylakoids in 2 and 5 mM Mg²⁺. We also tested for the effects of Mg²⁺ on detergent extractablity of pigment-protein complexes from intact membranes. Mg²⁺ sharply reduced the amount of LHI solubilized from membranes, while having little effect on the extractability of the light harvesting II complex (LHII) and the RC. Based on these results we suggest that LHI is the principal adhesion factor of R. palustris thylakoids.Abbreviations LHC light harvesting complex - OG octyl glucoside - RC reaction center This paper is dedicated to Professor G. Drews on the occasion of his 60th birthday     

Constitutive expression of human angiostatin in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by high-density cell culture

A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol−0.8% PTM4 to the growing culture for 60 h at 30°C. Dissolved oxygen level was maintained at 25–30% and pH was controlled at 5 by the addition of 7 M NH₄OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16 melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale expression of angiostatin and other heterologous proteins. Supported by the Natural Science Foundation of China (39670013) and “225” Science and Technology Program of Guangzhou Municipal Government of China (99-Z-004-001).     

A proteomics approach to cloning fenestrin from the nuclear exchange junction of tetrahymena

ABSTRACT. We set out to find the " fenestrin " gene, a gene whose protein is associated with numerous cellular apertures, including the nuclear exchange junction in mating Tetrahymena thermophila . First we developed protocols for imaging and isolating intact nuclear exchange junctions from conjugating cells. Proteins from these junctions were purified using SDS-PAGE, subjected to limited proteolysis, and precise molecular weights were determined by mass spectrometry. Using Protein Prospector^® software and the published Tetrahymena Genome Database, genes for 15 of the most abundant proteins found in our extracts were identified. The most promising candidate was cloned by PCR, fused to yellow fluorescent protein (YFP), and placed under the control of an inducible metallothionein promoter. YFP-localization within live Tetrahymena transformants strongly suggested that one of these genes encoded the fenestrin protein, a result that was subsequently confirmed by Western blotting.     

Effect of post-harvest heat treatment on proteome change of peach fruit during ripening

The extracted proteins from the heat-treated peach fruit (dipped in hot water at 48°C for 10min and then stored at room temperature (20°C-25°C) for up to 6 days) were used for proteomic analysis in order to understand the response of post-harvest peach fruit to heat treatment during ripening stage at proteomic level. After two dimensional gels electrophoresis (2-DE) was conducted, more than 600 protein spots were detected. Among them, 35 differently expressed spots (P<0.05) were selected to be excised and analyzed using MALDI-TOF/TOF, and finally 30 protein spots were confidently identified according to NCBI database. The results demonstrated that among the thirty protein spots expressed particularly induced by heat treatment, 43% were related to stress response, 17% to cell structure, 13% to protein fate, 7% to glycolytic pathway, 3% to ripening and senescence and 17% to unclassified. All of them are involved in the regulation of peach fruit development and ripening. All these indicated that the self-defense capability of peach fruit was improved by heat treatment. The study will enable future detailed investigation of gene expression and function linked with peach fruit ripening.     

FireStem2D – A Two-Dimensional Heat Transfer Model for Simulating Tree Stem Injury in Fires

FireStem2D, a software tool for predicting tree stem heating and injury in forest fires, is a physically-based, two-dimensional model of stem thermodynamics that results from heating at the bark surface. It builds on an earlier one-dimensional model (FireStem) and provides improved capabilities for predicting fire-induced mortality and injury before a fire occurs by resolving stem moisture loss, temperatures through the stem, degree of bark charring, and necrotic depth around the stem. We present the results of numerical parameterization and model evaluation experiments for FireStem2D that simulate laboratory stem-heating experiments of 52 tree sections from 25 trees. We also conducted a set of virtual sensitivity analysis experiments to test the effects of unevenness of heating around the stem and with aboveground height using data from two studies: a low-intensity surface fire and a more intense crown fire. The model allows for improved understanding and prediction of the effects of wildland fire on injury and mortality of trees of different species and sizes.