Conserving the small milkwort, <Emphasis Type="Italic">Comesperma polygaloides</Emphasis>, a vulnerable subshrub in a fragmented landscape

The conservation of remnant grassland vegetation on the Victorian volcanic plain (VVP) is crucial for the persistence of local biodiversity. Recent habitat loss has restricted the grassland to only a small percentage of its former range. Along with grassland habitats, species that occur on the VVP are in decline and many are legally protected. Comesperma polygaloides is a grassland species of the VVP that also occurs outside of the region in woodland habitats. We use 12 neutral microsatellite loci and two chloroplast regions to understand genotypic patterns of C. polygaloides in southeastern Australia. We found separate genetic clusters but they do not follow geographic boundaries. There are fewer alleles (2.96) and effective alleles (2.01) than expected from 12 microsatellite markers compared to other species. Even with the low number of alleles per locus there was a moderate level of genetic diversity detected (I = 0.69; H_o = 0.43; H_e = 0.40). Populations of the VVP could not be differentiated from populations elsewhere using neutral markers or chloroplast analyses. The genetic structure discovered was not consistent with the level of fragmentation observed. There may be several reasons for the observed lack of genetic structure: the species is more common than perceived, plants are long-lived and can reproduce clonally, and the bioregion is relatively young, geologically. Results indicate that restoration projects and long-term viability of C. polygaloides will be improved by composite seed sourcing, alleviating the risk of insufficient genetic diversity posed by an over-emphasis on local provenancing.     

Structural and functional consequences of tyrosine phosphorylation in the LRP1 cytoplasmic domain

The cytoplasmic domain of LRP1 contains two NPXY motifs that have been shown to interact with signaling proteins. In previous work, we showed that Tyr(4507) in the distal NPXY motif is phosphorylated by v-Src, whereas denaturation of the protein was required for phosphorylation of Tyr(4473) in the membraneproximal NPXY motif. Amide H/D exchange studies reveal that the distal NPXY motif is fully solvent-exposed, whereas the proximal one is not. Phosphopeptide mapping combined with in vitro and in vivo kinase experiments show that Tyr(4473) can be phosphorylated, but only if Tyr(4507) is phosphorylated or substituted with glutamic acid. Amide H/D exchange experiments indicate that solvent accessibility increases across the entire LRP1 cytoplasmic region upon phosphorylation at Tyr(4507); in particular the NPXY(4473) motif becomes much more exposed. This differential phosphorylation is functionally relevant: binding of Snx17, which is known to bind at the proximal NPXY motif, is inhibited by phosphorylation at Tyr(4473). Conversely, Shp2 binds most strongly when both of the NPXY motifs in LRP1 are phosphorylated.     

Regulation of gene expression by endogenous ABA in tomato plants

In response to water deficit, endogenous abscisic acid (ABA) accumulates in plants. This ABA serves as a signal for a multitude of processes, including regulation of gene expression. ABA accumulated in response to water deficit signals cellular as well as whole plant responses playing a role in the pattern of gene expression throughout the plant. Although the function of genes regulated by ABA during stress are currently poorly understood, a number of these genes may permit the plant to adapt to environmental stress.     

30 years of free‐air carbon dioxide enrichment (FACE): What have we learned about future crop productivity and its potential for adaptation?

Free‐air CO₂ enrichment (FACE) allows open‐air elevation of [CO₂] without altering the microclimate. Its scale uniquely supports simultaneous study from physiology and yield to soil processes and disease. In 2005 we summarized results of then 28 published observations by meta‐analysis. Subsequent studies have combined FACE with temperature, drought, ozone, and nitrogen treatments. Here, we summarize the results of now almost 250 observations, spanning 14 sites and five continents. Across 186 independent studies of 18 C₃ crops, elevation of [CO₂] by ca. 200 ppm caused a ca. 18% increase in yield under non‐stress conditions. Legumes and root crops showed a greater increase and cereals less. Nitrogen deficiency reduced the average increase to 10%, as did warming by ca. 2°C. Two conclusions of the 2005 analysis were that C₄ crops would not be more productive in elevated [CO₂], except under drought, and that yield responses of C₃ crops were diminished by nitrogen deficiency and wet conditions. Both stand the test of time. Further studies of maize and sorghum showed no yield increase, except in drought, while soybean productivity was negatively affected by early growing season wet conditions. Subsequent study showed reduced levels of nutrients, notably Zn and Fe in most crops, and lower nitrogen and protein in the seeds of non‐leguminous crops. Testing across crop germplasm revealed sufficient variation to maintain nutrient content under rising [CO₂]. A strong correlation of yield response under elevated [CO₂] to genetic yield potential in both rice and soybean was observed. Rice cultivars with the highest yield potential showed a 35% yield increase in elevated [CO₂] compared to an average of 14%. Future FACE experiments have the potential to develop cultivars and management strategies for co‐promoting sustainability and productivity under future elevated [CO₂].     

Variability,function and phylogenetic significance of periostracal microprojections in unionoid bivalves (Mollusca)

Microprojections of unionoid shells are virtually unstudied but could be important characters for resolving questions on the phylogeny and ecology of these bivalves. By investigating 26 unionoid and three species of their closest living relatives, the Trigonioida, using scanning electron microscopy, we identified three types of periostracal microprojections. (1) Microridges were present only in one species from each of the two unionoid families Mycetopodidae (Anodontites trapesialis) and Iridinidae (Chambardia bourguignati) and may represent a synapomorphy for the mycetopodid‐iridinid clade. In A. trapesialis, microridges were additionally equipped with (2)ensp;flag‐like projections (microfringes), possibly a synapomorphic character for the Mycetopodidae. Examination of partially bleached specimens indicated that both microridges and microfringes are predominantly or purely organic. In contrast, previously undescribed (3) spicule‐like spikes represent calcifications within the periostracum. These were found in 20 of the 29 species and four of the six unionoid families. Spikes were particularly large and abundant in umbonal (juvenile) shell regions and species characteristic of fast‐flowing habitats. These structures may thus serve in protecting the periostracum and shell underneath, and/or stabilizing life position by increasing shell friction. Microfringes and microridges, on the other hand, possibly aid in the orientation of the mussel within the sediment.     

Rheological and structural properties of starches from γ-irradiated and stored potatoes

Starch was extracted from irradiated and stored potato tubers and the properties were compared to CIPC (chlorpropham) treated tubers. The granule properties and dynamic viscoelasticity in temperature ramp and frequency sweep modes were studied while heating the samples. Starch structural characteristics were investigated by high performance anion exchange chromatography (HPAEC) and Fourier transform infrared spectroscopy (FTIR). Gamma-irradiation of potato tubers at a dosage of 0.1 kGy induced some degradation of starch molecules, resulting in earlier swelling of starch granules, and greater extents of amylose and total carbohydrate leaching. The early swelling phenomenon was also enhanced with tuber storage time. The retrogradation rate and extent for a concentrated starch gel also increased with tuber storage time whereas γ-irradiation delayed the gel retrogradation. Sprout inhibiting methods could be selected based on the specific processing and texture requirements of the end products.     

Metastasis-associated protein 3 (MTA3) regulates G2/M progression in proliferating mouse granulosa cells

Metastasis-associated protein 3 (MTA3) is a constituent of the Mi-2/nucleosome remodeling and deacetylase (NuRD) protein complex that regulates gene expression by altering chromatin structure and can facilitate cohesin loading onto DNA. The biological function of MTA3 within the NuRD complex is unknown. Herein, we show that MTA3 was expressed highly in granulosa cell nuclei of all ovarian follicle stages and at lower levels in corpora lutea. We tested the hypothesis that MTA3-NuRD complex function is required for granulosa cell proliferation. In the ovary, MTA3 interacted with NuRD proteins CHD4 and HDAC1 and the core cohesin complex protein RAD21. In cultured mouse primary granulosa cells, depletion of endogenous MTA3 using RNA interference slowed cell proliferation; this effect was rescued by coexpression of exogenous MTA3. Slowing of cell proliferation correlated with a significant decrease in cyclin B1 and cyclin B2 expression. Granulosa cell populations lacking MTA3 contained a significantly higher percentage of cells in G2/M phase and a lower percentage in S phase compared with control cells. Furthermore, MTA3 depletion slowed entry into M phase as indicated by reduced phosphorylation of histone H3 at serine 10. These findings provide the first evidence to date that MTA3 interacts with NuRD and cohesin complex proteins in the ovary in vivo and regulates G2/M progression in proliferating granulosa cells.     

Differential expression, function and response to inflammatory stimuli of 11β-hydroxysteroid dehydrogenase type 1 in human fibroblasts: a mechanism for tissue-specific regulation of inflammation

Stromal cells such as fibroblasts play an important role in defining tissue-specific responses during the resolution of inflammation. We hypothesized that this involves tissue-specific regulation of glucocorticoids, mediated via differential regulation of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Expression, activity and function of 11β-HSD1 was assessed in matched fibroblasts derived from various tissues (synovium, bone marrow and skin) obtained from patients with rheumatoid arthritis or osteoarthritis. 11β-HSD1 was expressed in fibroblasts from all tissues but mRNA levels and enzyme activity were higher in synovial fibroblasts (2-fold and 13-fold higher mRNA levels in dermal and synovial fibroblasts, respectively, relative to bone marrow). Expression and activity of the enzyme increased in all fibroblasts following treatment with tumour necrosis factor-α or IL-1β (bone marrow: 8-fold and 37-fold, respectively, compared to vehicle; dermal fibroblasts: 4-fold and 14-fold; synovial fibroblasts: 7-fold and 31-fold; all P < 0.01 compared with vehicle). Treatment with IL-4 or interferon-γ was without effect, and there was no difference in 11β-HSD1 expression between fibroblasts (from any site) obtained from patients with rheumatoid arthritis or osteoarthritis. In the presence of 100 nmol/l cortisone, IL-6 production – a characteristic feature of synovial derived fibroblasts – was significantly reduced in synovial but not dermal or bone marrow fibroblasts. This was prevented by co-treatment with an 11β-HSD inhibitor, emphasizing the potential for autocrine activation of glucocorticoids in synovial fibroblasts. These data indicate that differences in fibroblast-derived glucocorticoid production (via the enzyme 11β-HSD1) between cells from distinct anatomical locations may play a key role in the predeliction of certain tissues to develop persistent inflammation.     

3' end cDNA amplification using classic RACE

Having knowledge of the entire 3' sequence of a cDNA is often important because the non-coding terminal region can contain signals that regulate the stability or subcellular localization of the mRNA. Also, some messages use alternative genomic sites for cleavage and polyadenylation that can alter the above properties, or change the encoded protein. Full-length cDNAs can be obtained from complex mixtures of cellular mRNA using rapid amplification of cDNA ends (RACE) PCR as long as part of the mRNA sequence is known; adding non-specific tags to the ends of the cDNA allows the regions between the known parts of the sequence and the ends to be amplified. In 3' RACE, the poly(A) tail functions as a non-specific tag at the 3' end of the mRNA. cDNA ends can be obtained in 1-3 days using this protocol.