Regeneration niche of three epiphytic species of Gesneriaceae from Chilean rainforests: implications for the evolution of growth habits in Coronanthereae

Ecological and evolutionary studies of the epiphytic growth habit in angiosperms are limited. In this article, we assess the relationship between growth habit and regeneration niche in Coronanthereae (Gesneriaceae) and discuss its implications for the evolution of epiphytism in this lineage. In the temperate rainforest of southern Chile, we quantified the vertical distribution and experimentally examined the regeneration niche of three endemic species of Coronanthereae. One species was a holoepiphyte, which was more frequent in the upper canopy, and two species were secondary hemiepiphytes, which decreased in abundance with tree height. Seed germination of the holoepiphyte was higher on tree bark substrates and under open canopy than on forest soil and in the shade. In contrast, seed germination of both secondary hemiepiphytes did not differ between substrates (bark vs. soil) or light conditions (light vs. shade). Seedling survival percentage of secondary hemiepiphytes was higher on forest soil and under a closed canopy, thus behaving as shade‐tolerant species. In turn, the holoepiphyte behaved as a shade‐intolerant species. The reconstruction of the ancestral growth habits and regeneration niches on the inferred phylogenetic tree of Coronanthereae revealed that the specialized regeneration niche of Sarmienta repens, characterized by requirements of shade intolerance and germination on tree bark, was coupled with the evolution of the holoepiphytic growth habit. We conclude that differentiation in the regeneration niche is a key process in the evolution of epiphytic growth habits in Coronanthereae. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170 , 79–92.     

Concentration-dependent oligomerization and oligomeric arrangement of LptA

Gram-negative bacteria such as Escherichia coli have an inner membrane and an asymmetric outer membrane (OM) that together protect the cytoplasm and act as a highly selective permeability barrier. Lipopolysaccharide (LPS) is the major component of the outer leaflet of the OM and is essential for the survival of nearly all Gram-negative bacteria. Recent advances in understanding the proteins involved in the transport of LPS across the periplasm and into the outer leaflet of the OM include the identification of seven proteins suggested to comprise the LPS transport (Lpt) system. Crystal structures of the periplasmic Lpt protein LptA have recently been reported and show that LptA forms oligomers in either an end-to-end arrangement or a side-by-side dimer. It is not known if LptA oligomers bridge the periplasm to form a large, connected protein complex or if monomeric LptA acts as a periplasmic shuttle to transport LPS across the periplasm. Therefore, the studies presented here focus specifically on the LptA protein and its oligomeric arrangement and concentration dependence in solution using experimental data from several biophysical approaches, including laser light scattering, crosslinking, and double electron electron resonance spectroscopy. The results of these complementary techniques clearly show that LptA readily associates into stable, end-to-end, rod-shaped oligomers even at relatively low local protein concentrations and that LptA forms a continuous array of higher order oligomeric end-to-end structures as a function of increasing protein concentration.     

Relative stabilities of IgG1 and IgG4 Fab domains: Influence of the light-heavy interchain disulfide bond architecture

The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 C(H) 1 and IgG4 C(H) 1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L-H) bond, did not affect thermal stability. Introducing the IgG1 type L-H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L-H interchain DSB with the IgG4 type L-H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 C(H) 1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format.     

Thermal luminescence spectra of polyamides and their Maillard reaction with reducing sugars

Thermal luminescence (TL) spectra of polyamides were measured with a Fourier‐transform chemiluminescence spectrometer to elucidate the emission mechanism. A TL band of ε‐polylysine with a peak at 542 nm observed at 403 K was assigned to the emission due to the interaction of the –CO–NH– group with oxygen molecules by comparison with nylon‐6, polyglycine, and polyalanine. When the sample was kept at 453 K, the intensity of the TL band decreased and the wavelength of the peak shifted to 602 nm, which was assigned to the emission due to the interaction of the NH₂ group on the side chain with oxygen molecules by comparison with monomeric lysine. A weak emission with a peak at 668 nm was assigned to the advanced glycosylation end products (AGEs) yielded by the Maillard reaction with a catalytic amount of water. To understand this reaction and to examine the TL emission of AGEs, we measured TL spectra of mixtures of polylysine and reducing sugars such as glucose, maltose, lactose, and dextrin. The minimum temperature for TL emission, wavelength of the peak and the relative intensities of the TL emission were found to depend on the size of the sugars. Copyright © 2011 John Wiley & Sons, Ltd.     

Correlative light and electron microscopy (CLEM) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited. On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM), affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy. This powerful technique has been implemented to study many aspects of cell biology. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from ≤5 s up to several hours, depending on the nature of the microinjected sample.     

Adipose tissue inflammation and cancer cachexia: possible role of nuclear transcription factors

Cancer cachexia is a multifaceted syndrome whose aetiology is extremely complex and is directly related to poor patient prognosis and survival. Changes in lipid metabolism in cancer cachexia result in marked reduction of total fat mass, increased lipolysis, total oxidation of fatty acids, hyperlipidaemia, hypertriglyceridaemia, and hypercholesterolaemia. These changes are believed to be induced by inflammatory mediators, such as tumour necrosis factor-α (TNF-α) and other factors.Attention has recently been drawn to the current theory that cachexia is a chronic inflammatory state, mainly caused by the host’s reaction to the tumour. Changes in expression of numerous inflammatory mediators, notably in white adipose tissue (WAT), may trigger several changes in WAT homeostasis. The inhibition of adipocyte differentiation by PPARγ is paralleled by the appearance of smaller adipocytes, which may partially account for the inhibitory effect of PPARγ on inflammatory gene expression. Furthermore, inflammatory modulation and/or inhibition seems to be dependent on the IKK/NF-κB pathway, suggesting that a possible interaction between NF-κB and PPARγ is required to modulate WAT inflammation induced by cancer cachexia.In this article, current literature on the possible mechanisms of NF-κB and PPARγ regulation of WAT cells during cancer cachexia are discussed. This review aims to assess the role of a possible interaction between NF-κB and PPARγ in the setting of cancer cachexia as well as its significant role as a potential modulator of chronic inflammation that could be explored therapeutically.     

Flowering dynamics and pollination system of the sedge Rhynchospora ciliata (Vahl) Kükenth (Cyperaceae): does ambophily enhance its reproductive success?

Cyperaceae are characteristically anemophilous, but there are some reports of species re‐adapted to entomophily, such as Rhynchospora ciliata. Our objective was to investigate: (1) the distribution pattern of flowers in inflorescences of Rhynchospora ciliata; (2) the dynamics of its anthesis; and (3) whether R. ciliata is pollinated by bees, by wind or by both. Additionally, we tested the hypotheses: (i) the hypsophylls and/or anthers attract pollinators, and (ii) biotic vectors enhance the reproductive success of R. ciliata. We analysed floral biology, dynamics of anthesis, frequency and behaviour of insects visiting flowers; we also carried out experiments on flower attractiveness, pollination by wind and reproductive success. Rhynchospora ciliata has flowers with anemophilous attributes, including anthers exposed during anthesis; however, the anthers (here considered a mixed trait) together with the white hypsophylls can be considered as attributes that favour entomophily. Both wind and four species of bee were considered as pollen vectors of R. ciliata. Through flower attractiveness tests, we observed that the hypsophylls do not affect the frequency of pollinating bees and that the absence of exposed anthers affects the average number of visits, probably because pollen is the only floral resource. Reproductive tests indicate that R. ciliata is self‐incompatible and that ambophily enhances its reproductive success.     

Costs versus risks: Architectural changes with changing light quantity and quality in saplings of temperate rainforest trees of different shade tolerance

Light requirements and functional strategies of plants to cope with light heterogeneity in the field have a strong influence on community structure and dynamics. Shade intolerant plants often show a shade avoidance strategy involving a phytochrome‐mediated stem elongation in response to changes in red : far red ratio, while shade‐tolerant plants typically harvest light very efficiently. We measured plant size, stem diameter, internode and leaf lengths in randomly chosen saplings of 11 woody species differing in their shade tolerance in both a secondary forest and an old‐growth temperate evergreen rainforest in southern Chile. We also recorded the irradiance spectrum and the diffuse and direct light availabilities at each sampling point. Significant differences were found for the mean light environment of the saplings of each species, which also differed in basal stem diameter, internode length and leaf length, but not in plant height. Both plant slenderness (plant height/stem diameter) and mean internode length increased with increasing light availability, but no relationship was found between any of these two traits and red : far red ratio. The change in plant slenderness with light availability was of lesser magnitude with increasing shade tolerance of the species, while internode change with light availability increased with increasing shade tolerance of the species. Shade tolerators afford higher costs (thicker stems and plants), which render more biomechanically robust plants, and respond more to the light environment in a trait strongly influencing light interception (internode length) than shade intolerant species. By contrast, less shade‐tolerant plants afforded higher risks with a plastic response to escape from the understorey by making thinner plants that were biomechanically weaker and poorer light interceptors. Thus, species differing in their shade tolerances do differ in their plastic responses to light. Our results contribute to explain plant coexistence in heterogeneous light environments by improving our mechanistic understanding of species responses to light.     

长期弱光对苦草幼苗生长发育的影响

用遮光法研究弱光(5%、1%、0.5%、0.1%全光照)对苦草幼苗生长发育的影响,统计了苦草的生物学参数,测定了叶片叶绿素荧光参数。结果表明:1)0.1%组无新株萌发,随着实验时间的延长其余组新株萌发逐渐被抑制。2)随着实验时间增加和光照强度降低,老株叶片形成受到的抑制程度呈增大趋势;前20d时新株叶片形成未被抑制,但随着实验时间延长显著被抑制。3)老株、新株的叶宽均受到显著抑制。4)老株叶片的伸长显著被抑制,且随着光强降低叶片伸长的幅度呈显著降低趋势;前20天时新株叶长被促进,随着实验时间延长叶片伸长显著被抑制。5)随实验天数的增加,老株叶片光化学最大量子产量(Fv/Fm)呈显著降低趋势,第80天时相对电子传递速率(rETR)和非光化学淬灭(NPQ)显著降低。6)新、老植株根、茎、叶的鲜重均显著低于对照,且随着光照强度降低老株的茎重/株重和根重/株重呈增加趋势,而叶重/株重呈显著的降低趋势。第80天时苦草植株仍具有一定的光合能力,地下茎的生物量比例较高,因此,≤1%全光照下苦草植株具有较强的耐受能力。