Molecular physiology of glutamine and glutamate biosynthesis in developing seedlings of conifers

Nitrogen is a limiting factor in tree growth and development. The incorporation of ammonium ions in carbon skeletons is catalyzed by the sequential action of the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT). Most studies on nitrogen‐assimilating enzymes have been reported for annual crop plants. Knowledge of these enzymes in woody plants is much more limited, particularly at the molecular level. Here, we review current available information on glutamine/glutamate biosynthesis and chloroplast development in conifers.     

Global expression profiling applied to plant development

Plant development is controlled by both endogenous genetic programs and responses to exogenous signals. Microarray experiments are being used to identify the genes involved in these developmental processes. Most of the analyses conducted to date have been conducted on whole organs. Although these studies have provided valuable information, they are limited by the composite nature of plant organs that consist of multiple cell types. Technical advances that have made it possible to study global patterns of gene expression in individual cell types promise to increase greatly the information revealed by microarray experiments.     

Characterisation of bacterial polysaccharides: steps towards single-molecular studies

Techniques used in studies of polysaccharides, including chemical composition, linkage pattern, and higher order structures are in constant development. They provide information necessary for understanding of the polysaccharide properties and functions. Here, recent advancements in studies of the polysaccharides at the single-molecule level are highlighted. Over the last few years, single-molecule techniques such as force spectroscopy have improved in sensitivity and can today be used to detect forces in the pN range. In addition, these techniques can be used to investigate properties of single molecules close to physiological conditions. The challenges in the interpretation of the observations are aided by control experiments using well-characterised polysaccharides and by data provided by complementary methods. This field is expected to have increasing impact on the further advancement of the molecular understanding of the role of polysaccharides in various biological processes such as recognition and cell adhesion.     

Forecasting sites of future coastal marsh loss using topographical relationships and logistic regression

The last two assessments of the Intergovernmental Panel on Climate Change (IPCC) predict that rates of sea level rise will begin to accelerate by c. 2030–2040 CE. Considering that many marsh systems are already under threat from existing sea level trends, a dramatic upswing in rate only a few decades away poses critical questions about what the future may hold for these marshes (and those yet to manifest sea level impacts) requires coast-wide assessments. Extrapolations of detailed historical trends traditionally have provided the least equivocal way of providing such information, but the necessary data required for this approach are often lacking. In this paper, we describe how logistic regression analysis applied to spatial data on marsh loss and degradation—in this case derived from a validated Landsat-based marsh condition model—and its relation to such readily determined parameters as distance from shorelines or tidal creeks can be used to predict where future marsh losses may occur, even in those systems not presently affected. As such, it affords more targeted information for planning than can be had from general submergence versus accretion/elevation change models (e.g., SLAMM) that are limited by the paucity of vertical accretion data. The results also reinforce the concept that marsh adjustment to sea level rise can be broadly deduced at the landscape level, which in some respects is independent of marsh type and is responsive to tidal frame and coastal profile.     

Giardia, Cryptosporidium and the spectre of zoonosis: the Italian experience from land to sea

In the last decade, a major concern for the scientific community has been whether infected animals can serve as reservoirs of Giardia and Cryptosporidium infection for humans. Worldwide, prevalence studies and molecular tools have provided insights into the taxonomy and epidemiology of these protozoa in order to better understand such a relation. This paper presents data on the prevalence and molecular genotyping studies from several sample types from land to sea (humans, companion animals, sheep, cattle, goats, wastewaters, surface water, and shellfish) available in Italy. The contribution of Italian researchers to the international debate on the veterinary significance of these infections and their impact on public health is highlighted and the main objectives to be pursued in the future depicted.     

Mechanisms of vesicle formation: insights from the COP system.

The major cytosolic and membrane proteins that represent machinery of coat protein (COP)-coated transport vesicles within the secretory pathway are characterized to date. This has allowed investigation of the molecular mechanisms that underlie the formation of these vesicles. In vitro binding studies and reconstitution experiments have provided insights at the molecular level into the biogenesis of COPII- and COPI-coated vesicles.     

Nest sites as limiting resources for cavity‐nesting birds in mature forest ecosystems: a review of the evidence

ABSTRACT Assumptions that populations of cavity‐nesting birds are limited by access to nest sites have largely been based on anecdotal reports or correlative data. Nest‐box‐addition experiments or tree‐cavity‐blocking experiments are potentially rigorous ways to investigate how densities of breeding birds are affected by access to nest cavities. Experimental evidence indicates that natural tree holes are limited in human‐altered landscapes, but the possibility that cavity nests are limited in old growth (unmanaged) forests is less clear. I reviewed 31 nest‐cavity‐removal or addition experiments conducted with 20 species of cavity‐nesting birds in mature forests. Of these 31 experiments conducted with a variety of different species of birds, only 19% reported statistically significant changes in breeding densities. However, none of these studies included data about the reproductive history of individuals colonizing the boxes (i.e., whether birds using the boxes would have otherwise been floaters or that birds excluded from blocked cavities on the plots did not simply move elsewhere), so they provided no strong evidence that the number of breeding pairs was limited by availability of nest sites at the population scale. Although some studies indicate that nest sites are limited at local (plot) scales in old growth forests, there is still little empirical evidence for nest‐site limitation at the population‐ and landscape‐level in mature, unmanaged forests. I review the challenges in designing and interpreting box‐addition experiments and highlight the main gaps in knowledge that should be targeted in the future.     

Distribution of Alu and L1 repeats in human YAC recombinants

Evidence is accumulating that the two major families of interspersed repeated human DNA sequences, Alu and L1, are not randomly distributed. However, only limited information is available on their relative long-range distribution. We have analyzed a set of randomly selected, human Chromosome (Chr) 11-specific YAC recombinants constituting a total length of about 2 Mbp for the local and global distribution of Alu and L1 repeats: the data show a strong asymmetry in the distribution of these two repeat classes and give weight, at the long-range molecular level, to previous studies indicating their partition in the human genome; they also suggest a strong tendency for L1 repeats to cluster, with a higher proportion of full-length elements than expected.     

Collagen formation by fibroblasts of the chick embryo dermis

This investigation has sought to determine the relation between collagen fiber and fibroblast during fibrogenesis. Toward this end the surfaces of chick fibroblasts grown under in vitro conditions have been examined with the electron microscope after fixation in OsO(4). Supplementary information has been obtained from thin sections of fibroblasts fixed in situ during phases of fiber production. The evidence provided by these studies and by various conditions of the experiments indicates that the unit fibrils of collagen form in close association with the cell surface. They were never observed within the cell. When these unit fibrils form in bundles it appears as though templates of some nature, possibly coinciding with stress fibers within the cell cortex, influence the polymerization of the fibrils out of material available at the cell surface. From here the fibrils and bundles of them are shed into the intercellular spaces and there grow to limited diameters by accretion of materials from the general milieu.     

Towards reconstitution of membrane fusion mediated by SNAREs and other synaptic proteins

Abstract

Proteoliposomes have been widely used for in vitro studies of membrane fusion mediated by synaptic proteins. Initially, such studies were made with large unsynchronized ensembles of vesicles. Such ensemble assays limited the insights into the SNARE-mediated fusion mechanism that could be obtained from them. Single particle microscopy experiments can alleviate many of these limitations but they pose significant technical challenges. Here we summarize various approaches that have enabled studies of fusion mediated by SNAREs and other synaptic proteins at a single-particle level. Currently available methods are described and their advantages and limitations are discussed.     

Structural and Energetic Effects of A2A Adenosine Receptor Mutations on Agonist and Antagonist Binding

To predict structural and energetic effects of point mutations on ligand binding is of considerable interest in biochemistry and pharmacology. This is not only useful in connection with site-directed mutagenesis experiments, but could also allow interpretation and prediction of individual responses to drug treatment. For G-protein coupled receptors systematic mutagenesis has provided the major part of functional data as structural information until recently has been very limited. For the pharmacologically important A_2A adenosine receptor, extensive site-directed mutagenesis data on agonist and antagonist binding is available and crystal structures of both types of complexes have been determined. Here, we employ a computational strategy, based on molecular dynamics free energy simulations, to rationalize and interpret available alanine-scanning experiments for both agonist and antagonist binding to this receptor. These computer simulations show excellent agreement with the experimental data and, most importantly, reveal the molecular details behind the observed effects which are often not immediately evident from the crystal structures. The work further provides a distinct validation of the computational strategy used to assess effects of point-mutations on ligand binding. It also highlights the importance of considering not only protein-ligand interactions but also those mediated by solvent water molecules, in ligand design projects.     

Collagen Formation by Fibroblasts of the Chick Embryo Dermis

This investigation has sought to determine the relation between collagen fiber and fibroblast during fibrogenesis. Toward this end the surfaces of chick fibroblasts grown under in vitro conditions have been examined with the electron microscope after fixation in OsO₄. Supplementary information has been obtained from thin sections of fibroblasts fixed in situ during phases of fiber production. The evidence provided by these studies and by various conditions of the experiments indicates that the unit fibrils of collagen form in close association with the cell surface. They were never observed within the cell. When these unit fibrils form in bundles it appears as though templates of some nature, possibly coinciding with stress fibers within the cell cortex, influence the polymerization of the fibrils out of material available at the cell surface. From here the fibrils and bundles of them are shed into the intercellular spaces and there grow to limited diameters by accretion of materials from the general milieu.     

Isolation and characterization of proteolytic fragments of the sea urchin sperm receptor that retain species specificity

The sea urchin sperm receptor isolated from the eggs of Strongylocentrotus purpuratus is a high molecular weight proteoglycan-like molecule. Previous studies in our laboratory suggested that the sperm receptor has two functional components, glycosaminoglycan chains that are responsible for sperm binding and polypeptide chains that control species specificity in the binding process. We have investigated this idea further by generating fragments of the receptor by limited proteolytic digestion of the egg cell surface. The results of experiments with these receptor preparations support the hypothesis that the species specificity of inhibition of fertilization observed in a competitive bioassay is conferred by the polypeptide portion of the receptor molecule. Studies with various receptor preparations reveal that the presence of at least 30% of the polypeptide by weight is required to inhibit fertilization species specifically. Receptor preparations containing less than 10% protein lack species specificity and inhibit fertilization in both S. purpuratus and Arbacia punctulata.     

Heritable variation in heat shock gene expression: a potential mechanism for adaptation to thermal stress in embryos of sea turtles

The capacity of species to respond adaptively to warming temperatures will be key to their survival in the Anthropocene. The embryos of egg-laying species such as sea turtles have limited behavioural means for avoiding high nest temperatures, and responses at the physiological level may be critical to coping with predicted global temperature increases. Using the loggerhead sea turtle (Caretta caretta) as a model, we used quantitative PCR to characterise variation in the expression response of heat-shock genes (hsp60, hsp70 and hsp90; molecular chaperones involved in cellular stress response) to an acute non-lethal heat shock. We show significant variation in gene expression at the clutch and population levels for some, but not all hsp genes. Using pedigree information, we estimated heritabilities of the expression response of hsp genes to heat shock and demonstrated both maternal and additive genetic effects. This is the first evidence that the heat-shock response is heritable in sea turtles and operates at the embryonic stage in any reptile. The presence of heritable variation in the expression of key thermotolerance genes is necessary for sea turtles to adapt at a molecular level to warming incubation environments.     

Molecular signatures of lineage‐specific adaptive evolution in a unique sea basin: the example of an anadromous goby Leucopsarion petersii

Climate changes on various time scales often shape genetic novelty and adaptive variation in many biotas. We explored molecular signatures of directional selection in populations of the ice goby Leucopsarion petersii inhabiting a unique sea basin, the Sea of Japan, where a wide variety of environments existed in the Pleistocene in relation to shifts in sea level by repeated glaciations. This species consisted of two historically allopatric lineages, the Japan Sea (JS) and Pacific Ocean (PO) lineages, and these have lived under contrasting marine environments that are expected to have imposed different selection regimes caused by past climatic and current oceanographic factors. We applied a limited genome‐scan approach using seven candidate genes for phenotypic differences between two lineages in combination with 100 anonymous microsatellite loci. Neuropeptide Y (NPY) gene, which is an important regulator of food intake and potent orexigenic agent, and three anonymous microsatellites were identified as robust outliers, that is, candidate loci potentially under directional selection, by multiple divergence‐ and diversity‐based outlier tests in comparisons focused on multiple populations of the JS vs. PO lineages. For these outlier loci, populations of the JS lineage had putative signals of selective sweeps. Additionally, real‐time quantitative PCR analysis using fish reared in a common environment showed a higher expression level for NPY gene in the JS lineage. Thus, this study succeeded in identifying candidate genomic regions under selection across populations of the JS lineage and provided evidence for lineage‐specific adaptive evolution in this unique sea basin.     

Proteomics: addressing the challenges of multiple myeloma

Multiple myeloma (MM) is a malignancy of terminally differentiated B-lymphocytes that accounts for ~13% of all hematologic cancers. Despite a wealth of knowledge describing the molecular biology of MM as well as significant advances in therapeutics, this disease remains incurable. Since proteins govern the cellular structure and biological function, a wide selection of proteomic approaches holds great promise for increasing our understanding of this disease, such as by investigating the dynamic nature of protein expression, cellular and subcellular distribution, post-translational modifications, and interactions at both the cellular and subcellular levels. The aims of this review are to introduce the available and emerging proteomic technologies that have potential applications in the study of MM and to highlight the current status of proteomic studies of MM. To date, although there have been a limited number of proteomic studies in MM, those performed have provided valuable information with regard to MM diagnosis and therapy. The potential future application of proteomic technologies is expected to provide new avenues in MM diagnostics, individualized therapy design and therapy response surveillance for the clinician.     

Aluminum Toxicity: A Case Study on Tobacco (<i>Nicotiana tabacum</i> L.)

Aluminum is an abundant metal in the earth’s crust that turns out to be toxic in acidic environments. Many plants are affected by the presence of aluminum at the whole plant level, at the organ level, and at the cellular level. Tobacco as a cash crop (Nicotiana tabacum L.) is a widely cultivated plant worldwide and is also a good model organism for research. Although there are many articles on Al-phytotoxicity in the literature, reviews on a single species that are economically and scientifically important are limited. In this article, we not only provide the biology associated with tobacco Al-toxicity, but also some essential information regarding the effects of this metal on other plant species (even animals). This review provides information on aluminum localization and uptake process by different staining techniques, as well as the effects of its toxicity at different compartment levels and the physiological consequences derived from them. In addition, molecular studies in recent years have reported specific responses to Al toxicity, such as overexpression of various protective proteins. Besides, this review discusses data on various organelle-based responses, cell death, and other mechanisms, data on tobacco plants and other kingdoms relevant to these studies.