Genomic analysis of immunity in a Urochordate and the emergence of the vertebrate immune system: “waiting for Godot”

Genome-wide sequence analysis in the invertebrate chordate, Ciona intestinalis, has provided a comprehensive picture of immune-related genes in an organism that occupies a key phylogenetic position in vertebrate evolution. The pivotal genes for adaptive immunity, such as the major histocompatibility complex (MHC) class I and II genes, T-cell receptors, or dimeric immunoglobulin molecules, have not been identified in the Ciona genome. Many genes involved in innate immunity have been identified, including complement components, Toll-like receptors, and the genes involved in intracellular signal transduction of immune responses, and show both expansion and unexpected diversity in comparison with the vertebrates. In addition, a number of genes were identified which predicted integral membrane proteins with extracellular C-type lectin or immunoglobulin domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activation motifs (ITAMs) (plus their associated signal transduction molecules), suggesting that activating and inhibitory receptors have an MHC-independent function and an early evolutionary origin. A crucial component of vertebrate adaptive immunity is somatic diversification, and the recombination activating genes (RAG) and activation-induced cytidine deaminase (AID) genes responsible for the Generation of diversity are not present in Ciona. However, there are key V regions, the essential feature of an immunoglobulin superfamily VC1-like core, and possible proto-MHC regions scattered throughout the genome waiting for Godot.     

An automated method for the analysis of ‘particulate’ carbon and nitrogen in natural waters

A method is described for the measurement of the carbon and nitrogen content of particulate material in natural waters. Particulate material is separated by filtration through GF/C filters. The dried filter is encapsulated in silver foil using a purpose made press. Analysis is carried out using high temperature combustion with thermal conductivity detection of emission gasses. Analytical performance characteristics obtained with both standards and natural materials are given.     

Molecular analysis of the major cellulase (CelV) of Erwinia carotovora: evidence for an evolutionary “mix-and-match” of enzyme domains

The structural gene for the major cellulase of Erwinia carotovora subspecies carotovora (Ecc) was isolated and expressed in Escherichia coli. Sequencing of the gene (celV) revealed a typical signal sequence and two functional domains in the enzyme; a catalytic domain linked by a short proline/threonine-rich linker to a cellulose-binding domain (CBD). The deduced amino acid sequence of the catalytic domain showed homology with cellulases of Family A, including enzymes from Bacillus spp. and Erwinia chrysanthemi CelZ, whereas the CBD showed homology with cellulases from several diverse families, supporting a “mix-and-match” hypothesis for evolution of this domain. Analysis of the substrate specificity of CelV showed it to be an endoglucanase with some exoglucanase activity. The pH optimum is about 7.0 and the temperature optimum about 42°C. CelV is secreted by Ecc and by the taxonomically related Erwinia carotovora subspecies atroseptica (Eca) but not by E. coli. Overproduction of the enzyme from multicopy plasmids in Ecc appears to overload the secretory mechanism.     

Diachronic analysis of biodiversity: Study of a herbarium “reviewed” by Francesco Cupani (1657–1710) at the end of the 17th century

The subject of this paper is the study of the pre-Linnaean herbarium with the inventory number “VII F1 Hortus Botanicus Catinensis” belonging to the Department of Biological, Geological and Environmental Sciences (University of Catania). The volume examined comprises 186 pages (originally 190) with 1–9 specimens on each sheet. The herbarium samples mainly include angiosperms (386), but a few taxa belonging to gymnosperms (1) and pteridophytes (4) are also present. Unfortunately, some specimens are partially or entirely damaged or even missing; nonetheless, it has been possible to identify most of them. As far as the nomenclature is concerned, we find two kinds of handwriting (of which only that of Cupani can be identified), numerous corrections and/or integrations of the polylogus and different types of ink that were used. These elements allow us to state that we are speaking of a herbarium which was prepared by an unknown person and reviewed by Cupani.     

An analysis of the loss of acroporid corals at Looe Key,Florida, USA: 1983–2000

The Caribbean reef-building corals Acropora palmata and Acropora cervicornis have undergone widespread declines in the past two decades, leading to their designation as candidates for listing under the United States Endangered Species Act. Whole-reef censuses in 1983 and 2000 at Looe Key National Marine Sanctuary in the Florida Keys provide estimates of areal loss of live Acropora spp. cover. Area (square meters) of live coral cover was quantified from depiction on scaled base maps of extent of coral cover observed by a snorkeler on each reef spur at each census. Certain thickets appear to have been persistent (though none expanded), but the total area of live A. palmata at Looe Key is estimated to have declined by 93% and A. cervicornis by 98% during this 17-year interval. It is likely that acroporid populations may have already undergone substantial decline prior to our initial census in 1983.     

The polymorphism of desialyzed α2HS-glycoptein (AHS): isoelectric focusing in 2.5 M urea as a method for identification of genetic variants

Summary Desialyzed plasma specimens were phenotyped for ₂HS-glycoprotein (AHS) using polyacrylamide isoelectric focusing (IEF) in the range pH 5–6 in 2.5 M urea, followed by immunoblotting. The technique used in this study is easy to perform and can differentiate the gene products of all the currently known variants of ₂HS-glycoprotein except for AHS 4.     

The study of lysogeny at the Pasteur Institute (1950–1960): an epistemologically open system

Many historical studies have been devoted to the French school of molecular biology, in particular to the work of Jacques Monod on adaptive enzymes. By focusing on François Jacob’s studies on lysogeny between 1950 and 1960, we intend to redress the imbalance of historiography, as well as proposing a more fruitful point of view for understanding the relative importance of international contacts and local traditions in the genesis of the operon model.Elie Wollman and Jacob’s work on temperate bacteriophages rendered respectable a system that had been considered an artefact for more than two decades. They did this firstly by modelling their studies on those of the US phage group and secondly by basing these studies on a complex vision of the relations between bacteria and bacteriophages. The interaction between bacteria and temperate bacteriophages was considered ab initio as a biochemical process, the mechanisms of which would eventually be characterized. It was also considered as a “normal” phenomenon that could be used as a model to understand the process of differentiation, as well as the role of viruses in diseases and cancer. The temperate bacteriophage was a model system that was far more epistemologically open and, for this reason, in a sense more productive than the virulent phage studied by the US phage group.     

Topographical analysis of trypsin active site structure by means of the “inverse substrate” method

Trypsin-specific substrate analogs, “inverse substrates,” carrying the fluorescent dimethylaminonaphthalene group were synthesized. Preparation of acyl trypsins in which the fluorescent group is attached to the catalytic residue through a spacer group of various chain lengths was successfully carried out by use of these inverse substrates. The topographical structure of the trypsin active site vicinity was estimated on the basis of the fluorescence spectra of these acyl trypsins.     

Commercial production of β-glucuronidase (GUS): a model system for the production of proteins in plants

We have generated transgenic maize seed containing -glucuronidase(GUS) for commercial production. While many other investigators have demonstrated the expression of GUS as a scoreable marker, this is one of the first cases where a detailed characterization of the transgenic plants and the protein were performed which are necessary to use this as a commercial source of GUS. The recombinant -glucuronidase was expressed at levels up to 0.7% of water-soluble protein from populations of dry seed, representing one of the highest levels of heterologous proteins reported for maize. Southern blot analysis revealed that one copy of the gene was present in the transformant with the highest level of expression. In seeds, the majority of recombinant protein was present in the embryo, and subcellular localization indicated that the protein was dispersed throughout the cytoplasm. The purified recombinant -glucuronidase (GUS) was compared to native -glucuronidase using SDS-PAGE and western blot analysis. The molecular mass of both the recombinant and native enzymes was 68 000 Da. N-terminal amino acid sequence of the recombinant protein was similar to the sequence predicted from the cloned Escherichia coli gene except that the initial methionine was cleaved from the recombinant GUS. The recombinant and native GUS proteins had isoelectric points (pI) from 4.8 to 5.0. The purified proteins were stable for 30 min at 25, 37, and 50 ° C. Kinetic analysis of the recombinant and native GUS enzymes using 4-methylumbelliferyl glucuronide (MUG) as the substrate was performed. Scatchard analysis of these data demonstrated that the recombinant enzyme had a K_m of 0.20 mM and a V_max of 0.29 mM MUG per hour, and the native enzyme had a K_m and V_max of 0.21 mM and 0.22 mM/h respectively. Using D-saccharic acid 1,4-lactone, which is an inhibitor of -glucuronidase, the K_i of the native and recombinant enzymes was determined to be 0.13 mM. Thus, these data demonstrate that recombinant GUS is functionally equivalent to native GUS. We have demonstrated the expression of high levels of GUS can be maintained in stable germlines and have used an efficient recovery system where the final protein product, GUS, has been successfully purified. We describe one of the first model systems for the commercial production of a foreign protein which relies on plants as the bioreactor.     

Polyploids as a “model system” for the study of heterosis

Heterosis research over the past century has focused primarily on diploid plants and animals. This is despite the fact that most heterotic organisms contain polyploid events in their recent and/or ancient past and various important crop species are heterotic polyploids. We present an argument for the study of heterosis within polyploid systems and give examples of how their study can improve current hypotheses and generate new ones. Polyploid systems allow experiments not possible in diploids but the insights gained must be incorporated into models to explain heterosis at all levels.     

The digestive system of the “stick bug” Cladomorphus phyllinus (Phasmida,Phasmatidae): A morphological,physiological and biochemical analysis

This work presents a detailed morphofunctional study of the digestive system of a phasmid representative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas posterior midgut cells show a microapocrine secretion. A complex system of midgut tubules is observed in the posterior midgut which is probably related to the luminal alkalization of this region. Amaranth dye injection into the haemolymph and orally feeding insects with dye indicated that the anterior midgut is water-absorbing, whereas the Malpighian tubules are the main site of water secretion. Thus, a putative counter-current flux of fluid from posterior to anterior midgut may propel enzyme digestive recycling, confirmed by the low rate of enzyme excretion. The foregut and anterior midgut present an acidic pH (5.3 and 5.6, respectively), whereas the posterior midgut is highly alkaline (9.1) which may be related to the digestion of hemicelluloses. Most amylase, trypsin and chymotrypsin activities occur in the foregut and anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, while aminopeptidase occurs in the middle and posterior midgut in membrane bound forms. Both amylase and trypsin are secreted mainly by the anterior midgut through an exocytic process as revealed by immunocytochemical data.     

An “ancient” complexity? Evolutionary morphology of the circulatory system in Xiphosura

Horseshoe crabs (Xiphosura) have been an object of zoological research for almost 200 years. Although some morphological work on the circulatory system has been done, the three-dimensional structure of this complex organ system has never been shown satisfactorily and some crucial questions remain unanswered. Here, the circulatory systems of juveniles of the horseshoe crab taxa Limulus polyphemus and Carcinoscorpius rotundicauda were investigated using a combination of an injection method and micro-computed tomography. Data were processed and 3D-visualized using reconstruction software. Furthermore, the heart was examined using scanning electron microscopy. Additionally, the histology of some structures was investigated via light microscopy and transmission electron microscopy. The results show the high degree of complexity of the arterial and lacunar systems of Xiphosura and provide insights into their three-dimensional structure and relationship to other organ systems such as the central nervous system. We show that the major lacunae, previously described as vessel-like – though indeed highly ramified – can clearly be distinguished from arteries in histological sections because they have no distinct walls. Similarities and differences between the xiphosuran species and arachnids are highlighted and possible phylogenetic implications and evolutionary scenarios discussed.     

An analysis of the feasibility of using caudal vertebrae for ageing the spinetail devilray, Mobula japanica (Müller and Henle, 1841)

Age assessment of Mobula japanica may be possible using the caudal vertebrae, below the origin of the dorsal fin. This is supported by the significant linear relationship found between disc width (DW) and centrum radius (CR, n?=?55), the continuous record of growth bands in the vertebrae, the clarity to distinguish and count growth bands, and the precision of the band counts. Assuming an annual band formation, the preliminary assessment of the age suggests that M. japanica lives at least 14 years and has a low growth rate (K?=?0.28 year^?1). The minimum number of growth bands was one for spinetail devilrays with a 1,210–1,390 mm DW, while the maximum was 14 for a 2,300 mm DW devilray. While age validation is still required, results indicate the feasibility of the use of caudal vertebrae for age estimation. To provide robust estimates of validated age and growth for the spinetail devilray, the sampling coverage needed might imply an international cooperation.     

Isozyme analysis of intercontinental disjuncts within Styrax (Styracaceae): implications for the Madrean —Tethyan hypothesis

Various hypotheses have been put forward to explain the presence of sclerophyllous plant disjuncts between western North America and the Mediterranean region. The Madrean–Tethyan hypothesis postulates that the two regions were floristically connected in the Early to Middle Tertiary by way of a low-latitude migration route. Others deny the possibility of such a route, and instead postulate convergence to xerophytic conditions from more widespread mesophytic ancestors, or suggest long-distance dispersal scenarios. One example of a “Madrean–Tethyan link” between the two regions is composed of four species within the genus Styrax: S. officinalis subsp. officinalis from the Mediterranean region, S. officinalis subsp. redivivus and subsp. fulvescens from California, and three closely related species in Texas and northeastern Mexico (S. texanus, S. platanifolius, and S. youngiae). This group was examined with isozymes to assess whether patterns of genetic variation are consistent with those predicted by the Madrean–Tethyan hypothesis. Ten populations from California, six from the Mediterranean region, and three from Texas were sampled. Pairwise comparisons revealed mean genetic identity (I) estimates of 0.581 between Mediterranean and California populations, 0.470 between Mediterranean and Texas populations, and 0.640 between California and Texas populations. Two populations of a species thought by many to be the closest relative of S. officinalis on morphological grounds (S. jaliscanus) exhibited low I (0.299–0.321) relative to all other group comparisons. Intercontinentally disjunct populations of S. officinalis possessed an I value that warrants species status for the Californian and Mediterranean groups. Divergence time estimates between Madrean and Tethyan Styrax range from 5.0 to 13.8 Mya, too recent to be consistent with the Madrean–Tethyan hypothesis. However, alternative explanations for this disjunction are suboptimal in that they require the invocation of either long-distance dispersal, which appears unlikely in this group, or extinction. Nonetheless, the evidence presented here and in other recent studies casts substantial doubt on the Madrean–Tethyan hypothesis as a general explanation for the presence of Madrean and Tethyan taxa similar in overall appearance. More plants with Madrean–Tethyan distributions must be sampled before definitive conclusions regarding this aspect of Madrean and Tethyan vegetation can be reached.     

A novel modeling approach for the “end-to-end” analysis of marine ecosystems

There is a growing demand for “end-to-end” models, which are modeling tools used to analyze and understand the fundamental complexities of marine ecosystems and processes emerging from the interaction of individuals from different trophic groups with respect to the physical environment and, even, human activity. These models are valuable quantitative tools for ecosystem-based management. To explore potential answers to complex questions regarding ecosystems using these models, it is necessary to incorporate classical ontogenic changes through the life cycle of target individuals, in addition to inherited behavioral strategies, as an additional differentiating aspect, particularly when the behavior has a direct impact on the ecosystem phenomena under study. However, it is difficult to combine different fine scale time and spatial granularities to infer animal behavior and ontogenic development. This complexity has kept these two levels of analysis separated, because most current tools do not have the required computational resources and advanced software architecture. To address this issue, we propose an individual-based modeling framework that is capable of handling and unifying the two experimental categories with a comprehensive biological and behavioral model that strictly adheres to the physiological functions of ingestion, growth, and metabolism of organisms. In addition, this model incorporates the exchange and transfer of mass and energy through local interactions at all trophic levels (lower to higher), the physical environment, and anthropogenic activity. For the framework to model short time events, such as classical predator–prey interactions, while also generating long-term ecosystem emergent properties, a special interleaving scheduling engine and physical space computer model was devised, which optimizes memory and processing resources. The framework was tested through several experiments with a three-population ecosystem containing up to 40 thousand organisms evolving inside a 200,000 m² simulation environment during 12,000 model-hours; yet, requiring only a few hours of program execution on a regular personal computer. The model included various environmental physical elements, such as several hundred shelters, the number of which can be easily modified in each experiment to simulate substrate degradation and its impact on populations. With the aid of the quantitative and qualitative tools provided by the model, it was possible to observe a coupling between prey and predator population dynamics. In conclusion, we confirmed that the end-to-end model developed here could successfully generate detailed specific hypotheses about fish behavior and quantify impacts on population dynamics.