Isolation,Characterization, and Evolutionary Divergence of Mouse RNase 6: Evidence for Unusual Evolution in Rodents

The evolution of the ribonuclease A (RNase A) vertebrate-specific enzyme family is interesting in that specific gene lineages appear to be responding to unique selective pressures in wildly diverse manners to generate proteins that are capable of reducing the infectivity of viruses, killing systemic pathogens, and inducing the growth of blood vessels all while maintaining the signature motifs of a ribonuclease. In this paper, we present the DNA sequence and gene structure of Mus musculus RNase 6 and examine the expression pattern and enzymatic activity of the recombinant protein. M. musculus RNase 6 has a limited expression pattern compared to human RNase 6 and is an efficient ribonuclease, with a catalytic efficiency 17-fold higher than that of human protein. Evo- lutionary analysis reveals that RNase 6 was subject to unusual evolutionary forces (d_N/d_S=1.2) in an ancestral rodent lineage before the separation of Mus and Rattus. However, more recent evolution of rodent RNase 6 has been relatively conserved, with an average d_N/d_S of 0.66. These data suggest that the ancestral rodent RNase 6 was subject to accelerated evolution, resulting in the conserved modern gene, which most likely plays an important role in mouse physiology.Reviewing Editor: Dr. Lauren Ancel MeyersThe GenBank accession numbers for the new genes presented here are as follows: Mus musculus, AY545655; Rattus norvegicus, AY545654; Mus spicilegus, AY545653; Mus caroli, AY545651; and Mus pahari, AY545652.     

Occurrence, biosynthesis and functions of N-acylphosphatidylethanolamines (NAPE): not just precursors of N-acylethanolamines (NAE)

N-acylphosphatidylethanolamine (NAPE) is a minor phospholipid resulting from the transfer of an acyl chain from an acyl donor to the primary amine of the ethanolamine moiety of phosphatidylethanolamine (PE). Occurring in plant and animal kingdoms as well as in prokaryotic cells, it is synthesized in higher amounts in membranes during cellular stresses and tissue damage, and it is widely thought to be the precursor of the lipid mediator, N-acylethanolamine (NAE), which modulates the endocannabinoid signaling pathway and therefore regulates various physiological processes. However, recent studies have shown that NAPE is also a bioactive molecule that is involved in several physiological functions. The present paper reviews the occurrence of NAPE in animals and plants and focuses on the various properties of NAPE observed in vitro and in vivo. The different metabolic pathways promoting the synthesis and degradation of NAPE are also discussed and the differences between animals and plants are underlined.     

Holocene sea level changes and reef development in the southwestern Indian Ocean

 The sedimentological and chronological study of Holocene reef sequences recovered in drill cores through modern reefs of Mauritius, Réunion Island and Mayotte allows the reconstruction of sea level changes and reef growth patterns during the Holocene. The branching-coral facies systematically predominates over coral head facies throughout the Holocene reef sequences, and Acropora is the main frame builder among the branching forms. The reconstructed sea level curves, based both on identification of coral assemblages and on radiometric U/Th ages, are characterized by a rapid rise between 10 and 7.5 ky BP, followed by a clear inflection between 7.5 and 7 ky BP. The stabilization of sea level at its present level occurred between 2000 and 3000 years ago, probably without a higher sea level stand. Rates of vertical reef accretion range between 0.9 and 7 mm. y^-1. In Mauritius, and also probably in Réunion Island, the reef first tracked, then caught-up to sea level to reach an equilibrium position (“catch-up” growth), while the barrier reef margin off Mayotte has been able to keep pace with rising sea level (“keep-up” growth). Accepted: 1 March 1997     

Vegetation demographics in Earth System Models: A review of progress and priorities

Numerous current efforts seek to improve the representation of ecosystem ecology and vegetation demographic processes within Earth System Models (ESMs). These developments are widely viewed as an important step in developing greater realism in predictions of future ecosystem states and fluxes. Increased realism, however, leads to increased model complexity, with new features raising a suite of ecological questions that require empirical constraints. Here, we review the developments that permit the representation of plant demographics in ESMs, and identify issues raised by these developments that highlight important gaps in ecological understanding. These issues inevitably translate into uncertainty in model projections but also allow models to be applied to new processes and questions concerning the dynamics of real‐world ecosystems. We argue that stronger and more innovative connections to data, across the range of scales considered, are required to address these gaps in understanding. The development of first‐generation land surface models as a unifying framework for ecophysiological understanding stimulated much research into plant physiological traits and gas exchange. Constraining predictions at ecologically relevant spatial and temporal scales will require a similar investment of effort and intensified inter‐disciplinary communication.     

In Xenopus laevis, the product of a developmentally regulated mRNA is structurally and functionally homologous to a Saccharomyces cerevisiae protein involved in translation fidelity.

We have performed a differential screen of a Xenopus egg cDNA library and selected two clones (Cl1 and Cl2) corresponding to mRNA which are specifically adenylated and recruited into polysomes after fertilization. Sequence analysis of Cl1 reveals that the corresponding protein is 67.5% identical (83% similar) to the product of the Saccharomyces cerevisiae SUP45 (also called SUP1 or SAL4) gene. This gene, when mutated, is an omnipotent suppressor of nonsense codons. When expressed in a sup45 mutant, the Xenopus Cl1 cDNA was able to suppress sup45-related phenotypes, showing that the structural homology reflects a functional homology. Our discovery of a structural and functional homolog in Xenopus cells implies that the function of SUP45 is not restricted to lower eukaryotes and that the SUP45 protein may perform a crucial cellular function in higher eukaryotes.     

A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering

A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique – USER cloning – to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors.     

Late Holocene sea level indicators from twelve atolls in the central and eastern Tuamotus (Pacific Ocean)

New sea-level data for the late Holocene period are reported from twelve atolls of the Tuamotu archipelago: Faaite, Hikueru, Marokau, Hao, Amanu, Tatakoto, Pukarua, Nukutavake, Vairaatea, Tureia, Nukutipipi, and Hereheretue. The data come from coral conglomerate outcrops, coral colonies in growth position, in situ reef framework and marine notches; they give consistent results, and their ages are controlled by 29 radiocarbon datings. The Holocene MSL remained 0.8±0.2 m higher than at present, from before 4000 years BP until at least 1500 years BP, then dropped gradually to the present level. This pattern is very similar to that reported from the northwest Tuamotus, suggesting the absence of measurable differential vertical movements over a distance greater than 1300 km during the late Holocene. Effects of the 1983 cyclones on reef morphology are reported from some atolls and radiocarbon dates of some storm-generated reef blocks are given.     

Elevation of the Extracellular Concentrations of Glutamate and Aspartate in Rat Hippocampus During Transient Cerebral Ischemia Monitored by Intracerebral Microdialysis

Rats were implanted with 0.3-mm-diameter dialysis tubing through the hippocampus and subsequently perfused with Ringer's solution at a flow rate of 2 microliter/min. Samples of the perfusate representing the extracellular fluid were collected over 5-min periods and subsequently analyzed for contents of the amino acids glutamate, aspartate, glutamine, taurine, alanine, and serine. Samples were collected before, during, and after a 10-min period of transient complete cerebral ischemia. The extracellular contents of glutamate and aspartate were increased, respectively, eight- and threefold during the ischemic period; the taurine concentration also was increased 2.6-fold. During the same period the extracellular content of glutamine was significantly decreased (to 68% of the control value), whereas the concentrations of alanine and serine did not change significantly during the ischemic period. The concentrations of gamma-aminobutyric acid (GABA) were too low to be measured reliably. It is suggested that the large increase in the content of extracellular glutamate and aspartate in the hippocampus induced by the ischemia may be one of the causal factors in the damage to certain neurons observed after ischemia.