Production of transgenic goats by pronuclear microinjection of in vitro produced zygotes derived from oocytes recovered by laparoscopy

Oocytes collected by laparoscopic ovum pick-up (LOPU) were successfully used to produce transgenic goats by pronuclear microinjection of in vitro zygotes. Estrus cycles of 109 donor goats were synchronized using intravaginal sponges impregnated with 60 mg of medroxyprogesterone acetate and treatment with 70 mg NIH-FSH-P1 and 300 IU eCG to stimulate follicular development. Follicles were aspirated under laparoscopic observation. In vitro maturation (IVM) of oocytes was performed in M199 supplemented with hormones, kanamycin and 10% estrus goat serum. Following IVM, oocytes were cocultured with capacitated semen in TALP supplemented with 20% estrus goat serum for 15-20 h. The resulting zygotes were microinjected with a linear DNA fragment. In total, 3293 follicles were aspirated (15.7+/-9 follicles aspirated per donor) and 2823 oocytes were recovered (13.4+/-8 oocytes per donor). A total of 1366 zygotes were microinjected and transferred into 219 recipient goats by midventral laparotomy (average 6.2 embryos per recipient). A total of 150 kids were born, of which 9 (6 M: 3 F) were confirmed to be transgenic by PCR and Southern blotting analyses. These results demonstrate that acceptable transgenesis rates can be obtained in goats by DNA microinjection of in vitro produced zygotes.     

ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip data

Background  

Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) or ChIP followed by genome tiling array analysis (ChIP-chip) have become standard technologies for genome-wide identification of DNA-binding protein target sites. A number of algorithms have been developed in parallel that allow identification of binding sites from ChIP-seq or ChIP-chip datasets and subsequent visualization in the University of California Santa Cruz (UCSC) Genome Browser as custom annotation tracks. However, summarizing these tracks can be a daunting task, particularly if there are a large number of binding sites or the binding sites are distributed widely across the genome.     

Dead fish swimming: a review of research on the early migration and high premature mortality in adult Fraser River sockeye salmon Oncorhynchus nerka

Adult sockeye salmon Oncorhynchus nerka destined for the Fraser River, British Columbia are some of the most economically important populations but changes in the timing of their homeward migration have led to management challenges and conservation concerns. After a directed migration from the open ocean to the coast, this group historically would mill just off shore for 3-6 weeks prior to migrating up the Fraser River. This milling behaviour changed abruptly in 1995 and thereafter, decreasing to only a few days in some years (termed early migration), with dramatic consequences that have necessitated risk-averse management strategies. Early migrating fish consistently suffer extremely high mortality (exceeding 90% in some years) during freshwater migration and on spawning grounds prior to spawning. This synthesis examines multidisciplinary, collaborative research aimed at understanding what triggers early migration, why it results in high mortality, and how fisheries managers can utilize these scientific results. Tissue analyses from thousands of O. nerka captured along their migration trajectory from ocean to spawning grounds, including hundreds that were tracked with biotelemetry, have revealed that early migrants are more reproductively advanced and ill-prepared for osmoregulatory transition upon their entry into fresh water. Gene array profiles indicate that many early migrants are also immunocompromised and stressed, carrying a genomic profile consistent with a viral infection. The causes of these physiological changes are still under investigation. Early migration brings O. nerka into the river when it is 3-6° C warmer than historical norms, which for some late-run populations approaches or exceeds their critical maxima leading to the collapse of metabolic and cardiac scope, and mortality. As peak spawning dates have not changed, the surviving early migrants tend to mill in warm lakes near to spawning areas. These results in the accumulation of many more thermal units and longer exposures to freshwater diseases and parasites compared to fish that delay freshwater entry by milling in the cool ocean environment. Experiments have confirmed that thermally driven processes are a primary cause of mortality for early-entry migrants. The Fraser River late-run O. nerka early migration phenomenon illustrates the complex links that exist between salmonid physiology, behaviour and environment and the pivotal role that water temperature can have on population-specific migration survival.     

Reduced oviposition by Diaprepes abbreviatus (Coleoptera: Curculionidae) and growth enhancement of citrus by Surround particle film

Regularly applied sprays of a particle film, Surround WP, greatly enhanced the growth of citrus trees on a poorly drained Winder soil at Fort Pierce, FL. After 3 yr of applications every 3 or 4 wk, Surround-treated trees had at least 5 times the mass, 6 times the canopy volume, and approximately 4 times the cross-sectional area of the tree stems at the graft union compared with untreated trees. The larger Surround-treated trees attracted a higher number of adult weevil Diaprepes abbreviatus (L.) and to a lesser extent citrus root weevil, Pachnaeus litus (Germar), per tree, but there was an equivalent number of egg masses per tree compared with the control trees. The number of egg masses per female weevil oviposited on Surround-treated trees was significantly less than either the control trees or trees treated biannually with an entomopathogenic nematode, BioVector. The number of larvae per tree recovered from the roots of excavated trees was greater from trees treated with Surround once every 3 wk compared with control trees. The data suggest that Surround particle film greatly enhanced the growth of citrus trees grown in a poorly drained soil. The reduction in oviposition by D. abbreviatus was insufficient to significantly reduce the number of root weevil larvae per tree feeding on the roots. However, the more vigorous trees resulting from Surround applications may be more resistant or tolerant to root weevil feeding.     

Identification of a disulfide bridge linking the fourth and the seventh extracellular loops of the Na+/glucose cotransporter

The Na+/glucose cotransporter (SGLT1) is an archetype for the SLC5 family, which is comprised of Na+-coupled transporters for sugars, myo-inositol, choline, and organic anions. Application of the reducing agent dithriothreitol (DTT, 10 mM) to oocytes expressing human SGLT1 affects the protein's presteady-state currents. Integration of these currents at different membrane potentials (Vm) produces a Q-V curve, whose form was shifted by +25 mV due to DTT. The role of the 15 endogenous cysteine residues was investigated by expressing SGLT1 constructs, each bearing a single mutation for an individual cysteine, in Xenopus oocytes, using two-microelectrode voltage-clamp electrophysiology and fluorescent labeling. 12 of the 15 mutants were functional and could be separated into three distinct groups based on the effect of the mutation on the Q-V curve: four mutants did not perturb the transferred charge, six mutants shifted the Q-V curve towards negative potentials, and two mutants (C255A and C511A) produced a shift in the positive direction that was identical to the shift produced by DTT on the wild-type (wt) SGLT1. The double mutant C(255,511)A confirms that the effects of each single mutant on the Q-V curve were not additive. With respect to wt SGLT1, the apparent affinities for alpha-methylglucose (alphaMG) were increased in a similar manner for the single mutants C255A and C511A, the double mutant C(255,511)A as well as for wt SGLT1 treated with DTT. When exposed to a maleimide-based fluorescent probe, wt SGLT1 was not significantly labeled but mutants C255A and C511A could be clearly labeled, indicating an accessible cysteine residue. These residues are presumed to be C511 and C255, respectively, as the double mutant C(255,511)A could not be labeled. These results strongly support the hypothesis that C255 and C511 form a disulfide bridge in human SGLT1 and that this disulfide bridge is involved in the conformational change of the free carrier.     

Selective separation of cationic peptides from a tryptic hydrolysate of beta-lactoglobulin by electrofiltration

Electrofiltration (EF) was used to selectively separate cationic (basic) peptides contained in a tryptic beta-lactoglobulin (beta-LG) hydrolysate, with particular emphasis on the isolation of basic sequence beta-LG 142-148, which is a potential antihypertensive peptide. Both the influence of feed solution pH and operating parameters (transmembrane pressure, feed velocity) were assessed to find optimum conditions enabling the fractionation between peptides during EF. The cathode (-) was inserted in the permeate side to increase the separation of basic peptides contained in the tryptic beta-LG hydrolysate as compared to conventional NF. The highest separation factor between basic and neutral peptides was obtained at pH 9 using G-10 membrane with a molecular weight cut-off (MWCO) of 2,500 g/mol, at 5 V with the lowest transmembrane pressure (0.344 MPa) and feed velocity (0.047 m/s). The transmission behavior of the peptides during EF was better explained when taking into account the positive/negative charge ratio. Because of its 3+/1- charge ratio, beta-LG 142-148 had the highest transmission during EF. Consequently, its relative concentration was raised from 3.5% in the initial tryptic beta-LG hydrolysate up to 38% in the permeate. The electric field seemed more effective when the convective/shearing forces were minimized.     

Peculiar inhibition of human mitochondrial aspartyl-tRNA synthetaseby adenylate analogs

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), the enzymes which esterify tRNAs with the cognate specific amino acid, form mainly a different set of proteins than those involved in the cytosolic translation machinery. Many of the mt-aaRSs are of bacterial-type in regard of sequence and modular structural organization. However, the few enzymes investigated so far do have peculiar biochemical and enzymological properties such as decreased solubility, decreased specific activity and enlarged spectra of substrate tRNAs (of same specificity but from various organisms and kingdoms), as compared to bacterial aaRSs. Here the sensitivity of human mitochondrial aspartyl-tRNA synthetase (AspRS) to small substrate analogs (non-hydrolysable adenylates) known as inhibitors of Escherichia coli and Pseudomonas aeruginosa AspRSs is evaluated and compared to the sensitivity of eukaryal cytosolic human and bovine AspRSs. l-aspartol-adenylate (aspartol-AMP) is a competitive inhibitor of aspartylation by mitochondrial as well as cytosolic mammalian AspRSs, with K_i values in the micromolar range (4–27 μM for human mt- and mammalian cyt-AspRSs). 5′-O-[N-(l-aspartyl)sulfamoyl]adenosine (Asp-AMS) is a 500-fold stronger competitive inhibitor of the mitochondrial enzyme than aspartol-AMP (10 nM) and a 35-fold lower competitor of human and bovine cyt-AspRSs (300 nM). The higher sensitivity of human mt-AspRS for both inhibitors as compared to either bacterial or mammalian cytosolic enzymes, is not correlated with clear-cut structural features in the catalytic site as deduced from docking experiments, but may result from dynamic events. In the scope of new antibacterial strategies directed against aaRSs, possible side effects of such drugs on the mitochondrial human aaRSs should thus be considered.     

Reversal of the Mitochondrial Phenotype and Slow Development of Oxidative Biomarkers of Aging in Long-lived Mclk1+/? Mice

Although there is a consensus that mitochondrial function is somehow linked to the aging process, the exact role played by mitochondria in this process remains unresolved. The discovery that reduced activity of the mitochondrial enzyme CLK-1/MCLK1 (also known as COQ7) extends lifespan in both Caenorhabditis elegans and mice has provided a genetic model to test mitochondrial theories of aging. We have recently shown that the mitochondria of young, long-lived, Mclk1^+/− mice are dysfunctional, exhibiting reduced energy metabolism and a substantial increase in oxidative stress. Here we demonstrate that this altered mitochondrial condition in young animals paradoxically results in an almost complete protection from the age-de pend ent loss of mitochondrial function as well as in a significant attenuation of the rate of development of oxidative biomarkers of aging. Moreover, we show that reduction in MCLK1 levels can also gradually prevent the deterioration of mitochondrial function and associated increase of global oxidative stress that is normally observed in Sod2^+/− mutants. We hypothesize that the mitochondrial dysfunction observed in young Mclk1^+/− mutants induces a physiological state that ultimately allows for their slow rate of aging. Thus, our study provides for a unique vertebrate model in which an initial alteration in a specific mitochondrial function is linked to long term beneficial effects on biomarkers of aging and, furthermore, provides for new evidence which indicates that mitochondrial oxidative stress is not causal to aging.Because it is well known that the aging process is characterized by declines in basal metabolic rate and in the general performance of energy-dependent processes, many aging studies have focused on mitochondria because of their central role in producing chemical energy (ATP) by oxidative phosphorylation (1). Among the various theories of aging that have been proposed, the mitochondrial oxidative stress theory of aging is the most widely acknowledged and studied (2–4). It is based on the observation that mitochondrial energy metabolism produces reactive oxygen species (ROS),² that mitochondrial components are damaged by ROS, that mitochondrial function is progressively lost during aging, and that the progressive accumulation of global oxidative damage is strongly correlated with the aged phenotype. However, the crucial question of whether these facts mean that mitochondrial dysfunction and the related ROS production cause aging remains unproven (5–7). Furthermore, recent observations made in various species, including mammals, have begun to directly challenge this hypothesis, notably by relating oxidative stress to long (8) or increased (9) lifespans, by demonstrating that overexpression of the main antioxidant enzymes does not extend lifespan (10) as well as by showing that mitochondrial dysfunction could protect against age-related diseases (11).A direct and powerful approach to attempt to clarify this major question and to test the theory is to characterize the mitochondrial function of long-lived mutants (12). CLK-1/MCLK1 is an evolutionary conserved protein (13) and has been found to be located in the mitochondria of yeast (14), worms (15), and mice (16). The inactivation of the Caenorhabditis elegans gene clk-1 substantially increases lifespan (17). Moreover, the elimination of one functional allele of its murine orthologue also resulted in an extended longevity for Mclk1^+/− mice in three distinct genetic backgrounds (18). These findings have provided for an evolutionarily conserved pathways of animal aging that is affected by the function of a mitochondrial protein (19, 20). In mitochondria CLK1/MCLK1 acts as an hydroxylase and is implicated in the biosynthesis of ubiquinone (coenzyme Q or UQ), a lipid-like molecule primarily known as an electron carrier in the mitochondrial respiratory chain and as a membrane antioxidant but which is also associated with an increasing number of different aspects of cellular metabolism (20, 21). Taken together, these observations indicate that the long-lived Mclk1^+/− mouse is a model of choice for the understanding of the links between mitochondrial energy metabolism, oxidative stress, and the aging process in mammals.Previous analysis of Mclk1^+/− mice, which show the expected reduction of MCLK1 protein levels (22), have revealed that their tissues as well as their mitochondria contain normal levels of UQ at 3 months of age (23). Yet the same study also revealed a host of phenotypes induced by Mclk1 heterozygosity (see below). Thus, it appears that MCLK1 has an additional function that is unrelated to UQ biosynthesis but responsible for the phenotypes observed in young Mclk1^+/− mutants. This is consistent with several results from nematodes which also strongly suggest that CLK-1 has other functions (24, 25).In depth characterization of the phenotype of young Mclk1^+/− mutants has revealed that the reduction of MCLK1 levels in these animals profoundly alters their mitochondrial function despite the fact that UQ production is unaffected (23). In fact, we have shown that Mclk1 heterozygosity induces a severe impairment of mitochondrial energy metabolism as revealed by a reduction in the rates of mitochondrial electron transport and oxygen consumption as well as in ATP synthesis and ATP levels in both the mitochondria and the whole cell. ATP levels in several organs were surprisingly strongly affected with, for example, a 50% reduction of overall cellular ATP levels in the livers of Mclk1^+/− mutants (23). Moreover, we have found that the Mclk1^+/− mice sustain high mitochondrial oxidative stress by a variety of measurements, including aconitase activity, protein carbonylation, and ROS production (23). Additionally, we have shown that this early mitochondrial dysfunction is associated with a reduction in some aspects of cytosolic oxidative damage and global oxidative stress that can be measured via recognized plasma biomarkers such as 8-isoprostanes and 8-hydroxy-2-deoxyguanosine (8-OHdG). Considering that the accumulation of global oxidative damage is known to be tightly linked to the aging process (26), this latter result suggests that the anti-aging effect triggered by low MCLK1 levels might already act at a young age.To further investigate the clk-1/Mclk1-dependent mechanism of aging as well as to try to elucidate the still unclear relation between mitochondrial dysfunction, oxidative stress, and aging, we have now carefully analyzed the evolution of the phenotype of Mclk1^+/− mutants over time. We have also studied the effects of reduced MCLK1 levels on the phenotype of mice heterozygous for the mitochondrial superoxide dismutase (Sod2), which represent a well known model of mitochondrial oxidative stress (27). In addition of confirming the long lifespan phenotype of the Mclk1^+/− mutants in a mixed background (129S6 x BALB/c), we also report here a study of mutants and controls on a completely isogenic background where we find that the condition of Mclk1^+/− mutants unexpectedly results in protection against the age-dependent loss of mitochondrial function. Moreover, we found that the mutants are characterized by a significant attenuation of the age-associated increase in global oxidative stress normally observed in mammals. We also show that the Mclk1^+/− condition can gradually reverse the deterioration of mitochondrial function and the associated increase of global oxidative stress that is normally observed in Sod2^+/− mutants. Thus, this study provides for a unique vertebrate model in which reduced levels of a specific mitochondrial protein causes early mitochondrial dysfunction but has long term beneficial effects that slow down the rate of aging, as established with appropriate biomarkers, and can ultimately prolong lifespan in mice. Furthermore, in line with recent studies that have raised doubts about the validity of the mitochondrial oxidative stress theory of aging (4, 8, 10), our results, which relate to a recognized long-lived mice model, represent a novel and crucial indication that mitochondrial oxidative stress might not by itself be causal to aging.     

Phylogenetic and Structural Relationships of the PR5 Gene Family Reveal an Ancient Multigene Family Conserved in Plants and Select Animal Taxa

Pathogenesis-related group 5 (PR5) plant proteins include thaumatin, osmotin, and related proteins, many of which have antimicrobial activity. The recent discovery of PR5-like (PR5-L) sequences in nematodes and insects raises questions about their evolutionary relationships. Using complete plant genome data and discovery of multiple insect PR5-L sequences, phylogenetic comparisons among plants and animals were performed. All PR5/PR5-L protein sequences were mined from genome data of a member of each of two main angiosperm groups—the eudicots (Arabidoposis thaliana) and the monocots (Oryza sativa)—and from the Caenorhabditis nematode (C. elegans and C. briggsase). Insect PR5-L sequences were mined from EST databases and GenBank submissions from four insect orders: Coleoptera (Diaprepes abbreviatus and Biphyllus lunatus), Orthoptera (Schistocerca gregaria), Hymenoptera (Lysiphlebus testaceipes), and Hemiptera (Toxoptera citricida). Parsimony and Bayesian phylogenetic analyses showed that the PR5 family is paraphyletic in plants, likely arising from 10 genes in a common ancestor to monocots and eudicots. After evolutionary divergence of monocots and eudicots, PR5 genes increased asymmetrically among the 10 clades. Insects and nematodes contain multiple sequences (seven PR5-Ls in nematodes and at least three in some insects) all related to the same plant clade, with nematode and insect sequences separating as two clades. Protein structural homology modeling showed strong similarity among animal and plant PR5/PR5-Ls, with divergence only in surface-exposed loops. Sequence and structural conservation among PR5/PR5-Ls suggests an important and conserved role throughout the evolutionary divergence of the diverse organisms from which they reside. [Reviewing Editor: Dr. Rafael Zardoya]     

Anatomy of a red tide bloom off the southwest coast of Florida

A massive outbreak of Karenia brevis that had been ongoing for several months along the southwestern coast of Florida was sampled in early September 2005 off Sanibel Island to assess the utility of bio-optical features and ataxonomic analysis (quantification of eukaryotic and cyanobacterial picoplankton) by flow cytometry in monitoring red tide blooms. Sea-surface sampling followed aircraft visual location of discolored water. Within the most concentrated area of the bloom, chlorophyll a values exceeded 500 μg l⁻¹, and concentrations of nitrate (0.3 μM ± 0.0) and ammonium (<0.2 μM) were depleted compared to high concentrations of total dissolved nitrogen, total dissolved phosphorus, and soluble reactive phosphorus (141 ± 34 μM, 16.5 ± 2.5 μM, and 6.44 ± 0.57 μM, respectively). Low water clarity in the bloom (Secchi depth transparency 0.3 m, K_d estimated at 4.83 m⁻¹) was strongly influenced by attenuation from dinoflagellates as well as chromophoric dissolved organic matter (CDOM). The fact that the K. brevis bloom occurred in lower-salinity (30 psu), high-nutrient waters implicates riverine transport of land-based nutrients as a source of nutrient supplies that fueled or sustained the bloom. Throughout ongoing efforts to advance modeling and technological capabilities that presently lack reliable predictive capability, bio-optical remote sensing via aerial flyovers along with in-water sensor data can continue to provide accurate coverage of relatively large temporal and spatial features. Flow cytometry can provide conservative (because of some cell lysis), rapid, near-real-time validation of bloom components. The concentration and position of the organisms, along with water mass scalars, can also help to diagnose factors promoting K. brevis bloom development and dispersion.