Using quantitative real-time PCR to study competition and community dynamics among Delaware Inland Bays harmful algae in field and laboratory studies

The Delaware Inland Bays (DIB) have experienced harmful algal blooms of dinoflagellates and raphidophytes in recent years. We used quantitative polymerase chain reaction (QPCR) techniques to investigate the community dynamics of three DIB dinoflagellates (Karlodinium veneficum, Gyrodinium instriatum, and Prorocentrum minimum) and one raphidophyte (Heterosigma akashiwo) at a single site in the DIB (IR-32) in summer 2006 relative to salinity, temperature and nutrient concentrations. We also carried out complementary laboratory culture studies. New primers and probes were developed and validated for the 18S rRNA genes in the three dinoflagellates. K. veneficum, H. akashiwo, and G. instriatum were present in almost all samples throughout the summer of 2006. In contrast, P. minimum was undetectable in late June through September, when temperatures ranged from 20 to 30 °C (average 25.7 °C). Dissolved nutrients ranged from 0.1 to 2.8 μM PO₄³⁻ (median = 0.3 μM), 0.7–30.2 μM NO_x (median = 12.9 μM), and 0–19.4 μM NH₄⁺ (median = 0.7 μM). Dissolved N:P ratios covered a wide range from 2.6 to 177, with a median of 40. There was considerable variability in occurrence of the four species versus nutrients, but in general P. minimum and H. akashiwo were most abundant at higher (>40) N:P ratios and dissolved nitrogen concentrations, while K. veneficum and G. instriatum were most abundant at low dissolved N:P ratios (<20) and dissolved nitrogen concentrations < 10 μM. The semi-continuous laboratory competition experiment used mixed cultures of K. veneficum, P. minimum, and H. akashiwo grown at dissolved N:P ratios of 5, 16, and 25. At an N:P of 16 and 25 P. minimum was the dominant alga at the end of the experiment, even at a temperature that was much higher than that at which this alga was found to bloom in the field (27 °C). P. minimum and H. akashiwo had highest densities in the N:P of 25. K. veneficum grew equally well at all three N:P ratios, and was co-dominant at times at an N:P of 5. H. akashiwo had the lowest densities of the three algae in the laboratory experiment. Laboratory and field results showed both interesting similarities and significant differences in the influences of important environmental factors on competition between these harmful algal species, suggesting the need for more work to fully understand HAB dynamics in the DIB.     

L-arginine increases plasma homocysteine in apoE-/-/iNOS-/- double knockout mice.

Previous studies have shown that L-arginine (L-Arg) administration to apoE-/-/iNOS-/- double knockout mice (dKO) on a Western diet paradoxically results in an increase in atherosclerotic lesion size. We hypothesized that the potential beneficial effects of L-Arg could be offset, in part, by the byproducts of L-Arg catabolism, especially the atherogenic risk factor, homocysteine. In the kidney, L-Arg is converted to L-ornithine and guanidinoacetate (GAA) by L-arginine-glycine amidinotransferase. The efficient transmethylation of GAA by an S-adenosyl-methionine (SAM)-dependent methyltransferase in liver yields creatine and S-adenosylhomocysteine (SAH), which is readily hydrolyzed to homocysteine and adenosine. We, therefore, measured total plasma homocysteine in the dKO mice and control mice. We found that L-Arg supplementation caused a 37% increase in total plasma homocysteine (tHcy) levels in dKO mice compared to controls not treated with L-Arg (5.2+/-2.2 vs 3.8+/-1.5 microM Hcy, p<0.04). In a liver cell line, HepG2, addition of 10 and 50 microM GAA in the presence of 50 microM L-methionine (L-Met) increased tHcy production by approximately 1.47 (p<0.0001) and 2.3-fold (p<0.0001), respectively. In the presence of additional 100 microM L-Met, baseline homocysteine production was elevated by 20% (p<0.005), and 10 and 50 microM GAA augmented homocysteine production by an additional 1.88- (p<0.0001) and 3.4-fold (p<0.001), respectively, compared with 50 microM L-Met. These data suggest that increased concentrations of a methyl acceptor, such as L-Arg-derived GAA, drives SAM-dependent-methylation and consequent homocysteine formation. Furthermore, L-Met levels can also influence homocysteine production likely by regulating the synthesis of the methyl donor SAM. Epidemiological studies have suggested that homocysteine is a graded risk factor. In animal models, modestelevations of homocysteine can cause endothelial dysfunction and augment atherosclerosis. Our data suggest that L-arginine supplementation may contribute to vascular injury and atherogenesis under some circumstances by elevating homocysteine levels.     

An Attempt to Pinpoint the Phylogenetic Introduction of Glutaminyl-tRNA Synthetase Among Bacteria

Until recently it was believed that most Bacteria form Gln-tRNA^GLN by the amidation of Glu-tRNA^GLN, only a few members of the γ subdivision of Proteobacteria being able to charge tRNA^GLN directly. We undertook a phylogenetic study in an attempt to determine at what point the changeover to the direct system may have occurred. To this end, we selected a number of representative Proteobacteria to see if we could find a division point. We constructed degenerate primers and conducted PCR analysis to identify which Bacteria had Gln-tRNA synthetase, on the one hand, and which had the amidotransferase system, on the other. At the same time, we surveyed data banks of completely sequenced microbial genomes, as well as those for genomes in the process of being sequenced. These combined efforts revealed four Proteobacteria in a phylogenetically intermediate position which have the genetic potential for both mechanisms. Perplexingly, however, three distantly related bacteria were also found to have both enzymes. Received: 12 January 1999 / Accepted: 19 July 1999     

Chronic effects of copper exposure versus endocrine toxicity: two sides of the same toxicological process?

Chronic sub-lethal exposure to copper (Cu) causes a series of cellular and physiological changes in fish that enable the animal to survive. Copper is also an endocrine disrupting metal in the aquatic environment, and has a number of normal neuro-endocrine roles in vertebrates. This paper explores whether the chronic effects of Cu exposure can be explained by the effects of Cu on neuro-endocrine functions in fish. Chronic Cu exposure involves complex physiological adjustments in many body systems, including increased oxygen consumption, reduced mean swimming speed, up-regulation of ionic regulation, decreasing lymphocyte levels and increasing neutrophils, altered immunity, modulation of Cu-dependent and independent enzyme activities, and proliferation of epithelial cells in gills or intestine. These responses can occur with exposure via the food or the water and can be rationalised into three major categories: (1) up-regulation of enzymes/metabolism (2) altered haematopoietic responses and (3) altered cellularity (cell type, turnover or size) in tissues. Some of these responses can be explained by stimulation of general stress responses, including the adrenergic response and stimulated cortisol release via the hypothalamic-pituitary-interrenal axis. This can occur despite evidence of vacuolation and foci of necrosis in the brain, and increased macrophage activity, in the kidney of fish exposed to dietary Cu. In addition to generic stress responses, Cu regulates specific neuro-endocrine functions, including the loss of circadian rhythm during dietary Cu exposure that involves the failure to respond to circulating melatonin and a loss of circulating serotonin. We conclude that the chronic physiological effects of Cu and apparent endocrine disrupting effects of Cu are two sides of the same toxicological process.     

A specific segment of the transmembrane domain of Wbp1p is essential for its incorporation into the oligosaccharyl transferase complex

Wbp1p, a type I transmembrane protein, is an essential component of oligosaccharyl transferase (OT), which consists of nine different subunits in yeast. It has been proposed that three subunits, Wbp1p, Ost2p, and Swp1p, physically interact with each other, but the mechanism of these interactions is unknown. To explore the mode of interaction, we have focused on the single-transmembrane protein, Wbp1p, and made several deletions and mutations within the short cytosolic domain and the transmembrane domain. Our results show that the deletion of the cytosolic domain has no effect on cell growth, but mutation of all 17 amino acids in the transmembrane domain to 17 Leu residues or replacement of the transmembrane and cytosolic domains with the counterparts of Ost1p results in lethality. Immunoprecipitation experiments show that Wbp1p mutated in these two ways is not incorporated into the OT complex. This finding suggests that the transmembrane domain of Wbplp may mediate its association with the other subunits. A series of mutations of the transmembrane domain have revealed that block alterations in the half of the transmembrane domain facing the lumen of the endoplasmic reticulum (ER) impaired cell viability. Seven single-Lys mutants in the same domain were temperature sensitive for growth at 37 degrees C. In contrast, block mutations in the other half of the transmembrane domain facing the cytosol did not result in lethality and indicated that this portion of the transmembrane domain was not involved in stable incorporation of Wbp1p into the OT complex.     

Glutathione peroxidase-1 deficiency augments proinflammatory cytokine-induced redox signaling and human endothelial cell activation

Glutathione peroxidase-1 (GPx-1) is a crucial antioxidant enzyme, the deficiency of which promotes atherogenesis. Accordingly, we examined the mechanisms by which GPx-1 deficiency enhances endothelial cell activation and inflammation. In human microvascular endothelial cells, we found that GPx-1 deficiency augments intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression by redox-dependent mechanisms that involve NFκB. Suppression of GPx-1 enhanced TNF-α-induced ROS production and ICAM-1 expression, whereas overexpression of GPx-1 attenuated these TNF-α-mediated responses. GPx-1 deficiency prolonged TNF-α-induced IκBα degradation and activation of ERK1/2 and JNK. JNK or NFκB inhibition attenuated TNF-α induction of ICAM-1 and VCAM-1 expression in GPx-1-deficient and control cells, whereas ERK1/2 inhibition attenuated only VCAM-1 expression. To analyze further signaling pathways involved in GPx-1-mediated protection from TNF-α-induced ROS, we performed microarray analysis of human microvascular endothelial cells treated with TNF-α in the presence and absence of GPx-1. Among the genes whose expression changed significantly, dual specificity phosphatase 4 (DUSP4), encoding an antagonist of MAPK signaling, was down-regulated by GPx-1 suppression. Targeted DUSP4 knockdown enhanced TNF-α-mediated ERK1/2 pathway activation and resulted in increased adhesion molecule expression, indicating that GPx-1 deficiency may augment TNF-α-mediated events, in part, by regulating DUSP4.     

Alveolate phylogeny inferred using concatenated ribosomal proteins

Dinoflagellates and apicomplexans are a strongly supported monophyletic group in rDNA phylogenies, although this phylogeny is not without controversy, particularly between the two groups. Here we use concatenated protein-coding genes from expressed sequence tags or genomic data to construct phylogenies including "typical" dinophycean dinoflagellates, a parasitic syndinian dinoflagellate, Amoebophrya sp., and two related species, Oxyrrhis marina, and Perkinsus marinus. Seventeen genes encoding proteins associated with the ribosome were selected for phylogenetic analysis. The dataset was limited for the most part by data availability from the dinoflagellates. Forty-five taxa from four major lineages were used: the heterokont outgroup, ciliates, dinoflagellates, and apicomplexans. Amoebophrya sp. was included in this phylogeny as a sole representative of the enigmatic marine alveolate or syndinian lineage. The atypical dinoflagellate O. marina, usually excluded from rDNA analyses due to long branches, was also included. The resulting phylogenies were well supported in concatenated analyses with only a few unstable or weakly supported branches; most features were consistent when different lineages were pruned from the tree or different genes were concatenated. The least stable branches involved the placement of Cryptosporidium spp. within the Apicomplexa and the relationships between P. marinus, Amoebophrya sp., and O. marina. Both bootstrap and approximately unbiased test results confirmed that P. marinus, Amoebophrya sp., O. marina, and the remaining dinoflagellates form a monophyletic lineage to the exclusion of Apicomplexa.     

On the distribution of Na+ pump sites in the frog skin

Exposure of the outside of the isolated frog skin to a Ringer's solution, made hypertonic by the addition of mannitol, causes a rapid and sustained increase in transepithelial permeability through a structural distortion-a focal blistering-of the "tight" junctions of the outermost living cell layer. [(3)H]ouabain, used as an autoradiographic marker for the Na+-pump (Na+-K+-adenosine triphosphatase), is usually unable to penetrate the frog skin from the outside solution, but when added to a hypertonic mannitol- Ringer's solution in the outside bath it readily penetrates the epithelium, presumably through the opened shunt pathway. Radioautographic analysis of [(3)H]ouabain binding sites revealed that most of ouabain enters from the outside solution binds to the sites on the cell membranes of the stratum spinosum, as was the case when it was applied from the inside bath in an earlier study. The outer living cell layer, the first to be exposed to ouabain, does not appear to be the major site for the Na+-pump, and therefore, is not likely to be responsible for most of the active pumping of Na+. This result demonstrates that previous failure to show a high density of Na+-pump sites on the cells of the outermost layer, when [(3)H]ouabain was applied from the inside solution, was not due to the inability of the marker to reach these cells at a sufficient concentration to reveal all pump sites. These results provide further support for a model of Na+-transport across the frog skin which distributes the active pump step on the inward facing membranes of all living cells.     

Cdc37 has distinct roles in protein kinase quality control that protect nascent chains from degradation and promote posttranslational maturation

Cdc37 is a molecular chaperone that functions with Hsp90 to promote protein kinase folding. Analysis of 65 Saccharomyces cerevisiae protein kinases ( approximately 50% of the kinome) in a cdc37 mutant strain showed that 51 had decreased abundance compared with levels in the wild-type strain. Several lipid kinases also accumulated in reduced amounts in the cdc37 mutant strain. Results from our pulse-labeling studies showed that Cdc37 protects nascent kinase chains from rapid degradation shortly after synthesis. This degradation phenotype was suppressed when cdc37 mutant cells were grown at reduced temperatures, although this did not lead to a full restoration of kinase activity. We propose that Cdc37 functions at distinct steps in kinase biogenesis that involves protecting nascent chains from rapid degradation followed by its folding function in association with Hsp90. Our studies demonstrate that Cdc37 has a general role in kinome biogenesis.