Vertical Migration and Mortality of Marine Benthos in Dredged Material: A Synthesis

This account describes the comparative response of four species of benthic invertebrates to burial in terms of vertical migration and mortality, and provides a synthesis of studies and recommendations upon which to assess future impacts. The species featured were the bivalve Mercenaries mercenaria, the amphipod crustacean Parahaustorius longimerus, and the polychaetes Scoloplos fragilis and Nereis succinea. There was evidence of synergistic effects on burrowing activity and mortality with changes in time of burial, sediment depth, sediment type and temperature. In sediment with silt-clay, N. succinea was the most resistant species followed by M. mercenaria, S. fragilis and P. longimerus. In sediment without silt-clay the order of percent mortality was reversed. Studies of surface water chemistry and sediment geochemistry showed that dissolved oxygen decreased significantly and ammonia and sulfide increased significantly between the surface and below 2.0 cm within a 15-day period. Based on these results and physiological tolerances from the literature it was concluded that M. mercenaria and N. succinea would be relatively resistant to chemical effects of spoil disposal, whereas S. fragilis and P. longimerus would be less resistant to such effects. Vertical migration of benthic invertebrates through dredge disposal can be a viable mechanism of recolonization under certain conditions. Some effects of burial of benthos can be predicted based on morphology, behavior and physiology. These biological features were discussed with examples dealing with molluscs, crustaceans, and polychaetes. Finally, recommendations were made concerning the type of studies to provide additional data to aid management agencies in decision making about future dredging and disposal practices.     

Enhancement of Survival and Electricity Production in an Engineered Bacterium by Light-Driven Proton Pumping

Microorganisms can use complex photosystems or light-dependent proton pumps to generate membrane potential and/or reduce electron carriers to support growth. The discovery that proteorhodopsin is a light-dependent proton pump that can be expressed readily in recombinant bacteria enables development of new strategies to probe microbial physiology and to engineer microbes with new light-driven properties. Here, we describe functional expression of proteorhodopsin and light-induced changes in membrane potential in the bacterium Shewanella oneidensis strain MR-1. We report that there were significant increases in electrical current generation during illumination of electrochemical chambers containing S. oneidensis expressing proteorhodopsin. We present evidence that an engineered strain is able to consume lactate at an increased rate when it is illuminated, which is consistent with the hypothesis that proteorhodopsin activity enhances lactate uptake by increasing the proton motive force. Our results demonstrate that there is coupling of a light-driven process to electricity generation in a nonphotosynthetic engineered bacterium. Expression of proteorhodopsin also preserved the viability of the bacterium under nutrient-limited conditions, providing evidence that fulfillment of basic energy needs of organisms may explain the widespread distribution of proteorhodopsin in marine environments.Classic experiments in microbial bioenergetics used light-driven reactions from halobacterial bacteriorhodopsin or the photosynthetic reaction center to provide a temporary driving force for understanding transport and chemiosmotic coupling (6, 7, 19, 35). However, light-driven reactions have not been used in metabolic engineering to alter microbial physiology and production of chemicals. The recent discovery of proteorhodopsin (PR) in ocean microorganisms and the ease with which this membrane protein can be functionally expressed by recombinant bacteria have made possible many engineering strategies previously not available (1, 16). In this paper, we describe progress toward the goal of integrating light-driven reactions with biocatalysis.In contrast to the situation for established industrial microorganisms, such as Escherichia coli, our current understanding of less-studied algal and phototrophic bacteria may limit metabolic engineering strategies which require genetic manipulation. Metabolic engineering strategies using photosynthetic bacteria have focused largely on methods to increase hydrogen production, and improvements rely mainly on engineering of nitrogenase and hydrogenase to produce H₂. Algae appear to be suited to large-scale cultivation for lipid production, but so far little has been done to engineer these organisms (36). In principle, platform microbial hosts capable of producing a diverse range of products could be boosted by addition of light-driven processes from phototrophic metabolism.To demonstrate the feasibility of transferring a light-driven process into a nonphotosynthetic bacterium, we chose to study proteorhodopsin (PR) first because it is one of the simplest mechanisms for harnessing the energy from light. The proteorhodopsins are a group of transmembrane proteins that use the light-induced isomerization of retinal, the oxidative cleavage product of the carotenoid β-carotene, either to initiate signaling pathways or to catalyze the transfer of ions across cell membranes (8). PR was discovered by metagenomic analysis of marine samples (1) and is related to the well-studied bacteriorhodopsin of archaea (33) and rhodopsin (34), a eukaryotic light-sensing protein. The membrane potential generated by light-driven proton pumping by PR has been confirmed to drive ATP synthesis in a heterologous system (25). However, bacteria expressing heterologous PR were shown not to benefit from this pumping activity, as no significant increases in growth rates were observed (9). This led to the suggestion that PR may benefit the organism only under starvation conditions. In agreement with this hypothesis, Gomez-Consarnau et al. (10) have reported that the light-dependent growth rates of a marine flavobacterium that has a native PR are increased only when the organism is cultured under energy-limited conditions.Studies of both native and recombinant systems in which rhodopsins are expressed have generated light-dependent membrane potentials. In membrane vesicles isolated from a native host, the light-dependent membrane potential generated by bacteriorhodopsin provides the driving force for ATP synthesis (35) and uptake of leucine and glutamate (20, 22). More recently, studies of recombinant systems have coupled the membrane potential to other transport processes. In one example, the membrane potential-dependent export of specific toxic molecules increased when E. coli cells expressing both an archaeal rhodopsin and a specific efflux pump were exposed to light (17). In another experiment, starved E. coli cells expressing PR increased the swimming motion of their flagella when they were illuminated (44). Based upon measurements of flagellar motion as a function of light intensity and azide concentration, the proton motive force generated by PR was estimated to be −0.2 V, a value similar to the value for aerobic respiration in E. coli (42).As a nonphotosynthetic host for recombinant PR expression, we chose the dissimilatory metal-reducing bacterium Shewanella oneidensis strain MR-1, which is genetically tractable for engineering and is able to use a variety of terminal electron acceptors, including insoluble metal oxides (11, 30). Key to the ability of this bacterium to reduce metal oxides is a multicomponent extracellular respiratory pathway that transports electrons from menaquinol to cytochromes in the outer membrane. This pathway is composed of a cytoplasmic membrane tetraheme protein (CymA), a periplasmic decaheme protein (MtrA), an integral outer membrane protein (MtrB), and a decaheme lipoprotein (MtrC) that is associated with MtrB (14, 37, 40). The ability of S. oneidensis to reduce extracellular metal oxides has made it possible to harvest electrons from this organism by coupling it to an electrode which serves as the electron acceptor (21). The electron flow to the outer surface allows respiration rates to be measured directly by electrochemistry.In the current work, we introduced PR into an electricity-generating bacterium, S. oneidensis strain MR-1, and demonstrated that there was integration of a light-driven process into the metabolism of a previously nonphotosynthetic organism that resulted in a useful output. We show here that PR allows cells to survive for extended periods in stationary phase and that the presence of light results in an increase in electricity generation. A possible physiological model to explain these effects is discussed.     

Stimulation of oncogenic metabotropic glutamate receptor 1 in melanoma cells activates ERK1/2 via PKCepsilon

Metabotropic glutamate receptor 1 (Grm1, formerly mGluR1) is a G protein coupled receptor (GPCR) normally expressed and functional in the central nervous system. Studies of our transgenic mouse melanoma model (TG-3) revealed that ectopic expression of Grm1 in melanocytes is sufficient to induce melanoma development in vivo [P.M. Pollock, K. Cohen-Solal, R. Sood, J. Namkoong, J.J. Martino, A. Koganti, H. Zhu, C. Robbins, I. Makalowska, S.S. Shin, Y. Marin, K.G. Roberts, L.M. Yudt, A. Chen, J. Cheng, A. Incao, H.W. Pinkett, C.L. Graham, K. Dunn, S.M. Crespo-Carbone, K.R. Mackason, K.B. Ryan, D. Sinsimer, J. Goydos, K.R. Reuhl, M. Eckhaus, P.S. Meltzer, W.J. Pavan, J.M. Trent, S. Chen, Nat. Genet. 34 (2003) 108-112.]. We have established and characterized several cell lines in vitro from independent mouse melanoma tumors [Y.E. Marín, J. Namkoong, S.S. Shin, J. Raines, K. Degenhardt, E. White, S. Chen, Neuropharmacol. 49 (2005) 70-79.]. These cell lines are useful tools in the studies of signaling events that may be mediated by Grm1 in transformed melanocytes. Here we show that stimulation of Grm1 by l-quisqualate, a group I metabotropic glutamate receptor agonist, results in inositol triphosphate (IP3) accumulation, and the activation of ERK1/2 in these cell lines. IP3 accumulation and ERK1/2 activation were inhibited by pretreatment of the tumor cells with a Grm1-specific antagonist (LY367385) or by dominant negative mutants of Grm1, demonstrating the specificity of these events. We also show that ERK1/2 activation by Grm1 was PKC-dependent, but cAMP and PKA-independent. PKCepsilon was shown to play a pivotal role in Grm1-mediated ERK1/2 phosphorylation. Insights into the signaling cascades mediated by Grm1 in melanoma cells may aid in the identification of key molecular targets for the future design of combined therapies for melanoma.     

Circulating Insulin Concentrations,Smoking, and Alcohol Intake Are Important Independent Predictors of Leptin in Young Healthy Men

Objective : Leptin, an adipocyte-secreted hormone, has been shown to signal the status of energy stores to the brain, regulate energy homeostasis, and mediate the neuroendocrine response to food deprivation. Obesity is associated with increased leptin levels, and several hormones, including insulin and glucocorticoids, have been associated with leptin levels and expression in rodents. Although obesity has been strongly associated with increased leptin in humans, a significant percentage of leptin's variability remains unexplained. The role of endogenous hormones, demographic factors, or certain life-style factors in explaining the residual variability of leptin levels has not yet been clarified. We performed this cross-sectional study to document the relative importance of obesity, lifestyle factor, and endogenous hormones in determining serum leptin levels. Research Methods and Procedures : We measured serum concentrations of insulin, Cortisol, testosterone, growth hormone, and dehydroepiandrosterone sulfate; ascertained anthropometric, demographic, and lifestyle characteristics; and studied these variables in relationship to serum leptin concentrations in a sample of young healthy men. Results : Obesity and alcohol intake were independently and positively associated with circulating leptin concentrations. Additionally, cigarette smoking was negatively and independently associated with leptin concentrations. Finally, serum insulin concentration was an independent hormonal determinant of circulating leptin concentrations, whereas serum testosterone was negatively associated with leptin only by bivariate analysis. Discussion : We conclude that, in addition to obesity, cigarette smoking, alcohol intake, and serum insulin levels are associated with leptin levels in a population of healthy young men.     

Tuberculosis masquerading as malignancy: a multimodality approach to the correct diagnosis – a case report

BACKGROUND: Extrapulmonary tuberculosis is one of the great mimickers of medicine, and often masquerades as malignancy. As a result, patients may be referred to oncologists and surgeons for further evaluation and management, delaying the institution of appropriate anti-tuberculous drug therapy. CASE PRESENTATION: We present the case of a 21 year old man with tuberculous osteomyelitis, who was referred to the Bone and Soft Tissue Sarcoma Service at our institution with a provisional diagnosis of malignancy. Further investigation revealed extensive retroperitoneal abdominal and pelvic lymphadenopathy. The recognition of certain patterns on imaging, and finally the isolation of Mycobacterium tuberculosis from tissue samples obtained under image guidance, enabled the correct diagnosis to be made. CONCLUSION: This case highlights the importance of remaining cognisant of the protean manifestations of extrapulmonary tuberculosis, and illustrates the advantage of a clinically directed multi-modality imaging approach to diagnosis.     

FK506 binding protein 8 peptidylprolyl isomerase activity manages a late stage of cystic fibrosis transmembrane conductance regulator (CFTR) folding and stability

Cystic fibrosis (CF) is caused by mutations in the apical chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) with 90% of patients carrying at least one deletion of the F508 (ΔF508) allele. This mutant form of CFTR is characterized by a folding and trafficking defect that prevents exit from the endoplasmic reticulum. We previously reported that ΔF508 CFTR can be recovered in a complex with Hsp90 and its co-chaperones as an on-pathway folding intermediate, suggesting that Δ508 CF disease arises due to a failure of the proteostasis network (PN), which manages protein folding and degradation in the cell. We have now examined the role of FK506-binding protein 8 (FKBP8), a component of the CFTR interactome, during the biogenesis of wild-type and ΔF508 CFTR. FKBP8 is a member of the peptidylprolyl isomerase family that mediates the cis/trans interconversion of peptidyl prolyl bonds. Our results suggest that FKBP8 is a key PN factor required at a post-Hsp90 step in CFTR biogenesis. In addition, changes in its expression level or alteration of its activity by a peptidylprolyl isomerase inhibitor alter CFTR stability and transport. We propose that CF is caused by the sequential failure of the prevailing PN pathway to stabilize ΔF508-CFTR for endoplasmic reticulum export, a pathway that can be therapeutically managed.     

Fast Diffusion of Very Long Chain Saturated Fatty Acids across a Bilayer Membrane and Their Rapid Extraction by Cyclodextrins: IMPLICATIONS FOR ADRENOLEUKODYSTROPHY*

Abnormalities in the transport of saturated very long chain fatty acids (VLCFA; >C18:0) contribute to their toxic levels in peroxisomal disorders of fatty acid metabolism, such as adrenoleukodystrophy and adrenomyeloneuropathy. We previously showed that VLCFA desorb much slower than normal dietary fatty acids from both albumin and protein-free lipid bilayers. The important step of transbilayer movement (flip-flop) was not measured directly as a consequence of this very slow desorption from donors, and the extremely low aqueous solubility of VLCFA precludes addition of unbound VLCFA to lipid membranes. We have overcome these limitations using methyl-β-cyclodextrin to solubilize VLCFA for rapid delivery to “acceptor” phosphatidylcholine vesicles (small and large unilamellar) and to cells. VLCFA binding was monitored in real time with the fluorescent probe fluorescein-labeled phosphatidylethanolamine in the outer membrane leaflet, and entrapped pyranine was used to detect flip-flop across the membrane. The upper limit of the rate of flip-flop across the membrane was independent of temperature and media viscosity and was similar for model raft and non-raft membranes as well as living cells. We further showed that cyclodextrins can extract VLCFA rapidly (within seconds) from vesicles and cells, which have implications for the mechanism and potential alternative approaches to treat adrenoleukodystrophy. Because VLCFA diffuse through the lipid bilayer, proteins may not be required for their transport across the peroxisomal membrane.     

A new species of Trichogramma (Hymenoptera: Trichogrammatidae) parasitic on eggs of the alderfly Sialis melania (Neuroptera: Sialidae) from Japan,with comments on its phylogeny and male wing polymorphism

A new species of Trichogramma that parasitizes Sialis melania eggs is described as Trichogramma tajimaense Yashiro, Hirose and Honda, sp. nov. from Japan. Its phylogenetic position is based on a DNA‐based analysis, and data regarding its male wing polymorphism are also presented. The view that T. tajimaense is closely related to T. semblidis, another parasitoid of Sialis eggs, is supported by the results of a phylogenetic analysis, as well as by the biological and morphological similarities between both species. Trichogramma tajimaense is also similar in male wing polymorphism to T. kurosuae, a gregarious egg parasitoid of the lepidopteran Ivela auripes, as both Trichogramma species exhibit male wing trimorphism (fully alate, brachypterous and apterous forms) in contrast to the male wing dimorphism (fully alate and apterous forms) of T. semblidis. However, no phylogenetic analysis reveals a close relationship between T. tajimaense and T. kurosuae, and a difference exists between these two species in the mean percentage of flightless (brachypterous and apterous) males that emerge from a host egg mass; 96% of T. tajimaense males are incapable of flight, whereas about 50% of T. kurosuae males are flightless. Because all or almost all males of T. semblidis parasitizing Sialis eggs are apterous, T. tajimaense is more similar to T. semblidis than to T. kurosuae in the proportion of flightless males. In addition, male wing polymorphisms of Trichogramma in relation to mating systems could also show a similarity between T. tajimaense and T. semblidis when considering both species as quasi‐gregarious parasitoids of Sialis eggs.     

Poliovirus entry into human brain microvascular cells requires receptor-induced activation of SHP-2

Viruses use specific receptor molecules to bind selectively to target cells. Receptors have often been considered as mere docking sites, but they may also possess intrinsic signaling capacities that serve to prime the cell for entry and infection. Poliovirus (PV) initiates infection by binding to the PV receptor (PVR) and causes paralytic poliomyelitis by replicating within motor neurons of the brain and spinal cord. We have examined the process by which PV enters cultured human brain microvascular endothelial cells (HBMEC), an in vitro model of the blood-brain barrier. We found that PV enters HBMEC by dynamin-dependent caveolar endocytosis, and that entry depends on intracellular signals triggered by virus attachment to PVR. Tyrosine kinase and RhoA GTPase activation initiated by PVR ligation were both essential. Virus attachment also induced tyrosine phosphorylation of PVR; this permitted the association of PVR with SHP-2, a protein tyrosine phosphatase whose activation was required for entry and infection. The results indicate that receptor-induced signals promote virus entry and suggest a role for tyrosine phosphatases in viral pathogenesis.     

Amelioration of Bacterial Genomes: Rates of Change and Exchange

Although bacterial species display wide variation in their overall GC contents, the genes within a particular species' genome are relatively similar in base composition. As a result, sequences that are novel to a bacterial genome—i.e., DNA introduced through recent horizontal transfer—often bear unusual sequence characteristics and can be distinguished from ancestral DNA. At the time of introgression, horizontally transferred genes reflect the base composition of the donor genome; but, over time, these sequences will ameliorate to reflect the DNA composition of the new genome because the introgressed genes are subject to the same mutational processes affecting all genes in the recipient genome. This process of amelioration is evident in a large group of genes involved in host-cell invasion by enteric bacteria and can be modeled to predict the amount of time required after transfer for foreign DNA to resemble native DNA. Furthermore, models of amelioration can be used to estimate the time of introgression of foreign genes in a chromosome. Applying this approach to a 1.43-megabase continuous sequence, we have calculated that the entire Escherichia coli chromosome contains more than 600 kb of horizontally transferred, protein-coding DNA. Estimates of amelioration times indicate that this DNA has accumulated at a rate of 31 kb per million years, which is on the order of the amount of variant DNA introduced by point mutations. This rate predicts that the E. coli and Salmonella enterica lineages have each gained and lost more than 3 megabases of novel DNA since their divergence. Received: 7 July 1996 / Accepted: 27 September 1996