3H-leucine Incorporation Research Articles

Diel changes in bulk and single‐cell bacterial heterotrophic activity in winter surface waters of the northwestern Mediterranean Sea

Two diel cycle studies were conducted to determine the effect of day—night light changes on winter bacterial activity in the coastal Mediterranean (Blanes Bay Microbial Observatory). Bacterial abundances, bacterial heterotrophic activity, and flagellate grazing counts were determined at 4‐h intervals during two 3‐d periods separated by 2 d. Twice a day, the single‐cell activity of major bacterial groups was further analyzed by applying microautoradiography combined with catalyzed reporter deposition—fluorescence in situ hybridization. During the first cycle, all the measured parameters (including the number of active cells in most groups) varied synchronously, with higher values at night and lower during the day. An episode of strong winds between the two studied periods disrupted this periodicity at the onset of the second cycle. The bulk incorporation of 3H‐leucine recovered the diel pattern after 2 d, mostly driven by the activity of Gammaproteobacteria. Among the possibilities considered, the observed nighttime increases in the grazing activity of heterotrophic nanoflagellates appeared to partially drive the activity of all bacterial taxa through potential periodic release of dissolved organic matter.

Mercury and flooding cycles in the Tapajós river basin, Brazilian Amazon: The role of periphyton of a floating macrophyte ( Paspalum repens)

Methylmercury (MeHg) increases mercury (Hg) toxicity and is biomagnified in the trophic chain contaminating riverine Amazon populations. Freshwater macrophyte roots are a main site of Hg methylation in different Brazilian environments. Paspalum repens periphyton was sampled in four floodplain lakes during the dry, rainy and wet seasons for measurement of total Hg (THg), MeHg, Hg methylation potentials, %C, %N, δ 13C, δ 15N and bacterial heterotrophic production as 3H-leucine incorporation rate. THg concentration varied from 67 to 198 ng/g and the potential of Me 203Hg formation was expressive (1–23%) showing that periphyton is an important matrix both in the accumulation of Hg and in MeHg production. The concentration of MeHg varied from 1 to 6 ng/g DW and was positively correlated with Me 203Hg formation. Though methylmercury formation is mainly a bacterial process, no significant correlation was observed between the methylation potentials and bacterial production. The multiple regressions analyses suggested a negative correlation between THg and %C and %N and between methylation potential and δ 13C. The discriminant analysis showed a significant difference in periphyton δ 15N, δ 13C and THg between seasons, where the rainy season presented higher δ 15N and the wet period lighter δ 13C, lower THg values and higher Me 203Hg formation. This exploratory study indicates that the flooding cycle could influence the periphyton composition, mercury accumulation and methylmercury production.

Large-scale distribution and production of bacterioplankton in the Adriatic Sea

Two oceanographic cruises encompassing the whole Adriatic Sea were carried out during February and October 2008. Selected stations were sampled at several depths to determine total prokaryotes and picocyanobacteria abundance using epifluorescence microscopy, and to estimate prokaryotic carbon production by 3H-leucine incorporation. Biological data were related to physical parameters including temperature, salinity and fluorescence, and an attempt to associate bacterial dynamics to water mass characteristics was performed. In both seasons prokaryotic distribution and production showed a decreasing latitudinal gradient likely dependent on riverine inputs highlighted by a strong negative correlation with salinity ( P < 0.001). A vertical gradient with higher cell numbers at the surface and lower values at the bottom layer was also always detected. In the southern basin in February, however, picocyanobacteria were retrieved also in deep waters, probably linked to higher nutrient loads carried by the Levantine Intermediate Waters and/or the deep water ventilation known to occur in this area. From an oceanographic point of view, we sampled within four different water types, but no relationship between these water types and bacterioplankton abundances was found. The present work contributes to the acquisition of a more holistic overview of prokaryotic distribution and production in the Adriatic Sea, both on a spatial and temporal scale.

Enhanced Expression of G(alpha)q and PLCβ1 in Aortic Vascular Smooth Muscle Cells from Spontaneously Hypertensive Rats: Role of Endogenous Angiotensine II and Endothelin‐1

Gαq and Gαq associated signaling play an important role in the regulation of vascular tone and cardiac hypertrophy. We have earlier shown that hyperglycemia, induced enhanced expression of Gαq and PLCβ proteins in vscular smooth muscle cells (VSMC) which was attributed to the endogenous levels of angiotensin II (Ang II) and endothelin‐1 (ET‐1). The present study was undertaken to investigate if the expression of Gαq and PLCβ1 proteins is upregulated in aortic VSMC from 16 week‐old spontaneously hypertensive rats (SHR) that has been shown to exhibit cardiac hypertrophy and to further explore the implication of endogenous Ang II and ET‐1 in this upregulation. The expression of Gαq and PLCβ proteins in VSMC was determined by Western Blotting analysis, whereas vascular hypertrophy and prolifeartion was determined by 3H‐leucine and 3H‐thymidine incorporation respectively. The expression of Gαq and PLCβ1 proteins was significantly increased in VSMC from 16‐week old SHR and not from 12‐week old SHR as compared to their age mached WKY rats. The incresead expression of Gαq and PLCβ1 was attenuated by AT1 receptor antagonist, losartan, and ETA receptor antagonist, BQ123, but not by ETB receptor antagonist, BQ788. Protein and DNA synthesis was significantly augmented in VSMC from 16 week‐old SHR as compared to WKY rats. However, the antisense of Gαq attenuated the hypertrophy but not the prolifeartion. These results suggest that VSMC from 16‐week old SHR exhibit enhanced expression of Gαq and PLCβ1 which may be attributed to enhanced levels of Ang II and ET‐1 and may be responsible for the vascular hypertrophy observed in 16 week‐ald SHR.

Adenovirus‐mediated overexpression of cardiac troponin I‐interacting kinase promotes cardiomyocyte hypertrophy

1. Cardiac troponin I-interacting kinase (TNNI3K) is a novel cardiac-specific kinase gene. Quantitative real-time reverse transcription polymerase chain reaction analysis showed a significant increase in TNNI3K mRNA expression in hypertrophic cardiomyocytes induced by endothelin-1 (ET-1). The aim of the present study was to investigate the effects of TNNI3K on neonate rat cardiomyocyte hypertrophy induced by ET-1. 2. Adenoviruses were amplified in 293A cells. To determine a reasonable adenovirus infection dose cardiomyocytes were infected with an adenovirus carrying human TNNI3K (Ad-TNNI3K) at varying multiplicity of infection (MOI) and the expression of TNNI3K was analysed by western blot. 3. Cardiomyocytes were infected with either a control adenovirus carrying green fluorescent protein (Ad-GFP) or Ad-TNNI3K. Compared with Ad-GFP, the Ad-TNNI3K induced an increase in sarcomere organization, cell surface area, (3) H-leucine incorporation and β-MHC re-expression. This type of hypertrophic phenomenon is similar to that observed in Ad-GFP-infected hypertrophic cardiomyocytes induced by ET-1. To determine the functional role of TNNI3K in ET-1-induced hypertrophic cardiomyocytes, the cells were infected with Ad-GFP or Ad-TNNI3K. Ad-TNNI3K induced an increase in sarcomere organization, cell surface area and (3) H-leucine incorporation compared with Ad-GFP. 4. These results suggest that TNNI3K overexpression induces cardiomyocytes hypertrophy and accelerates hypertrophy in hypertrophic cardiomyocytes. Therefore, TNNI3K might be an interesting target for the clinical treatment of hypertrophy.

Diversity in UV sensitivity and recovery potential among bacterioneuston and bacterioplankton isolates

To assess the variability in UV-B (280-320 nm) sensitivity of selected bacterial isolates from the surface microlayer and underlying water of the Ria de Aveiro (Portugal) estuary and their ability to recover from previous UV-induced stress. Bacterial suspensions were exposed to UV-B radiation (3·3 W m⁻²). Effects on culturability and activity were assessed from colony counts and (3) H-leucine incorporation rates, respectively. Among the tested isolates, wide variability in UV-B-induced inhibition of culturability (37·4-99·3%) and activity (36·0-98·0%) was observed. Incubation of UV-B-irradiated suspensions under reactivating regimes (UV-A, 3·65 W m⁻²; photosynthetic active radiation, 40 W m⁻²; dark) also revealed diversity in the extent of recovery from UV-B stress. Trends of enhanced resistance of culturability (up to 15·0%) and enhanced recovery in activity (up to 52·0%) were observed in bacterioneuston isolates. Bacterioneuston isolates were less sensitive and recovered more rapidly from UV-B stress than bacterioplankton isolates, showing enhanced reduction in their metabolism during the irradiation period and decreased culturability during the recovery process compared to bacterioplankton. UV exposure can affect the diversity and activity of microbial communities by selecting UV-resistant strains and alter their metabolic activity towards protective strategies.

Response of the microbial community to coral spawning in lagoon and reef flat environments of Hawaii, USA

The response of the microbial community to coral spawning was investigated over a period of 18 mo, from January 2006 to July 2007, in reef flat and lagoon environments of a sub- tropical embayment (Kaneohe Bay, Oahu, Hawaii, USA). The composition of the bacterioplankton community was characterized using terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial small-subunit (SSU) ribosomal RNA genes in parallel with measurements of microbial cell abundances, bacterial production via 3 H-leucine incorporation, and seawater bio- chemical parameters. We observed a variable bacterioplankton community structure and 2- to 3-fold changes in the cellular abundance of microorganisms, concentrations of chlorophyll a, and rates of bacterial carbon production at both sites during non-spawning conditions. While shifts in the structure of the bacterioplankton community were evident for both environments following coral spawning, microbial abundances and rates of bacterial production remained largely unchanged from pre-spawning levels. Thus, it appeared that only a small fraction of the microbial community responded to the presence of coral-produced organic matter. Differences in the composition of the bacterioplankton community, cellular abundances of microorganisms, and rates of bacterial produc- tion were evident between the lagoon and reef flat sites during non-spawning conditions, probably signifying the importance of the surface flow regime for coastal reef microbial communities. Our observations indicate that the Kaneohe Bay microbial community may be more significantly affected by physical mixing processes than by organic matter loading from coral spawning.

Flow cytometric assessment of specific leucine incorporation in the open Mediterranean

Abstract. The surface of the Mediterranean Sea is a low-phosphate-low-chlorophyll marine area where marine heterotrophic prokaryotes significantly contribute to the biogeochemical cycles of all biogenic elements such as carbon, notably through the mineralization of dissolved organic compounds. Cell-specific leucine incorporation rates were determined in early summer in the open stratified Mediterranean Sea. The bulk leucine incorporation rate was on average 5 ± 4 pmol leu l−1 h−1 (n=30). Cell-specific 3H-leucine incorporation rates were assayed using flow cytometry coupled to cell sorting. Heterotrophic prokaryotes (Hprok) were divided into cytometric groups according to their side scatter and green fluorescence properties: high nucleic acid containing cells (HNA) with high scatter (HNA-hs) and low scatter (HNA-ls) and low nucleic acid containing cells (LNA). Cell-specific leucine incorporation rates of these cytometric groups ranged from 2 to 54, 0.9 to 11, and 1 to 12 × 10-21 mol cell−1 h−1, respectively. LNA cells represented 45 to 63% of the Hprok abundance, and significantly contributed to the bulk leucine incorporation rates, from 12 to 43%. HNA/LNA ratios of cell-specific leucine incorporation were on average 2.0 ± 0.7 (n=30). In surface layers (from 0 m down to the deep chlorophyll depth, DCM), cell-specific rates of HNA-hs were elevated (7 and 13 times greater than LNA and HNA-ls, respectively). Nevertheless, on average HNA-hs (26%) and LNA (27%) equally contributed to the bulk leucine incorporation in these layers. Prochlorococcus cells were easily sorted near the DCM and displayed cell-specific leucine incorporation rates ranging from 3 to 55 × 10-21 mol leu cell−1 h−1, i.e. as high as HNA-hs'. These sorted groups could therefore be defined as key-players in the process of leucine incorporation into proteins. The mixotrophic features of certain photosynthetic prokaryotes and the high contribution of LNA cells to leucine incorporation within the microbial communities of the Mediterranean could be reinforced.

Targeted Inhibition of mTORC1 and mTORC2 by the Active-Site mTOR Inhibitors, PP-242 and OSI-027, Has Cytotoxic Effects In T-Cell Acute Lymphoblastic Leukemia

AbstractAbstract 3242T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia characterized by uncontrolled proliferation of lymphoblasts committed to the T-cell lineage. Despite improvements in therapy, 25% of children and 50%–70% of adults with T-ALL relapse and develop a polychemotherapy-resistant disorder, which carries a poor prognosis. Thus, major efforts are being made to develop targeted molecules against deregulated signaling pathways that sustain T-ALL cell growth and survival. The mTOR serine/threonine kinase belongs to two separate multi-protein complexes, referred to as mTORC1 and mTORC2. The mTORC1 complex, which is inhibited by rapamycin/rapalogs, controls mRNA translation through the phosphorylation of the translation repressor 4E-BP1, while mTORC2 could be involved in regulating proliferation and survival through phosphorylation of Akt on Ser 473. mTORC2 is usually considered to be rapamycin-resistant. Both mTORC1 and mTORC2 are activated in most of T-ALL patients underscoring these complexes as major targets for T-ALL treatment. However, the allosteric inhibition of mTORC1 by rapamycin/rapalogs has only modest effects in T-ALL cells. This could be due, among other things, to the fact that rapamycin/rapalogs are mainly cytostatic and do not dephosphorylate 4E-BP1 in preclinical models of T-ALL. Recently, however, ATP-competitive inhibitors specific for the mTOR kinase active site have been synthesized. These compounds target both mTORC1 and mTORC2 and are more active than rapamycin/rapalogs against p-4E-BP1. Here, we have explored the therapeutic potential of two active-site mTOR inhibitors, PP-242 and OSI-027, against both T-ALL cell lines and primary samples from T-ALL patients displaying activation of mTORC1 and mTORC2. OSI-027 is being now tested in clinical trials in patients with solid malignancies. PP-242 and OSI-027 affected T-ALL cell line viability as documented by MTT assays. IC50 for PP-242 ranged from 0.15 to 0.50 μM at 24 hours, whereas OSI-027 was less potent (IC50 ranging from 0.6 to 1.3 μM at 48 h). Of note, both the drugs were effective against CEM T-ALL cells overexpressing 170-kDa P-glycoprotein, one of the major determinants of drug-resistance. Overall, PP-242 was slightly more powerful than the dual PI3K/mTOR inhibitor, PI-103, whose IC50 ranged from 0.2 to 1.0 μM. When T-ALL patient samples were examined, PP-242 displayed an IC50 of 0.5–1.0 μM, while the IC50 for OSI-027 ranged between 2.3 and 4.8 μM. Both the inhibitors affected to a much lower extent the proliferation of peripheral blood T-lymphocytes from healthy donors stimulated with phytohemagglutinin and interleukin-2. Remarkably, PP-242 targeted the side population of T-ALL cell lines, which might correspond to leukemic stem cells. The drugs induced both cell cycle arrest in G0/G1 phase and apoptosis, as documented by flow cytometric analysis, western blot analysis for cleaved caspase-3, and transmission electron microscopy. Western blot analysis demonstrated that the inhibitors induced a dose- and time-dependent dephosphorylation of Akt on Ser 473 (indicative of mTORC2 inhibition) as well as a dephosphorylation of mTORC1 downstream targets including Thr 389 p70S6 kinase, Ser 235/236 S6 ribosomal protein, Thr 37/46 4E-BP1, and Ser 209 eIF4E. Also a dephosphorylation of the Akt downstream target GSK3β on Ser 21/9 was observed. In contrast, rapamycin failed to dephosphorylate Thr 37/46 4E-BP1 and Ser 209 eIF4E. At variance with rapamycin, we also found a marked inhibition of mRNA translation in T-ALL cell lines treated with active-site mTOR inhibitors, as attested by a reduction in the incorporation of 3H-leucine and a shift from large to small polysomes.In MOLT-4 and Jurkat cells, the inhibitors, when used at subcytotoxic concentrations, strongly synergized with vincristine (combination index = 0.2–0.6), a traditional chemotherapeutic drug currently used for treating T-ALL patients, and with the Bcl-2 inhibitor, ABT-263 (combination index =0.15-0.19). In conclusion, both the inhibitors displayed a remarkable anti-leukemic activity through inhibition of mTORC1 and mTORC2 activity. The lower cytotoxicity against healthy T-lymphocytes ex-vivo suggests a favourable therapeutic index, which emphasizes the development of active-site mTOR inhibitors as clinical candidates for therapy in T-ALL. Disclosures:No relevant conflicts of interest to declare.

AMP-Dependent Kinase/mTOR Complex 1 Signaling In T-Cell Acute Lymphoblastic Leukemia: Therapeutical Implications

AbstractAbstract 3225Over the past 20 years, survival rates of T-ALL patients have improved, mainly because of advances in chemotherapy protocols. Despite these improvements, we still need novel and less toxic treatment strategies targeting aberrantly activated signaling intermediates which increase proliferation, survival, and drug-resistance of T-ALL cells. One such intermediate is represented by the mammalian target of rapamycin (mTOR). mTOR exists as two complexes, referred to as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Allosteric mTOR inhibitors (rapamycin and its analogs) mainly target mTORC1 and have displayed some promising effects in preclinical models of T-ALL. However, mTORC1 activity can be inhibited by activating AMP-dependent kinase (AMPK). AMPK activators include the antidiabetic drug, metformin. It is now emerging that metformin possesses antineoplastic activity in preclinical settings of solid tumors and its efficacy is being currently evaluated in cancer patients. Therefore, there is a strong rationale for further investigating the role of AMPK/mTORC1 signaling in malignant hematological disorders, as this pathway could represent a target for innovative treatments. Here, we have analyzed the therapeutic potential of metformin in T-ALL cell lines and pediatric patient lymphoblasts. We have used a panel of T-ALL cell lines including CEM-R [which overexpresses high levels of the membrane transporter, 170-kDa P-glycoprotein], Jurkat, and RPMI-8402 cells. Cell lines and fresh leukemia patients samples displayed mTORC1 activation as documented by the levels of Thr 37/46 p-4E-BP1 and Ser 235/236 p-S6 ribosomal protein. MTT assays demonstrated that metformin affected the viability of T-ALL cells. The IC50 for metformin at 48 h ranged from 1.6 to 5.6 mM in cell lines. When T-ALL patient samples were studied, metformin IC50 was 0.6–0.9 mM at 96 h. Remarkably, metformin targeted the side population (identified by flow cytometric analysis of Hoechst 33342 staining and ABCG2 expression) of T-ALL cell lines, which might correspond to leukemia initiating cells. Metformin induced apoptosis, as documented by western blot analysis with an antibody to caspase-8 and transmission electron microscopy analysis. Consistently with mTORC1 signaling inhibition, metformin caused autophagy, as demonstrated by transmission electron microscopy and western blot analysis with antibodies to beclin 1 and LC3B. Western blotting documented increased levels of Thr 172 p-AMPKα in CEM-R and RPMI8402 cells treated with metformin, indicating AMPKα activation. In contrast, no AMPKα activation was observed in Jurkat cells. Consistently with the activation of PKCζ/LKB1/AMPKα signaling, increased phosphorylation of Thr 410/403 p-PKCζ was detected in CEM-R and RPMI8402 cells. As a further proof of AMPKα activation by metformin, Ser 792 p-Raptor phosphorylation was seen in CEM-R and RPMI8402 cells. However, we detected decreased phosphorylation levels of Thr 37/46 p-4E-BP1, Ser 235/235 p-S6 ribosomal protein, Thr 389 p-p70S6K, and Ser 209 eIF4E in all the cell lines treated with metformin, implying inhibition of mTORC1 signaling. Decreased levels of Thr 37/46 p-4E-BP1 and Ser 235/235 p-S6 ribosomal protein were detected also in patient lymphoblasts. Interestingly, Ser 473 p-Akt levels decreased in Jurkat cells treated with metformin, suggesting that in this cell line, mTORC1 inhibition could be due to downregulation of IRS1/PI3K/Akt signaling, as recently demonstrated in breast cancer cell lines. Unlike rapamycin, metformin caused a marked inhibition of mRNA translation in T-ALL cell lines, as attested by a reduction in the incorporation of 3H-leucine and a shift from large to small polysomes. Our findings demonstrated that metformin was cytotoxic to T-ALL cell lines and patients lymphoblasts, activated AMPK through different mechanisms, downregulated mTORC1 signaling, and targeted mRNA translation more efficiently than rapamycin. The metformin concentrations which were cytotoxic to T-ALL cells are similar to the effective concentrations reported for solid tumors and could be obtained in human tissues and cells in vivo. The data could have a significant impact on our knowledge of both the relevance of AMPK/mTORC1 signaling as an innovative therapeutic target in T-ALL and the efficacy of metformin in this clinical setting. Disclosures:No relevant conflicts of interest to declare.

Dendritic cells restrict the transformation ofHistoplasma capsulatumconidia into yeasts

Infections due to Histoplasma capsulatum occur as a result of the inhalation of airborne microconidia of the mold into the alveoli of the lungs. In this study we quantified the transformation over time of conidia into yeast-like cells within macrophages (MΦ) and dendritic cells (DC). Conidia from strain G217B which had been surface labeled with carboxy-fluorescein succinimidyl ester (CFSE), or conidia from strain G217B that expresses green fluorescent protein (GFP) only in the yeast phase, were used to infect MΦ and DC. At various time points, numbers of intracellular conidia or yeasts were quantified via phase-contrast and fluorescent microscopy. Transformation of conidia from non-GFP-expressing G217B also was quantified by their incorporation of ³H-leucine. In both human and murine MΦ, numerous yeast-like cells appeared by day 3 post-infection. The time course of conidia transformation into yeasts in culture medium was the same as in MΦ. However, transformation of conidia to yeasts was significantly restricted in human DC and murine lung DC. In DC, significant numbers of yeasts did not appear until 5 days post-infection. Further, MΦ monolayers were destroyed by day 6-7 post-infection, whereas DC monolayers remained intact throughout the study period. These data suggest that in vivo, conidia may transform into yeast-like cells efficiently whether or not they are phagocytosed by MΦ, but not when ingested by DC.

Effects of Angiotensin III on Protein, DNA, and Collagen Synthesis of Neonatal Cardiomyocytes and Cardiac Fibroblasts In Vitro

This study compared angiotensin II (Ang II) and angiotensin III (Ang III) for their effects on rat neonatal cardiomyocytes and cardiac fibroblasts in vitro and discussed the possible role of Ang III in the pathogenesis of cardiac remodeling. To do so, protein synthesis, cardiac fibroblast proliferation, collagen synthesis, and secretion in response to treatment with Ang III and Ang II were investigated. Protein synthesis rate was assessed by (3)H-Leucine ((3)H-Leu) incorporation; the content of DNA was defined by (3)H-thymidine ((3)H-TdR) incorporation; and collagen synthesis and secretion were assessed by ( 3)H-proline ((3)H-Pro) incorporation. In neonatal cardiomyocytes, Ang III stimulated protein synthesis in a concentration-dependent manner, whereas in neonatal cardiac fibroblasts, DNA synthesis as well as collagen synthesis and secretion were increased in a concentration-dependent manner. Treatment with captopril, selective aminopeptidase A (APA) inhibitor (EC33), or selective aminopeptidase N inhibitor (PC18) had no effect on these outcomes. Treatment with losartan significantly decreased the effects of Ang III, except for cardiomyocyte protein synthesis. Compared with Ang II, Ang III could stimulate cardiomyocyte protein synthesis, cardiac fibroblast proliferation, and collagen synthesis and secretion. Furthermore, 10(-7) mol/L Ang II but not Ang III significantly increased APA activity in both cardiomyocytes and fibroblasts. All these results show the bioactive effects of Ang III on myocardial cells and suggest that Ang III could be an important independent factor besides Ang II in the regulation of cardiac function and may affect the pathogenesis of cardiac remodeling.

Bacterial Growth Response to Photoactive Quinones

Quinones are known producers of reactive oxygen species (ROS) that may be toxic in natural aquatic environments. In this study, the effects of parent quinones and their photodegradation products on bacterial growth were determined, and photochemical ROS formation rates were measured. Using (3)H-leucine incorporation to measure growth of the bacterium Pseudomonas aeruginosa and natural seawater bacterioplankton, growth inhibition was observed when samples were exposed to dichlone, chloranil and sodium anthraquinone-2-sulfonate (AQ2S). For seawater, compared with other quinones tested, dichlone showed the greatest toxicity in the dark, and AQ2S toxicity was greatest during simultaneous exposure to sunlight. Photodegraded chloranil and dichlone showed decreased toxicity compared with nonirradiated samples. For P. aeruginosa, AQ2S and its photodegradation products showed the greatest toxicity during simultaneous exposure to sunlight. Chloranil photodegradation products showed reduced toxicity compared with the parent compound during simultaneous exposure to sunlight. Dichlone was the only compound to show any toxicity to P. aeruginosa in the dark, and its photodegradation products were more toxic than the parent compound. Based on the results of dark and light controlled experiments measuring bacterial growth and estimated ROS production rates, ROS alone does not account for relative differences in toxicity between these quinones.

Toxicity of five protein synthesis inhibiting antibiotics and their mixture to limnic bacterial communities

Antibiotics are common contaminants of aquatic environments, the protein synthesis inhibitors being one frequently detected class. Even though antibiotics target bacteria, little is known about the sensitivity of naturally occurring freshwater bacteria to these compounds. Limnic bacterial communities were therefore systematically exposed to five common protein synthesis inhibiting antibiotics, each representing a particular subgroup: streptomycin, chloramphenicol, fusidic acid, rifampicin and chlortetracycline. Full concentration–response curves and No Observed Effect Concentrations (NOECs) were determined for all antibiotics, using the 3Hleucine incorporation method. All test compounds were toxic to planktonic communities of limnic bacteria, with EC50s ranging from 0.138 μmol/L for chlortetracycline to 79.1 μmol/L for streptomycin. The order of toxicity was chlortetracycline > rifampicin > fusidic acid > chloramphenicol > streptomycin, based on the individual EC50 values. A comparison to reported chemical monitoring data shows that environmental concentrations of chlortetracycline are in a range that clearly inhibits the protein biosynthesis activity of planktonic bacterial communities. All compounds show exceptionally flat concentration–response relationship, for fusidic acid the ratio of EC50 to EC05 exceeds four orders of magnitude. This challenges the standard assessment factors of 10–100 for the extrapolation from high to low doses. Environmental exposure situations are often characterized by the presence of mixtures of antibiotics, e.g. in sewage effluents or river systems. Hence, also combined effects of the five antibiotics were determined, and compared to mixture toxicity predictions based on Concentration Addition and Independent Action. Concentration Addition slightly underestimated the observed EC50 by a factor of 1.5, independent on whether the prediction was based on single substance data that were recorded in parallel or whether historical data were used. Independent Action predicted higher mixture toxicity than Concentration Addition due to the flatness of the individual concentration–response curves. Implications of these findings for the environmental risk assessment of antibiotics and their mixtures are discussed.

MAPK and PI3K activities are required for leptin stimulation of protein synthesis in human trophoblastic cells

Leptin, the LEP gene product, is produced in placenta where it has been found to be an important autocrine signal for trophoblastic growth during pregnancy. Thus, we have recently described the antiapoptotic and trophic effect of leptin on choriocarcinoma cell line JEG-3, stimulating DNA and protein synthesis. We have also demonstrated the presence of leptin receptor and leptin signaling in normal human trophoblastic cells, activating JAK-STAT, PI3K and MAPK pathways. In the present work we have employed dominant negative forms of MAPK and PKB constructs to find out the signaling pathways that specifically mediates the effect of leptin on protein synthesis. As previously shown, leptin stimulates protein synthesis as assessed by 3H-leucine incorporation. However, both dominant negative forms of MAPK and PKB inhibited protein synthesis in JEG-3 choriocarcinoma cells. The inhibition of PKB and MAPK activity by transfection with the dominant negative kinases prevented the leptin stimulation of p70 S6K, which is known to be an important kinase in the regulation of protein synthesis. Moreover, leptin stimulation of phosphorylation of EIF4EBP1 and EIF4E, which allows the initiation of translation was also prevented by MAPK and PI3K dominant negative constructs. Therefore, these results demonstrate that both PI3K and MAPK are necessary to observe the effect of leptin signaling that mediates protein synthesis in choriocarcinoma cells JEG-3.

Temperature and Solar Radiation Interactions on 3H‐leucine Incorporation by Bacterioplankton in a Subtropical Estuary

Although the effects of UV radiation are thought to be temperature independent, the photoinhibition of aquatic bacteria may be temperature dependent owing to enzymatic repair kinetics, an important consideration for climate change analyses. We examined the interactions between temperature and solar radiation in water samples collected from the Blackwater River, Pensacola Bay, and the coastal Gulf of Mexico (Florida) in July 2008. Subsamples were incubated in the dark for 20 h at either the in situ temperature, +5 degrees C from in situ or -5 degrees C from in situ after which they were amended with (3)H-leucine and irradiated in full sunlight at their respective temperatures and compared to samples incubated simultaneously in the dark. Temperature and light significantly affected (3)H-leucine incorporation at all locations and interactive effects between temperature and sunlight were found for Pensacola Bay and the Gulf. Generally, warmer waters reduced photoinhibition. The -5 degrees C treatment was always significantly more inhibited than the +5 degrees C treatment, but the in situ temperature and +5 degrees C and -5 degrees C treatments were not always significantly different. Photoinhibition reduction at warmer temperatures suggests specific effects on photobiology not observed in general cellular activity may be important in determining interactive ecosystem effects of climate change.

Major contribution of autotrophy to microbial carbon cycling in the deep North Atlantic’s interior

Abstract Current estimates point to a mismatch of particulate organic carbon supply derived from the surface ocean and the microbial organic carbon demand in the meso- and bathypelagic realm. Based on recent findings that chemoautotrophic Crenarchaeota are abundant in the mesopelagic zone, we quantified dissolved inorganic carbon (DIC) fixation in the meso- and bathypelagic North Atlantic and compared it with heterotrophic microbial activity. Measuring 14C-bicarbonate fixation and 3H-leucine incorporation revealed that microbial DIC fixation is substantial in the mesopelagic water masses, ranging from 0.1 to 56.7 μmol C m−3 d−1, and is within the same order of magnitude as heterotrophic microbial activity. Integrated over the dark ocean’s water column, DIC fixation ranged from 1–2.5 mmol C m−2 d−1, indicating that chemoautotrophy in the dark ocean represents a significant source of autochthonously produced ‘new organic carbon’ in the ocean’s interior amounting to about 15–53% of the phytoplankton export production. Hence, chemoautotrophic DIC fixation in the oxygenated meso- and bathypelagic water column of the North Atlantic might substantially contribute to the organic carbon demand of the deep-water microbial food web.

Does indoxyl sulfate, a uraemic toxin, have direct effects on cardiac fibroblasts and myocytes?

Indoxyl sulfate (IS) is a uraemic toxin found at high concentration in patients with chronic kidney disease (CKD) co-morbid with chronic heart failure (CHF). The aim of this study was to determine direct effects of IS on cardiac cells as well as the pro-inflammatory effect of IS. Indoxyl sulfate significantly increased neonatal rat cardiac fibroblast collagen synthesis (by 145.7% vs. control, P < 0.05) and myocyte hypertrophy (by 134.5% vs. control, P < 0.001) as determined by (3)H-proline or (3)H-leucine incorporation, respectively. Indoxyl sulfate stimulated tumour necrosis factor-alpha, interleukin-6 (IL-6), and IL-1beta mRNA expression in THP-1 cells as quantified by RT-PCR. Both p38 (RWJ-67657) and MEK1/2 (U0126) inhibitors suppressed all these effects by IS. Furthermore, western blot analysis showed that IS activated mitogen-activated protein kinase (MAPK) (p38, p42/44) and nuclear factor-kappa B (NFkappaB) pathways. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that IS exerted its effects without affecting cell viability. This study has, for the first time, demonstrated that IS has pro-fibrotic, pro-hypertrophic, and pro-inflammatory effects, indicating that IS might play an important role in adverse cardiac remodelling mediated via activation of the p38 MAPK, p42/44 MAPK, and NFkappaB pathways. Targeting reduction of IS and/or the pathways it activates may represent a novel therapeutic approach to the management of CHF with concomitant CKD.

Mechanism underlying carbon tetrachloride-inhibited protein synthesis in liver

To study the mechanism underlying carbon tetrachloride (CCl(4)) -induced alterations of protein synthesis in liver. Male Sprague-Dawley rats were given CCl(4) (1 mL/100 g body weight) and (3)H-leucine incorporation. Malondialdehyde (MDA) level in the liver, in vitro response of hepatocyte nuclei nucleotide triphosphatase (NTPase) to free radicals, and nuclear export of total mRNA with 3'-poly A(+) were measured respectively. Survival response of HepG2 cells to CCl(4) treatment was assessed by methyl thiazolyl tetrazolium. Km and Vmax values of nuclear envelope NTPase activity in liver of rats treated with CCl(4) were assayed by a double-reciprocal plot. The protein synthesis was inhibited while the MDA level was significantly increased in liver of rats treated with CCl(4). In addition, CCl(4) decreased the NTPase binding capacity of nuclear envelope (Km value) in cultured HepG2 cells. Moreover, in vitro ferrous radicals from Fenton's system suppressed the NTPase activity of liver nuclear envelope in a dose-dependent manner. Down-regulation of the nuclear envelope NTPase activity indicated a lower energy provision for nucleocytoplasmic transport of mRNA molecules, an evidence in CCl(4)-treated HepG2 cells correspondingly supported by the nuclear sequestration of poly (A)(+) mRNA molecules in morphological hybridization research. Inhibition of mRNA transport, suggestive of decreased NTPase activity of the nuclear envelope, may be involved in carbon tetrachloride-inhibited protein synthesis in liver.

Synthesis and Antimicrobial Evaluation of Amphiphilic Neamine Derivatives

The aminoglycoside antibiotics bind to the 16S bacterial rRNA and disturb the protein synthesis. One to four hydroxyl functions of the small aminoglycoside neamine were capped with phenyl, naphthyl, pyridyl, or quinolyl rings. The 3',4'- (6), 3',6- (7a), and the 3',4',6- (10a) 2-naphthylmethylene derivatives appeared to be active against sensitive and resistant Staphylococcus aureus strains. 10a also showed marked antibacterial activities against Gram (-) bacteria, including strains expressing enzymes modifying aminoglycosides, efflux pumps, or rRNA methylases. 7a and 10a revealed a weak and aspecific binding to a model bacterial 16S rRNA. Moreover, as compared to neomycin B, 10a showed a lower ability to decrease (3)H leucine incorporation into proteins in Pseudomonas aeruginosa. All together, our results suggest that the 3',4',6-tri-2-naphthylmethylene neamine derivative 10a should act against Gram (-) bacteria through a mechanism different from inhibition of protein synthesis, probably by membrane destabilization.