Fundulus as the premier teleost model in environmental biology: opportunities for new insights using genomics

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.     

Determination of roxithromycin in human plasma by HPLC with fluorescence and UV absorbance detection: application to a pharmacokinetic study

A selective HPLC method with fluorescence detection for the determination of roxithromycin (ROX) in human plasma was described. After solid-phase extraction (SPE), ROX and erythromycin (internal standard, I.S.) were derivatized by treatment with 9-fluorenylmethyl chloroformate (FMOC-Cl). Optimal resolution of fluorescence derivatives of ROX and I.S. was obtained during one analytical run using reversed phase, C(18) column. The mobile phase was composed of potassium dihydrogenphosphate solution, pH 7.5 and acetonitrile. Fluorescence of the compounds was measured at the maximum excitation, 255 nm and emission, 313 nm, of ROX derivatives. Validation parameters of the method were also established. After SPE, differences in recoveries of ROX and erythromycin from human plasma were observed. The linear range of the standard curve of ROX in plasma was 0.5-10.0 mg/l. The validated method was successfully applied for pharmacokinetic studies of ROX after administration of a single tablet of ROX.     

Urine profiling by SELDI-TOF/MS: monitoring of the critical steps in sample collection, handling and analysis

The topic of this study is the impact of several pre-analytical and analytical variables on proteomic profiling of human urine by surface enhanced laser desorption/ionization time of flight-mass spectrometry (SELDI-TOF-MS) in healthy subjects. Urine storage at room temperature caused a progressive degradation of proteins, which was prevented by the addition of protease inhibitors only up to 2 h from the collection. The timing of collection over the day had only a minor impact on protein profile, although influencing the intensity of peaks. Repeated freeze/thaw cycles (up to five) did not affect either the number or the intensity of the peaks. A comparison of the protein profile from eight different healthy individuals showed fairly consistent inter-subject similarities, along with between-subject differences, which were markedly dependent on the sex and the type of ProteinChip array used. The addition of a variety of denaturing agents improved the quality of the spectra with all the chips tested (CM10, Q10 and H50), but not with the copper-coated IMAC-30 chip. Finally, SPA matrix allowed to achieve a better performance of SELDI-TOF/MS spectrum, as compared with CHCA, regardless of the ProteinChip array used and even in the low m/z range (2500-10,000). In conclusion, we suggest that a careful choice of a number of pre-analytical and analytical conditions is required to accomplish and define a unifying protocol for the analysis of human urine by SELDI-TOF/MS, in physiological and in pathological states.     

Determination of treosulfan in plasma and urine by HPLC with refractometric detection; pharmacokinetic studies in children undergoing myeloablative treatment prior to haematopoietic stem cell transplantation

A direct and selective HPLC method with refractometric detection was worked out for determination of treosulfan in plasma and urine of children. Before injection onto reverse phase column plasma samples with treosulfan and barbital (I.S.) were clarified using filtration. The mobile phase was composed of phosphate buffer, pH 5 and acetonitrile. The linear range of the standard curve of treosulfan spanned concentrations of 10.0-2000.0 microg/ml and 50.0-10000.0 microg/ml in plasma and urine, respectively, and covered the levels found in biological fluids after infusion of the drug. The limit of detection amounted to 5 microg/ml for plasma and 25 microg/ml for urine. Intra- and inter-day precision and accuracy of the measurement fulfilled analytical criteria accepted in pharmacokinetic studies. Recovery of treosulfan as well as stability in biological fluids was also calculated. The validated method was successfully applied in pharmacokinetic studies of treosulfan administered to children prior to haematopoietic stem cell transplantation. Differences between pharmacokinetics of treosulfan in children and adults were also studied.     

Metabolic profile and quantification of deoxyribose synthesis pathways in HepG2 cells

Traditional tracer studies of cell proliferation fail to distinguish between label enrichment due to increased DNA repair versus DNA replication. We used the emerging stable (non-radiating) isotope-based dynamic metabolic profiling technique on HepG2 cells to determine synthesis pathways of nucleic acids from glucose and rates of proliferation using CG-MS assay of RNA and DNA enrichment. Comparing the isotopic enrichment curve in DNA with the theoretical curve based on cell growth, we observed that the measured tracer enrichment was significantly higher, indicating that surplus label was acquired during DNA repair. In particular, after the first duplication (3 days), 80.13% of the total enrichment observed corresponds to duplication and 19.87% corresponds to DNA repair as calculated from the [1, 2-¹³C₂]-glucose incorporation curve. Our data indicate contemporary measurements of cell proliferation rates relying on tracer incorporation may be overestimated. ¹³C label was distributed between m1 (m1/Σm = 80) and m2 (m2/Σm = 14) of deoxyribose, indicating that most of the glucose carbon was acquired via direct glucose oxidation in the pentose cycle. The stable isotope technique distinguishes rates of DNA synthesis and repair via the oxidative and non-oxidative pentose cycle, separately, in one test, without inhibition of either process. The contribution of DNA repair in malignant cells to isotope accumulation in deoxyribose remains to be investigated.     

Unexpected link between metal ion deficiency and autophagy in Aspergillus fumigatus

Autophagy is the major cellular pathway for bulk degradation of cytosolic material and is required to maintain viability under starvation conditions. To determine the contribution of autophagy to starvation stress responses in the filamentous fungus Aspergillus fumigatus, we disrupted the A. fumigatus atg1 gene, encoding a serine/threonine kinase required for autophagy. The ΔAfatg1 mutant showed abnormal conidiophore development and reduced conidiation, but the defect could be bypassed by increasing the nitrogen content of the medium. When transferred to starvation medium, wild-type hyphae were able to undergo a limited amount of growth, resulting in radial expansion of the colony. In contrast, the ΔAfatg1 mutant was unable to grow under these conditions. However, supplementation of the medium with metal ions rescued the ability of the ΔAfatg1 mutant to grow in the absence of a carbon or nitrogen source. Depleting the medium of cations by using EDTA was sufficient to induce autophagy in wild-type A. fumigatus, even in the presence of abundant carbon and nitrogen, and the ΔAfatg1 mutant was severely growth impaired under these conditions. These findings establish a role for autophagy in the recycling of internal nitrogen sources to support conidiophore development and suggest that autophagy also contributes to the recycling of essential metal ions to sustain hyphal growth when exogenous nutrients are scarce.     

Soil CO2 efflux in a mixed pine-oak forest in Valsaín (central Spain)

Soil-surface CO2 efflux and its spatial and temporal variation were investigated in a southern Mediterranean, mixed pine-oak forest ecosystem on the northern slopes of the Sierra de Guadarrama in Spain from February 2006 to July 2006. Measurements of soil CO2 efflux, soil temperatures, and moisture were conducted in nine 1963-m2 sampling plots distributed in a gradient around the ecotone between Pinus sylvestris L. and Quercus pyrenaica Lam. forest stands. Total soil organic matter, Walkey-Black C, particulate organic matter, organic matter fraction below 53 microm, total soil nitrogen content, total soil organic carbon content, and pH were also measured under three representative mature oak, pine, and mixed pine-oak forest stands. Soil respiration showed a typical seasonal pattern with minimums in winter and summer, and maximums in spring, more pronounced in oak and oak-pine stands. Soil respiration values were highest in pine stands during winter and in oak stands during spring and summer. Soil respiration was highly correlated with soil temperatures in oak and pine-oak stands when soil moisture was above a drought threshold of 15%. Below this threshold value, soil moisture was a good predictor of soil respiration in pine stands. Greater soil organic matter, particulate organic matter, Walkey-Black C, total organic C, and total N content in pine compared to oak sites potentially contributed to the greater total soil CO2 efflux in these stands during the winter. Furthermore, opposing trends in the organic matter fraction below 53 microm and soil respiration between plots suggest that in oak stands, the C forms are less affected by possible changes in use. The effects of soil properties on soil respiration were masked by differences in soil temperature and moisture during the rest of the year. Understanding the spatial and temporal variation even within small geographic areas is essential to assess C budgets at ecosystem level accurately. Thus, this study bears important implications for the study of large-scale ecosystem dynamics, particularly in response to climatic change.     

A proteomics analysis of cell signaling alterations in colorectal cancer

To gain further insight into alterations in cellular pathways, tumor profiling, and marker discovery in colorectal cancer (CRC) we used a new antibody microarray specific for cell signaling. Soluble protein extracts were prepared from paired tumor/normal biopsies of 11 patients diagnosed with colorectal carcinoma at different stages; four liver carcinomas were used as a reference. Antibody microarray analysis identified 46 proteins that were differentially expressed between normal colorectal epithelium and adenocarcinoma. These proteins gave a specific signature for CRC, different from other tumors, as well as a panel of novel markers and potential targets for CRC. Twenty-four proteins were validated by using a specific colorectal cancer tissue microarray and immunoblotting analysis. Together with some previously well known deregulated proteins in CRC (beta-catenin, c-MYC, or p63), we found new potential markers preferentially expressed in CRC tumors: cytokeratin 13, calcineurin, CHK1, clathrin light chain, MAPK3, phospho-PTK2/focal adhesion kinase (Ser-910), and MDM2. CHK1 antibodies were particularly effective in discriminating between tumoral and normal mucosa in CRC. Moreover a global picture of alterations in signaling pathways in CRC was observed, including a significant up-regulation of different components of the epidermal growth factor receptor and Wnt/beta-catenin pathways and the down-regulation of p14(ARF). The experimental approach described here should be applicable to other pathologies and neoplastic processes.