Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

iMet-Q: A User-Friendly Tool for Label-Free Metabolomics Quantitation Using Dynamic Peak-Width Determination

Efficient and accurate quantitation of metabolites from LC-MS data has become an important topic. Here we present an automated tool, called iMet-Q (intelligent Metabolomic Quantitation), for label-free metabolomics quantitation from high-throughput MS1 data. By performing peak detection and peak alignment, iMet-Q provides a summary of quantitation results and reports ion abundance at both replicate level and sample level. Furthermore, it gives the charge states and isotope ratios of detected metabolite peaks to facilitate metabolite identification. An in-house standard mixture and a public Arabidopsis metabolome data set were analyzed by iMet-Q. Three public quantitation tools, including XCMS, MetAlign, and MZmine 2, were used for performance comparison. From the mixture data set, seven standard metabolites were detected by the four quantitation tools, for which iMet-Q had a smaller quantitation error of 12% in both profile and centroid data sets. Our tool also correctly determined the charge states of seven standard metabolites. By searching the mass values for those standard metabolites against Human Metabolome Database, we obtained a total of 183 metabolite candidates. With the isotope ratios calculated by iMet-Q, 49% (89 out of 183) metabolite candidates were filtered out. From the public Arabidopsis data set reported with two internal standards and 167 elucidated metabolites, iMet-Q detected all of the peaks corresponding to the internal standards and 167 metabolites. Meanwhile, our tool had small abundance variation (≤0.19) when quantifying the two internal standards and had higher abundance correlation (≥0.92) when quantifying the 167 metabolites. iMet-Q provides user-friendly interfaces and is publicly available for download at http://ms.iis.sinica.edu.tw/comics/Software_iMet-Q.html.     

Using spatially explicit commodity flow and truck activity data to map urban material flows

To analyze and promote resource efficiency in urban areas, it is important to characterize urban metabolism and particularly, material flows. Material flow analysis (MFA) offers a means to capture the dynamism of cities and their activities. Urban‐scale MFAs have been conducted in many cities, usually employing variants of the Eurostat methodology. However, current methodologies generally reduce the study area into a “black box,” masking details of the complex processes within the city's metabolism. Therefore, besides the aggregated stocks and flows of materials, the movement of materials—often embedded in goods or commodities—should also be highlighted. Understanding the movement and dispersion of goods and commodities can allow for more detailed analysis of material flows. We highlight the potential benefits of using high‐resolution urban commodity flows in the context of understanding material resource use and opportunities for conservation. Through the use of geographic information systems and visualizations, we analyze two spatially explicit datasets: (1) commodity flow data in the United States, and (2) Global Positioning System‐based commercial vehicle (truck) driver activity data in Singapore. In the age of “big data,” we bring advancements in freight data collection to the field of urban metabolism, uncovering the secondary sourcing of materials that would otherwise have been masked in typical MFA studies. This brings us closer to a consumption‐based, finer‐resolution approach to MFA, which more effectively captures human activities and its impact on urban environments.     

Germanium oxide inhibits the transition from G2 to M phase of CHO cells

We report here for the first time that germanium oxide (GeO(2)) blocks cell progression. GeO(2) is not genotoxic to Chinese hamster ovary (CHO) cells and has limited cytotoxicity. However, GeO(2) arrests cells at G2/M phase. The proportion of cells stopped at G2/M phase increased dose-dependently up to 5 mM GeO(2) when treated for 12 h, but decreased at GeO(2) concentration was greater than 5 mM. Analysis of 5-bromodeoxyuridine-labeled cells indicated that GeO(2) delayed S phase progression in a dose-dependent manner, and blocked cells at G2/M phase. Microscopic examination confirmed that GeO(2) treatment arrested cells at G2 phase. Similar to several other events that cause G2 block, the GeO(2)-induced G2 block can also be ameliorated by caffeine in a dose- and time-dependent manner. To explore the mechanism of G2 arrest by GeO(2), cyclin content and cyclin-dependent kinase activity were examined. Cyclin B1 level was not affected after GeO(2) treatment in CHO cells. However, GeO(2) decreased p34(cdc2) kinase (Cdk1) activity. The kinase activity recovered within 9 h after GeO(2) removal and correlated with the transition of G2/M-G1 phase of the cells. This result suggests that GeO(2) treatment reduces Cdk1 activity and causing the G2 arrest in CHO cells.     

The "five-sites" rule and the evolution of red and green color vision in mammals

Amino acid changes S180A (S-->A at site 180), H197Y, Y277F, T285A, and A308S are known to shift the maximum wavelength of absorption (lambda max) of red and green visual pigments toward blue, essentially in an additive fashion. To test the generality of this "five-sites" rule, we have determined the partial amino acid sequences of red and green pigments from five mammalian orders (Artiodactyla, Carnivora, Lagomorpha, Perissodactyla, and Rodentia). The result suggests that cat (Felis catus), dog (Canis familiaris), and goat (Capra hircus) pigments all with AHYTA at the five critical sites have lambda max values of approximately 530 nm, whereas rat (Rattus norvegicus) pigment with AYYTS has a lambda max value of approximately 510 nm, which is accurately predicted by the five-sites rule. However, the observed lambda max values of the orthologous pigments of European rabbit (Oryctolagus cuniculus), white-tailed deer (Odocoileus virginianus), gray squirrel (Sciurus carolinensis), and guinea pig (Cavia procellus) are consistently more than 10 nm higher than the predicted values, suggesting the existence of additional molecular mechanisms for red and green color vision. The inferred amino acid sequences of ancestral organisms suggest that the extant mammalian red and green pigments appear to have evolved from a single ancestral green-red hybrid pigment by directed amino acid substitutions.      

A new quantitative LC tandem mass spectrometry assay for serum 25-hydroxy vitamin D

BackgroundThe accurate measurement of 25-hydoxy vitamin D (25OH-D) in serum has been a challenge for many years. We developed a liquid chromatography tandem mass spectrometry (LC Tandem MS) assay for the quantitative determination of 25OH-D₂ and 25OH-D₃ in serum. The new method was compared with two widely used commercially available immunoassays.MethodsSample preparation involved protein precipitation with acetonitrile containing deuterated forms of the target species as internal standards. An API 5000 mass spectrometer coupled with a photoionization source was used for quantitation. The performance of the new LC Tandem MS assay was compared with a radioimmunoassay (RIA, Diasorin) and a chemiluminescence immunoassay (ECLIA, Roche Diagnostics), analysing serum obtained from 152 individuals.ResultsUsing 100 μl of serum, the LC Tandem MS assay had a limit of quantitation of 1.3 nmol/L for both 25OH-D₂ and 25OH-D₃ with a linear response between 1.3 and 625 nmol/L and accuracy of between 95 and 124%. Intra- and inter-assay precision were ≤7% and ≤4%, respectively. Measurement of 25OH-D levels in 152 serum samples gave run averages of 71, 56 and 62 nmol/L for LC Tandem MS, ECLIA and RIA, respectively. Correlations between the various methods were: LC Tandem MS vs. RIA: r = 0.931; LC Tandem MS vs. ECLIA: r = 0.784; RIA vs. ECLIA: r = 0.787. The LC Tandem MS method had a positive proportional bias of 26% over the RIA, whereas the ECLIA showed variable differences.ConclusionThe new LC Tandem MS assay is accurate and precise at physiologically relevant 25OH-D concentrations, and compares favourably with the RIA. In contrast, the ECLIA shows variable bias with the other assays tested.     

In situ forming and rutin-releasing chitosan hydrogels as injectable dressings for dermal wound healing

An in situ gel-forming system composed of rutin- and tyramine-conjugated chitosan derivatives, horseradish peroxidase (HRP), and hydrogen peroxide (H(2)O(2)) was prepared and applied to dermal wound repair. Rutin was employed to enhance production and accumulation of extracellular matrix in the healing process. In vitro study demonstrates that released rutin significantly enhanced cell proliferation as compared with media without rutin. In vivo wound healing study was performed by injecting hydrogels on rat dorsal wounds with a diameter of 8 mm for 14 days. Histological results demonstrated that rutin-conjugated hydrogel exhibited enhancement of wound healing as compared with treatments with PBS, hydrogel without rutin, and a commercialized wound dressing (Duoderm). More specifically, rutin-conjugated hydrogels induced better defined formation of neo-epithelium and thicker granulation, which is closer to the original epithelial tissue. As a result, this study suggests that the in situ gel-forming system can be a promising injectable gel-type wound dressing.     

Cross-regulation among disparate antibiotic biosynthetic pathways of Streptomyces coelicolor

A complex programme of regulation governs gene expression during development of the morphologically and biochemically complex eubacterial genus Streptomyces. Earlier work has suggested a model in which 'higher level' pleiotropic regulators activate 'pathway-specific' regulators located within chromosomal gene clusters encoding biosynthesis of individual antibiotics. We used mutational analysis and adventitious overexpression of key Streptomyces coelicolor regulators to investigate functional interactions among them. We report here that cluster-situated regulators (CSRs) thought to be pathway-specific can also control other antibiotic biosynthetic gene clusters, and thus have pleiotropic actions. Surprisingly, we also find that CSRs exhibit growth-phase-dependent control over afsR2/afsS, a 'higher level' pleiotropic regulatory locus not located within any of the chromosomal gene clusters it targets, and further demonstrate that cross-regulation by CSRs is modulated globally and differentially during the S. coelicolor growth cycle by the RNaseIII homologue AbsB. Our results, which reveal a network of functional interactions among regulators that govern production of antibiotics and other secondary metabolites in S. coelicolor, suggest that revision of the currently prevalent view of higher-level versus pathway-specific regulation of secondary metabolism in Streptomyces species is warranted.     

Susceptibilities to Amphotericin B, Fluconazole and Voriconazole of Trichosporon Clinical Isolates

A total of 35 Trichosporon isolates were collected from the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) project from 1999 to 2006, and their identifications as well as drug susceptibilities were determined. The most frequently isolated species was T. asahii (62.9%), and the most common clinical sample that yielded Trichosporon isolates was urine (37.1%). The etiology of all seven invasive trichosporonosis was T. asahii. For the 22 T. asahii isolates, the MIC(50) and MIC(90) for amphotericin B were 0.25 and 1 μg/mL, respectively. Those for fluconazole were 2 and 4 μg/mL, respectively, and for voriconazole 0.031 and 0.063 μg/mL, respectively. When the intraclass correlation coefficients (ICCs) and agreements were calculated, we found that the MICs of fluconazole obtained from different methods were similar and the inter-method discrepancies were low. Nevertheless, no unanimous MIC of amphotericin B and voriconazole was obtained among different methods.