Quantitative analysis of sinistrin in serum with high-performance liquid chromatography for renal function testing

Sinistrin, as inulin, is widely used as a marker for renal function testing. A reliable and accurate method with a simple sample preparation for the quantitative determination of sinistrin would be of advantage. We developed a high-performance liquid chromatography (HPLC)-based method with electrochemical detection for the quantitative measurement of sinistrin in serum and plasma. Sample preparation is easy and includes enzymatic removal of glucose, deproteinization, acid hydrolysis of sinistrin, and HPLC separation of fructose. The recovery of sinistrin from serum is the same as that from water and is near 100%. The method presented has a linear range up to 500 mg/L. The results from our method are in agreement with a fully enzymatic quantification (regression coefficient 1.01, coefficient of variation 0.97). Sinistrin concentrations in aqueous solutions can be measured down to 2mg/L with a coefficient of variation of 5.7%. Quantification in serum is primarily limited by its physiological fructose content. The sensitivity of the described method is sufficient for its use in renal function testing. We describe a method for quantification of sinistrin which allows accurate measurements especially at low concentrations and low sample volumes. This laboratory method may be used for obtaining sinistrin pharmacokinetics in renal function testing.     

Ecology of deepwater rice-fields in Bangladesh 3. Associated algae and macrophytes

1. Aquatic macrophytes formed dense beds in fallow areas during the four and a half months of the flood season in all but one deepwater rice-growing location in Bangladesh; these included several types of life-form, but the fine-leaved species, Myriophyllum sp., Najas indica, Utricularia stellaris were often especially abundant. The same species grew inside deepwater rice fields, but at much lower densities. A similar contrast occurred for the algae, although deepwater rice often developed dense masses of epiphytes on aquatic roots, stems and leaf sheaths, when plants were growing in isolated, well-illuminated situations.
2. Two widespread algae, Aulosira fertilissima and Scytonema mirabile, were equally successful on soil in the period prior to the arrival of floodwaters and floating on the surface of the water during the flood season. Other species common during the flood season differed from those common on soil.
3. Most blue-green algae inside deepwater rice fields were heterocystous; the only species not so, but forming distinct colonies, was Aphanothece stagnina. However only non-heterocystous forms were found at one location in south Bangladesh (Phaltita) and a change from heterocystous to non-heterocystous forms was noted at the main research site (near Sonargaon) during late September in at least one year. The water column at the former was almost entirely anoxic, while the change at the latter occurred at a time when the water sometimes became anoxic during the night. It is suggested that differences in ability to tolerate anoxic periods may be a key factor in determining the success of the algal and vascular plant species in the different micro-habitats within these DWR-growing areas.
4. Although diatoms were quantitatively only a minor component of the algal biomass, they became more frequent later in the season when the water became microaerobic or anoxic for part of the day. Navicula confervacea was overall the most abundant species at the two main research locations.

     

Biosorption of dyes using dead macro fungi: effect of dye structure, ionic strength and pH

Biosorbents prepared from dead macro fungi, namely Fomes fomentarius and Phellinus igniarius, were applied for the uptake of Methylene Blue (MB) and Rhodamine B (RB). Equilibrium isotherm data could be well described by the Langmuir and Freundlich models. Methylene Blue was found to be more adsorbable than Rhodamine B. Langmuir monolayer coverage was determined as 204.38-232.73 mg/g and 25.12-36.82 mg/g for MB and RB, respectively. Molecular structure and ionic radius of dyes were found to be responsible for differences in their uptakes. Results showed that sorption of MB increased while that of RB decreased as pH of respective dye solutions changed from 3 to 11. An increase in ionic strength also exhibited an adverse effect on dye sorption capacity. Ionic strength and pH affected the sorption of MB more as compared to the sorption of RB. The presence of carboxylic (-ve) and amino (+ve) groups in RB could explain the lower sorption of RB compared to MB.     

Premature Translation of oskar in Oocytes Lacking the RNA-Binding Protein Bicaudal-C

Bicaudal-C (Bic-C) is required during Drosophila melanogaster oogenesis for several processes, including anterior-posterior patterning. The gene encodes a protein with five copies of the KH domain, a motif found in a number of RNA-binding proteins. Using antibodies raised against the BIC-C protein, we show that multiple isoforms of the protein exist in ovaries and that the protein, like the RNA, accumulates in the developing oocyte early in oogenesis. BIC-C protein expressed in mammalian cells can bind RNA in vitro, and a point mutation in one of the KH domains that causes a strong Bic-C phenotype weakens this binding. In addition, oskar translation commences prior to posterior localization of oskar RNA in Bic-C⁻ oocytes, indicating that Bic-C may regulate oskar translation during oogenesis.     

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

The generation of transgenic cell lines is acquired by facilitating the uptake and integration of DNA. Unfortunately, most of the systems generating stable expression systems are cost and time-consuming and transient expression is optimized to generate milligram amounts of the recombinant protein. Therefore we improved and compared two transfection systems, one based on cationic liposomes consisting of DOTAP/DOPE and the second one on polyethylenimine (PEI). Both systems have been used as chemically defined transfection systems in combination with serum-free cultivated host cell line. At first we had determined the toxicity and ideal ratio of DNA to PEI followed by determination of the optimal transfection conditions in order to achieve maximum transfection efficiency. We then directly compared DOTAP/DOPE and PEI in transient transfection experiments using enhanced green fluorescence protein (EGFP) and a human monoclonal antibody, mAb 2F5, as a model protein. The results which were achieved in case of EGFP were more than 15% transfectants at a viability of 85%. Despite the fact that expression of the mAb was found negligible we used both techniques to generate stable mAb 2F5 expressing cell lines that underwent several cycles of screening and amplification with methotrexate, and resulted in cell lines with similar volumetric production titers. These experiments serve to demonstrate the potential of stable cell lines even in case where the transient systems did not show satisfying results.     

The Importance of Intraoperative Selenium Blood Levels on Organ Dysfunction in Patients Undergoing Off-Pump Cardiac Surgery: A Randomised Controlled Trial

Introduction

Cardiac surgery is accompanied by an increase of oxidative stress, a significantly reduced antioxidant (AOX) capacity, postoperative inflammation, all of which may promote the development of organ dysfunction and an increase in mortality. Selenium is an essential co-factor of various antioxidant enzymes. We hypothesized a less pronounced decrease of circulating selenium levels in patients undergoing off-pump coronary artery bypass (OPCAB) surgery due to less intraoperative oxidative stress.

Methods

In this prospective randomised, interventional trial, 40 patients scheduled for elective coronary artery bypass grafting were randomly assigned to undergo either on-pump or OPCAB-surgery, if both techniques were feasible for the single patient. Clinical data, myocardial damage assessed by myocard specific creatine kinase isoenzyme (CK-MB), circulating whole blood levels of selenium, oxidative stress assessed by asymmetric dimethylarginine (ADMA) levels, antioxidant capacity determined by glutathionperoxidase (GPx) levels and perioperative inflammation represented by interleukin-6 (IL-6) levels were measured at predefined perioperative time points.

Results

At end of surgery, both groups showed a comparable decrease of circulating selenium concentrations. Likewise, levels of oxidative stress and IL-6 were comparable in both groups. Selenium levels correlated with antioxidant capacity (GPx: r = 0.720; p<0.001) and showed a negative correlation to myocardial damage (CK-MB: r = −0.571, p<0.001). Low postoperative selenium levels had a high predictive value for the occurrence of any postoperative complication.

Conclusions

OPCAB surgery is not associated with less oxidative stress and a better preservation of the circulating selenium pool than on-pump surgery. Low postoperative selenium levels are predictive for the development of complications.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01409057","term_id":"NCT01409057"}}NCT01409057     

Differential regulation of the three methanol methyltransferase isozymes in Methanosarcina acetivorans C2A

Genetic analysis of the three methanol-specific methyltransferase 1 operons (mtaCB1, mtaCB2, and mtaCB3) in Methanosarcina acetivorans led to the suggestion that each of them has a discrete function during growth on methanol, which might be reflected in differential gene regulation (Pritchett and Metcalf, Mol. Microbiol. 56:1183-1194, 2005). To test this suggestion, reporter gene fusions were constructed for each of the three operons, and their expression was examined under various growth conditions. Expression of the mtaCB1 and mtaCB2 fusions was 100-fold and 575-fold higher, respectively, in methanol-grown cells than in trimethylamine (TMA)-grown cells. The mtaCB3 fusion was expressed at low levels on methanol, TMA, and dimethylamine but was significantly upregulated on monomethylamine and acetate. When TMA- or acetate-grown cultures were shifted to methanol, the mtaCB1 fusion was expressed most highly during exponential phase, whereas the mtaCB2 fusion, although strongly induced prior to mtaCB1 expression, did not reach full expression levels until stationary phase. The mtaCB3 fusion was transiently expressed prior to entry into exponential phase during a TMA-to-methanol substrate shift experiment. When acetate-grown cells were shifted to medium containing both TMA and methanol, TMA utilization commenced prior to utilization of methanol; however, these two substrates were consumed simultaneously later in growth. Under these conditions expression of the mtaCB2 and mtaCB3 fusions was delayed, suggesting that methylamines may repress their expression.     

Phosphatidylinositol 3-kinase-dependent membrane association of the Bruton's tyrosine kinase pleckstrin homology domain visualized in single living cells

Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) has been proposed to act as a second messenger to recruit regulatory proteins to the plasma membrane via their pleckstrin homology (PH) domains. The PH domain of Bruton's tyrosine kinase (Btk), which is mutated in the human disease X-linked agammaglobulinemia, has been shown to interact with PI(3,4,5)P3 in vitro. In this study, a fusion protein containing the PH domain of Btk and the enhanced green fluorescent protein (BtkPH-GFP) was constructed and utilized to study the ability of this PH domain to interact with membrane inositol phospholipids inside living cells. The localization of expressed BtkPH-GFP in quiescent NIH 3T3 cells was indistinguishable from that of GFP alone, both being cytosolic as assessed by confocal microscopy. In NIH 3T3 cells coexpressing BtkPH-GFP and the epidermal growth factor receptor, activation of epidermal growth factor or endogenous platelet-derived growth factor receptors caused a rapid (<3 min) translocation of the cytosolic fluorescence to ruffle-like membrane structures. This response was not observed in cells expressing GFP only and was completely inhibited by treatment with the PI 3-kinase inhibitors wortmannin and LY 292004. Membrane-targeted PI 3-kinase also caused membrane localization of BtkPH-GFP that was slowly reversed by wortmannin. When the R28C mutation of the Btk PH domain, which causes X-linked agammaglobulinemia, was introduced into the fluorescent construct, no translocation was observed after stimulation. In contrast, the E41K mutation, which confers transforming activity to native Btk, caused significant membrane localization of BtkPH-GFP with characteristics indicating its possible binding to PI(4,5)P2. This mutant, but not wild-type BtkPH-GFP, interfered with agonist-induced PI(4,5)P2 hydrolysis in COS-7 cells. These results show in intact cells that the PH domain of Btk binds selectively to 3-phosphorylated lipids after activation of PI 3-kinase enzymes and that losing such binding ability or specificity results in gross abnormalities in the function of the enzyme. Therefore, the interaction with PI(3,4,5)P3 is likely to be an important determinant of the physiological regulation of Btk and can be utilized to visualize the dynamics and spatiotemporal organization of changes in this phospholipid in living cells.