Chromatographic detection of residual cellular DNA on short monolithic columns

Analysis of crude samples from biotechnological processes is often required to demonstrate that residual host cell impurities are reduced or eliminated during purification. Current knowledge suggests that a continuous-cell-line DNA can be considered a cellular contaminant rather than a significant risk factor requiring removal to extremely low levels. Anion-exchange chromatography is one of the most important methods used in the downstream processing and analysis of different biomolecules. In this article, an application using Convective Interaction Media monolithic columns to improve the detection of residual cellular DNA is described.     

Cutting edge: the ontogeny and function of Va14Ja18 natural T lymphocytes require signal processing by protein kinase C theta and NF-kappa B

The rapid and robust immunoregulatory cytokine response of Va14Ja18 natural T (iNKT) cells to glycolipid Ags determines their diverse functions. Unlike conventional T cells, iNKT lymphocyte ontogeny absolutely requires NF-kappa B signaling. However, the precise role of NF-kappa B in iNKT cell function and the identity of upstream signals that activate NF-kappa B in this T cell subset remain unknown. Using mice in which iNKT cell ontogeny has been rescued despite inhibition of NF-kappa B signaling, we demonstrate that iNKT cell function requires NF-kappa B in a lymphocyte-intrinsic manner. Furthermore, the ontogeny of functional iNKT cells requires signaling through protein kinase C theta, which is dispensable for conventional T lymphocyte development. The unique requirement of protein kinase C theta implies that signals emanating from the TCR activate NF-kappa B during iNKT cell development and function. Thus, we conclude that NF-kappa B signaling plays a crucial role at distinct levels of iNKT cell biology.     

The role of rat Crry, a complement regulatory protein, in proliferation of thymocytes

In our previous work we showed that 3F10 monoclonal antibody (mAb), which recognizes the rat complement receptor 1-related/gene protein y (Crry), induces homotipic aggregation of thymocytes. In this work we studied the effect of 3F10 mAb on proliferation of rat thymocytes stimulated with concanavalin A (ConA) or by cross-linking the T cell receptor (TCR) by anti-alphabetaTCR mAb (R73), in vitro, and the mechanisms involved in the process. Our results show that 3F10 mAb stimulates proliferation of total thymocytes triggered by suboptimal concentrations of ConA or TCR cross-linking, in a dose-dependent manner. Maximal stimulation was observed using 10 microg/ml and 20 microg/ml of 3F10 mAb, respectively. The 3F10-induced stimulation of thymocytes proliferation in the presence of ConA, that was followed by increased production of interleukin-2 (IL-2), up-regulation of the expression of IL-2 receptor alpha (IL-2Ralpha) and was inhibited by anti-CD11a and anti-CD18 mAbs. Purified thymocytes did not respond by proliferation to 3F10 mAb, either alone or in combination with R73 mAb or ConA. Proliferation of these cells was achieved only in the presence of OX-6+ antigen-presenting cells (APC) and additional signals transmitted by TCR or ConA. These results suggest that Crry is involved in the LFA-1 dependent proliferation of thymocytes, a phenomenon that has not been recognized so far.     

Comparative study of the antimutagenic potential of Vitamin E in different E. coli strains

The antimutagenic potential of Vitamin E due to its antioxidative properties was studied. The new Escherichia coli K12 assay-system designed in our laboratory was employed in order to detect the antimutagenic potential of Vitamin E and to determine its molecular mechanisms of action. The assay is composed of three tests. In Test A, we examine the influence of the antioxidant on induced oxidative mutagenesis in a repair-proficient strain. Spontaneous mutagenesis is monitored in Test B, which is performed with two mutator strains, one mismatch repair-deficient (mutS) and another deficient in 8-oxo-dGTP-ase activity (mutT). In Test M, a repair-proficient strain and its mismatch repair-deficient counterpart (mutH), both carrying a plasmid with microsatellite sequences, are used to measure the level of microsatellite instability. To examine the antimutagenic potential of Vitamin E we also used the WP2 antimutagenicity test. Protective properties of Vitamin E against oxidative mutagenesis were detected in all tests with the E. coli K12 assay-system as well as in the WP2 antimutagenicity test. This study confirms that mismatch repair is essential for repair of oxidative DNA damage. The results obtained indicate that Vitamin E prevents the formation of DNA adducts by lipid peroxidation products rather than those formed by direct oxidation of DNA bases. Moreover, it can reduce microsatellite instability. After further validation, the new E. coli K12 assay-system can be used to test the antimutagenic potential of antioxidants.     

Near-infrared fluorescent deoxyglucose analogue for tumor optical imaging in cell culture and living mice

2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has extensively been used for clinical diagnosis, staging, and therapy monitoring of cancer and other diseases. Nonradioactive glucose analogues enabling the screening of the glucose metabolic rate of tumors are of particular interest for anticancer drug development. A nonradioactive fluorescent deoxyglucose analogue may have many applications for both imaging of tumors and monitoring therapeutic efficacy of drugs in living animals and may eventually translate to clinical applications. We found that a fluorescent 2-deoxyglucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), can be delivered in several tumor cells via the glucose transporters (GLUTs). We therefore conjugated D-glucosamine with a near-infrared (NIR) fluorphor Cy5.5 and tested the feasibility of the Cy5.5-D-glucosamine (Cy5.5-2DG) conjugate for NIR fluorescence imaging of tumors in a preclinical xenograft animal model. Cy5.5-2DG was prepared by conjugating Cy5.5 monofunctional N-hydroxysuccinimide ester (Cy5.5-NHS) and D-glucosamine followed by high-performance liquid chromatography purification. The accumulation of Cy5.5-2DG and Cy5.5-NHS in different tumor cell lines at 37 and 4 degrees C were imaged using a fluorescence microscope. Tumor targeting and retention of Cy5.5-2DG and Cy5.5-NHS in a subcutaneous U87MG glioma and A375M melanoma tumor model were evaluated and quantified by a Xenogen IVIS 200 optical cooled charged-coupled device system. Fluorescence microscopy imaging shows that Cy5.5-2DG and Cy5.5-NHS are taken up and trapped by a variety of tumor cell lines at 37 degrees C incubation, while they exhibit marginal uptake at 4 degrees C. The tumor cell uptake of Cy5.5-2DG cannot be blocked by the 50 mM D-glucose, suggesting that Cy5.5-2DG may not be delivered in tumor cells by GLUTs. U87MG and A375M tumor localization was clearly visualized in living mice with both NIR fluorescent probes. Tumor/muscle contrast was clearly visible as early as 30 min postinjection (pi), and the highest U87MG tumor/muscle ratios of 2.81 +/- 0.10 and 3.34 +/- 0.23 were achieved 24 h pi for Cy5.5-2DG and Cy5.5-NHS, respectively. While as a comparison, the micropositron emission tomography imaging study shows that [18F]FDG preferentially localizes to the U87MG tumor, with resulting tumor/muscle ratios ranging from 3.89 to 4.08 after 30 min to 2 h postadministration of the probe. In conclusion, the NIR fluorescent glucose analogues, Cy5.5-2DG and Cy5.5-NHS, both demonstrate tumor-targeting abilities in cell culture and living mice. More studies are warranted to further explore their application for optical tumor imaging. To develop NIR glucose analogues with the ability to target GLUTs/hexokinase, it is highly important to select NIR dyes with a reasonable molecular size.     

A microsatellite-based genetic linkage map of the waterflea, Daphnia pulex: On the prospect of crustacean genomics

We describe the first genetic linkage map for Daphnia pulex using 185 microsatellite markers, including 115 new markers reported in this study. Our approach was to study the segregation of polymorphisms in 129 F2 progeny of one F1 hybrid obtained by crossing two genetically divergent lineages of Daphnia isolated from two Oregon populations. The map spanned 1206 Kosambi cM and had an average intermarker distance of 7 cM. Linkage groups ranged in size from 7 to 185 cM and contained 4 to 27 markers. The map revealed 12 linkage groups corresponding to the expected number of chromosomes and covers approximately 87% of the genome. Tests for random segregation of alleles at individual loci revealed that 21% of the markers showed significant transmission ratio distortion (primarily homozygote deficiency) likely due to markers being linked to deleterious recessive alleles. This map will become the anchor for the physical map of the Daphnia genome and will serve as a starting point for mapping single and quantitative trait loci affecting ecologically important phenotypes. By mapping 342 tentative orthologous gene pairs (Daphnia/Drosophila) into the Daphnia linkage map, we facilitate future comparative projects.     

Autotaxin and its product lysophosphatidic acid suppress brown adipose differentiation and promote diet-induced obesity in mice

Brown adipose tissue is a thermogenic organ that dissipates stored energy as heat to maintain body temperature. This process may also provide protection from development of diet-induced obesity. We report that the bioactive lipid mediator lysophosphatidic acid (LPA) markedly decreases differentiation of cultured primary brown adipocyte precursors, whereas potent selective inhibitors of the LPA-generating enzyme autotaxin (ATX) promote differentiation. Transgenic mice overexpressing ATX exhibit reduced expression of brown adipose tissue-related genes in peripheral white adipose tissue and accumulate significantly more fat than wild-type controls when fed a high-fat diet. Our results indicate that ATX and its product LPA are physiologically relevant negative regulators of brown fat adipogenesis and are consistent with a model in which a decrease in mature peripheral brown adipose tissue results in increased susceptibility to diet-induced obesity in mice.     

Hierarchical recruitment into nascent ribosomes of assembly factors required for 27SB pre-rRNA processing in Saccharomyces cerevisiae

To better define the roles of assembly factors required for eukaryotic ribosome biogenesis, we have focused on one specific step in maturation of yeast 60 S ribosomal subunits: processing of 27SB pre-ribosomal RNA. At least 14 assembly factors, the ‘B-factor’ proteins, are required for this step. These include most of the major functional classes of assembly factors: RNA-binding proteins, scaffolding protein, DEAD-box ATPases and GTPases. We have investigated the mechanisms by which these factors associate with assembling ribosomes. Our data establish a recruitment model in which assembly of the B-factors into nascent ribosomes ultimately leads to the recruitment of the GTPase Nog2. A more detailed analysis suggests that this occurs in a hierarchical manner via two largely independent recruiting pathways that converge on Nog2. Understanding recruitment has allowed us to better determine the order of association of all assembly factors functioning in one step of ribosome assembly. Furthermore, we have identified a novel subcomplex composed of the B-factors Nop2 and Nip7. Finally, we identified a means by which this step in ribosome biogenesis is regulated in concert with cell growth via the TOR protein kinase pathway. Inhibition of TOR kinase decreases association of Rpf2, Spb4, Nog1 and Nog2 with pre-ribosomes.     

Biomarkers of mild hyperthermia related to flashover training in firefighters

Flashover phenomenon occurs when surfaces exposed to thermal radiation reach the ignition temperature, and the fire rapidly spreads in enclosed area. Flashover training (FOT) performed by firefighters is a simulation of flashover phenomenon under controlled conditions. The study aimed to test thermal and physical strain in male firefighters and instructors attending FOT and its influence on DNA damage, exhaled breath condensate (EBC) pH, and fraction of exhaled nitric oxide (FeNO). DNA damage markers were analyzed in 51 attendees and 7 instructors, and EBC pH and FeNO in 40 respiratory healthy non-smoking subjects (34 attendees and 6 instructors).The average body temperature and pulse increase was 1.1 °C and 30 beats per minute, respectively. A prominent increase in the alkali-labile sites' level has been observed in instructors' peripheral leukocytes compared to first-time attendees (tail length p=0.050, % of DNA in tail p=0.005). FOT was related only to physiological FVC and FEV₁ increase (by 4% and 2.7% on average), and FeNO dropped after the exercise by 2 ppb in comparison with basal values (P=0.034). EBC pH did not change during FOT, but FeNO was inversely correlated to EBC pH after the exercise (Spearman's rho=−0.66, P=0.013). With respect to the thermal and physical strain, FOT is considered to be a safe training procedure for healthy firefighters. The increase rate in primary DNA damage found in the instructors' peripheral leukocytes requires further examination in a larger sample size.