Isolation by fluorescence-activated cell sorting of Chinese hamster ovary cell lines with pleiotropic, temperature-conditional defects in receptor recycling.

We have isolated several Chinese hamster ovary cell lines with temperature-sensitive defects in the recycling of receptors after endocytosis. These cell lines were selected using fluorescence-activated cell sorting for retention of a pulse of labeled transferrin after a chase in the presence of unlabeled transferrin. One of these cell lines, TfT1.11, was selected for further characterization. In TfT1.11 the trapping of transferrin within the cells is paralleled by a loss of cell surface transferrin receptors. Within 4 h after the shift from 33 to 41 degrees C the surface binding of transferrin is reduced to 18% of parental cells at 41 degrees C. The trapping of transferrin and the loss of transferrin receptor from the cell surface are caused by a temperature-conditional 5.5-fold decrease in the initial rate of transferrin recycling. TfT1.11 cells also rapidly lose 89% of their ability to take up alpha 2-macroglobulin after the temperature shift to 41 degrees C. These data indicate that the TfT1.11 cell line has a pleiotropic defect in receptor recycling.     

A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell.     

An Aqueous Two-Phase System for the Concentration and Extraction of Proteins from the Interface for Detection Using the Lateral-Flow Immunoassay

The paper-based immunoassay for point-of-care diagnostics is widely used due to its low cost and portability over traditional lab-based assays. Lateral-flow immunoassay (LFA) is the most well-established paper-based assay since it is rapid and easy to use. However, the disadvantage of LFA is its lack of sensitivity in some cases where a large sample volume is required, limiting its use as a diagnostic tool. To improve the sensitivity of LFA, we previously reported on the concentration of analytes into one of the two bulk phases of an aqueous two-phase system (ATPS) prior to detection. In this study, we preserved the advantages of LFA while significantly improving upon our previous proof-of-concept studies by employing a novel approach of concentrating gold nanoparticles, a common LFA colorimetric indicator. By conjugating specific antibodies and polymers to the surfaces of the particles, these gold nanoprobes (GNPs) were able to capture target proteins in the sample and subsequently be concentrated within 10 min at the interface of an ATPS solution comprised of polyethylene glycol, potassium phosphate, and phosphate-buffered saline. These GNPs were then extracted and applied directly to LFA. By combining this prior ATPS interface extraction with LFA, the detection limit of LFA for a model protein was improved by 100-fold from 1 ng/μL to 0.01 ng/μL. Additionally, we examined the behavior of the ATPS system in fetal bovine serum and synthetic urine to more closely approach real-world applications. Despite using more complex matrices, ATPS interface extraction still improved the detection limit by 100-fold within 15 to 25 min, demonstrating the system’s potential to be applied to patient samples.     

Uptake of tritium-labelled biogenic amines by the prostomium of the polychaete Nereis virens (Sars) (Annelida)

The uptake of tritium-labelled 5-HT, noradrenaline, 5-hydroxytrytophan, DOPA and dopamine by the cerebral ganglion and prostomial nervous system of the polychaete Nereis virens has been examined using radioautography at the level of the light microscope. Pronounced uptake of (3)H-5HT occurred in the antennal, palpal, tegumentary and nuchal nerves as well as in ganglionic nuclei 13, 14, 15, 16, 17, 20, 24 and 25, the mid-brain neuropile, the neurosecretory neuropil and the infracerebral organ; (3)H-NA uptake was observed in small cells in the prostomial epidermis, and the infracerebral organ; (3)H-dopamine only in one of two common types of epidermal mucus cells. Prostomial muscles labelled generally with (3)H-NA and at specific sites with (3)H-5HT. These observations support the concept of an efferent serotonergic system originating in several cerebral ganglionic nuclei and serving prostomial muscle and epidermis. Evidence for an afferent adrenergic system is less substantial. The role of dopamine remains obscure.     

The BH3-only protein bid is dispensable for DNA damage- and replicative stress-induced apoptosis or cell-cycle arrest

Bid, a caspase-activated proapoptotic BH3-only protein, is essential for Fas-induced hepatocyte destruction. Recent studies published in Cell produced conflicting results, indicating that loss of Bid either protects or enhances apoptosis induced by DNA damage or replicative stress. To resolve this controversy, we generated novel Bid-deficient mice on an inbred C57BL/6 background and removed the drug-selection cassette from the targeted locus. Nine distinct cell types from these Bid-deficient mice underwent cell-cycle arrest and apoptosis in a manner indistinguishable from control WT cells in response to DNA damage or replicative stress. Moreover, we found that even cells from the original Bid-deficient mice responded normally to these stimuli, indicating that differences in genetic background or the presence of a strong promoter within the targeted locus are unlikely to explain the differences between our results and those reported previously. We conclude that Bid has no role in DNA damage- or replicative stress-induced apoptosis or cell-cycle arrest.     

Complete and integrated pyrene degradation pathway in Mycobacterium vanbaalenii PYR-1 based on systems biology

Mycobacterium vanbaalenii PYR-1 was the first bacterium isolated by virtue of its ability to metabolize the high-molecular-weight polycyclic aromatic hydrocarbon (PAH) pyrene. We used metabolic, genomic, and proteomic approaches in this investigation to construct a complete and integrated pyrene degradation pathway for M. vanbaalenii PYR-1. Genome sequence analyses identified genes involved in the pyrene degradation pathway that we have proposed for this bacterium. To identify proteins involved in the degradation, we conducted a proteome analysis of cells exposed to pyrene using one-dimensional gel electrophoresis in combination with liquid chromatography-tandem mass spectrometry. Database searching performed with the M. vanbaalenii PYR-1 genome resulted in identification of 1,028 proteins with a protein false discovery rate of <1%. Based on both genomic and proteomic data, we identified 27 enzymes necessary for constructing a complete pathway for pyrene degradation. Our analyses indicate that this bacterium degrades pyrene to central intermediates through o-phthalate and the beta-ketoadipate pathway. Proteomic analysis also revealed that 18 enzymes in the pathway were upregulated more than twofold, as indicated by peptide counting when the organism was grown with pyrene; three copies of the terminal subunits of ring-hydroxylating oxygenase (NidAB2, MvanDraft_0817/0818, and PhtAaAb), dihydrodiol dehydrogenase (MvanDraft_0815), and ring cleavage dioxygenase (MvanDraft_3242) were detected only in pyrene-grown cells. The results presented here provide a comprehensive picture of pyrene metabolism in M. vanbaalenii PYR-1 and a useful framework for understanding cellular processes involved in PAH degradation.     

A quantitative structure activity analysis on the relative sensitivity of the olfactory and the nasal trigeminal chemosensory systems

We have applied a quantitative structure-activity relationship (QSAR) approach to analyze the chemical parameters that determine the relative sensitivity of olfaction and nasal chemesthesis to a common set of volatile organic compounds (VOCs). We used previously reported data on odor detection thresholds (ODTs) and nasal pungency thresholds (NPTs) from 64 VOCs belonging to 7 chemical series (acetate esters, carboxylic acids, alcohols, aliphatic aldehydes, alkylbenzenes, ketones, and terpenes). The analysis tested whether NPTs could be used to separate out "selective" chemosensory effects (i.e., those resting on the transfer of VOCs from the gas phase to the receptor phase) from "specific" chemosensory effects in ODTs. Previous work showed that selective effects overwhelmingly dominate chemesthetic potency whereas both selective and specific effects control olfactory potency. We conclude that it is indeed possible to use NPTs to separate out selective from specific effects in ODTs. Among the series studied, aldehydes and acids, except for formic acid, show clear specific effects in their olfactory potency. Furthermore, for VOCs whose odor potency rests mainly on selective effects, we have developed a QSAR equation that can predict their ODTs based on their NPTs.