Validation of a gas chromatography-mass spectrometry method for the determination of pg/ml levels of 17beta-estradiol and 17beta-trenbolone in bovine serum

A method for the quantitation of pg/ml levels of 17beta-estradiol and 17beta-trenbolone in bovine serum by gas chromatography/electron-capture mass spectrometry has been developed and validated. Using the area ratios of the integrated molecular-ion peaks of the analytes to their corresponding deuterated internal standards, [2,4,16,16-2H4] 17beta-estradiol (17beta-estradiol-d(4)) and [16,16-2H2] 17beta-trenbolone (17beta-trenbolone-d(2)), and non-weighted linear regression, two calibration curves per analyte; 5-50 and 50-500 pg/ml for 17beta-estradiol in sera, and 25-250 and 250-2500 pg/ml for 17beta-trenbolone in sera, respectively, were constructed. Splitless injection of 200 fg 17beta-estradiol and 1000 fg 17beta-trenbolone could be detected and quantified. Tested batches of control bovine sera did not exhibit interference for 17beta-trenbolone, and showed expected background presence of endogenous 17beta-estradiol. Intra-day residual errors did not exceed 20%, and regression correlations were greater than 0.99. Intra-day precision data was similar to inter-day precision data. Using this method, 16 samples can be processed within one working day.     

FlyGEM, a full transcriptome array platform for the Drosophila community

We have constructed a DNA microarray to monitor expression of predicted genes in Drosophila. By using homotypic hybridizations, we show that the array performs reproducibly, that dye effects are minimal, and that array results agree with systematic northern blotting. The array gene list has been extensively annotated and linked-out to other databases. Incyte and the NIH have made the platform available to the community via academic microarray facilities selected by an NIH committee.     

Allelic variation at alcohol metabolism genes (<Emphasis Type="Italic">ADH1B</Emphasis>,<Emphasis Type="Italic"> ADH1C</Emphasis>,<Emphasis Type="Italic"> ALDH2</Emphasis>) and alcohol dependence in an American Indian population

Enzymes encoded by two gene families, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), mediate alcohol metabolism in humans. Allelic variants have been identified that alter metabolic rates and influence risk for alcoholism. Specifically, ADH1B*47His (previously ADH2-2) and ALDH2-2 have been shown to confer protection against alcoholism, presumably through accumulation of acetaldehyde in the blood and a resultant 'flushing response' to alcohol consumption. In the current study, variants at ADH1B (previously ADH2), ADH1C (previously ADH3), and ALDH2 were assayed in DNA extracts from participants belonging to a Southwest American Indian tribe (n=490) with a high prevalence of alcoholism. Each subject underwent a clinical interview for diagnosis of alcohol dependence, as well as evaluation of intermediate phenotypes such as binge drinking and flushing response to alcohol consumption. Detailed haplotypes were constructed and tested against alcohol dependence and related intermediate phenotypes using both association and linkage analysis. ADH and ALDH variants were also assayed in three Asian and one African population (no clinical data) in order to provide an evolutionary context for the haplotype data. Both linkage and association analysis identified several ADH1C alleles and a neighboring microsatellite marker that affected risk of alcohol dependence and were also related to binge drinking. These data strengthen the support for ADH as a candidate locus for alcohol dependence and suggest further productive study.     

Amino-terminal and midmolecule parathyroid hormone-related protein,phosphatidylcholine, and type II cell proliferation in silica-injured lung

Acute silica lung injury is marked by alveolar phospholipidosis and type II cell proliferation. Parathyroid hormone-related protein (PTHrP) 1-34 could have a regulatory role in this process because it stimulates phosphatidylcholine secretion and inhibits type II cell growth. Other regions of the PTHrP molecule may have biological activity and can also exert pulmonary effects. This study examined the temporal pattern for expression of several regions of PTHrP after silica lung injury and evaluated the effects of changes in expression on cell proliferation and lung phospholipids. Expression of all PTHrP regions fell at 4 days after injury. Reversing the decline in PTHrP 1-34 or PTHrP 67-86 with one intratracheal dose and four daily subcutaneous doses of PTHrP 1-34 or PTHrP 67-86 stimulated bronchoalveolar lavage disaturated phosphatidylcholine (DSPC) levels. Cell culture studies indicate that the peptides exerted direct effects on DSPC secretion by type II cells. Neither peptide affected type II cell proliferation with this dosing regimen, but addition of an additional intratracheal dose resulted in significant inhibition of growth, consistent with previous effects of PTHrP 1-34 in hyperoxic lung injury. These studies establish a regulatory role for PTHrP 1-34 and PTHrP 67-86 in DSPC metabolism and type II cell proliferation in silica injury. Growth inhibitory effects of PTHrP could interact with phospholipid stimulation by affecting type II cell numbers. Further studies are needed to explore the complex interactions of PTHrP-derived peptides and the type II cell response at various stages of silica lung injury.     

Cutting edge: cross-presentation as a mechanism for efficient recruitment of tumor-specific CTL to the brain

The number and localization of effector cells to the tumor site are crucial elements for immune rejection of solid tumors. However, for cerebral malignancies, antitumor responses need to be finely tuned to avoid neuropathologic consequences. In this study, we determine factors that regulate CTL localization and tumoricidal function after intracerebral implantation of tumors expressing model Ag. H-2(bxd) mice implanted with a CW3(+) murine glioma lacking H-2K(d) molecules necessary to present the CW3(170-179) epitope demonstrate cross-priming of H-2K(d)-restricted CTL, and moreover, Ag-dependent accumulation of functional H-2K(d)/CW3(170-179)-specific CTL within the tumor bed. This implicates a role for cross-presentation not only in priming, but also in retention of fully differentiated CTL in the tumor stroma at the effector stage of the response. Modulating cross-presentation of Ag may be the key in regulating specific immune responses in the brain: either by augmenting protective responses or by down-modulating destructive autoimmune reactions.     

An attenuating mutation in nsP1 of the Sindbis-group virus S.A.AR86 accelerates nonstructural protein processing and up-regulates viral 26S RNA synthesis

The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.     

Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis

Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.     

Quantification of hepatic carbohydrate metabolism in conscious mice using serial blood and urine spots

In vivo studies of hepatic carbohydrate metabolism in (genetically modified) conscious mice are hampered by limitations of blood and urine sample sizes. We developed and validated methods to quantify stable isotope dilution and incorporation in small blood and urine samples spotted onto filter paper. Blood glucose and urinary paracetamol-glucuronic acid were extracted from filter paper spots reproducibly and with high yield. Fractional isotopomer distributions of glucose and paracetamol-glucuronic acid when extracted from filter paper spots were almost identical to those isolated from the original body fluids. Rates of infusion of labeled compounds could be adjusted without perturbing hepatic glucose metabolism. This approach was used in mice to find the optimal metabolic condition for the study of hepatic carbohydrate metabolism. In fed mice, no isotopic steady state was observed during a 6-h label-infusion experiment. In 9-h-fasted mice, isotopic steady state was reached after 3 h of label infusion and important parameters in hepatic glucose metabolism could be calculated. The rate of de novo glucose-6-phosphate synthesis was 143 +/- 17 micromol kg(-1) min(-1) and partitioning to plasma glucose was 79.0 +/- 5.2%. In 24-h-fasted mice, abrupt changes were noticed in whole body and in hepatic glucose metabolism at the end of the experiment.     

Extraction and reconstitution of calponin and consequent contractile ability in permeabilized smooth muscle fibers

We demonstrate reduction and restoration of contractile ability in response to protein extraction and reconstitution in Triton X-100/glycerol-permeabilized smooth muscle fibers. Through significant reduction in the content of caldesmon (CaD), calponin (CaP), and the 20-kDa regulatory light chain (RLC) of myosin, but not other contractile proteins in "chemically skinned" fibers, we substantially reduced the contractile ability of these fibers, as measured by their ability to generate isometric force and to hydrolyze ATP by actomyosin Mg2+ ATPase. When the protein-depleted fibers were then reconstituted (either with a mixture of purified protein standards of CaD, CaP, and myosin RLC or with a protein extract from the demembranized muscle fibers containing CaD, CaP, and myosin RLC plus several low-molecular-mass proteins), all proteins used for reincorporation returned nearly to control levels, as did isometric force generation and rate of ATP hydrolysis. The fact that the low-molecular-mass proteins do not affect contractility in this model system indicates that our methods for reversible modulation of the content of CaP and CaD may provide a valuable tool for studying the thin-filament-based regulation of contractility.