Kinetic analysis of recombinant mammalian α<Subscript>1</Subscript> and α<Subscript>1</Subscript>β glycine receptor channels

To analyze the influence of the beta-subunit on the kinetic properties of GlyR channel currents, alpha(1)-subunits and alpha(1)beta-subunits were transiently expressed in HEK 293 cells. A piezo dimorph was used for fast application of glycine to outside-out patches. The rise time of activation was dose dependent for both receptors and decreased with increasing glycine concentrations. Subunit composition had no effect on the time course of activation. Coexpression of alpha(1)- and beta-subunits resulted in a significantly lower EC(50) and a reduced slope of the dose-response curve of glycine compared with expression of alpha(1)-subunits alone. For both receptor subtypes, the time course of desensitization was concentration dependent. Desensitization was best fitted with a single time constant at 10-30 micro M, with two at 0.1 mM, and at saturating concentrations (0.3-3 mM) with three time constants. Desensitization of homomeric alpha(1)-receptor channels was significantly slower than that of alpha(1)beta-receptor channels. The time course of current decay after the end of glycine pulses was tested at different pulse durations of 1 mM glycine. It was best fitted with two time constants for both alpha(1) and alpha(1)beta GlyR channels, and increased significantly with increasing pulse duration.     

Standardizing global gene expression analysis between laboratories and across platforms

To facilitate collaborative research efforts between multi-investigator teams using DNA microarrays, we identified sources of error and data variability between laboratories and across microarray platforms, and methods to accommodate this variability. RNA expression data were generated in seven laboratories, which compared two standard RNA samples using 12 microarray platforms. At least two standard microarray types (one spotted, one commercial) were used by all laboratories. Reproducibility for most platforms within any laboratory was typically good, but reproducibility between platforms and across laboratories was generally poor. Reproducibility between laboratories increased markedly when standardized protocols were implemented for RNA labeling, hybridization, microarray processing, data acquisition and data normalization. Reproducibility was highest when analysis was based on biological themes defined by enriched Gene Ontology (GO) categories. These findings indicate that microarray results can be comparable across multiple laboratories, especially when a common platform and set of procedures are used.     

An LC-MS-MS method for the determination of cyclizine in human serum

Cyclizine is a piperazine derivative with anti-emetic activity that is useful in the prevention and treatment of nausea and vomiting associated with motion sickness. A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method is presented for the quantitation of cyclizine in serum. Sample pretreatment involved liquid-liquid extraction of 200 microl of serum with dichloromethane after the addition of 100 microl each of ammonium hydroxide and internal standard solutions. The extracts were analyzed by HPLC on a Luna C18 reversed-phase column and an ion-trap mass spectrometer with an electrospray interface. A limit of detection of 1 ng/ml was determined which allowed for the reliable measurement of cyclizine in the serum of human subjects. The method was found to be linear over the calibration range of 2.5-100 ng/ml. The applicability of this method was demonstrated by the analysis of serum obtained from a human volunteer following administration of a single 50 mg cyclizine hydrochloride tablet. The reported method was observed to have the necessary sensitivity, selectivity, precision and accuracy for monitoring cyclizine concentrations in human subjects following oral administration.     

Doxycycline regulation in a single retroviral vector by an autoregulatory loop facilitates controlled gene expression in liver cells

The tetracycline system has limitations in liver cells, such as toxic effects and low controllability. We generated different retroviral vectors for controlled gene expression in liver cells, in which the regulatory elements were arranged in different patterns. Only the organization of the tetracycline system in an autoregulatory loop in the sense orientation results in high retroviral titres and in tight regulation of gene expression in highly differentiated hepatoma cells. Because of the toxicity of the transactivator tTA, it was impossible to establish doxycycline-dependent stable HepG2 cell lines. To avoid sequelching-related toxicity in liver cells, we replaced tTA with new non-toxic transactivators. By using tTA2, tTA3 and tTA4, we observed tight doxycycline-dependent gene expression in 23, 49 and 45% of the isolated clones. The tTA4 vector was used to transduce hepatocytes of mice in vivo. Tight doxycycline-controllable gene regulation was also observed in the liver of mice, confirming our hypothesis that retroviral vectors with autoregulatory loops of the tetracycline system facilitate inducible gene expression in the liver in vivo. Our new retroviral vector system allows rapid isolation of controllable clones in a very high yield and should make the tetracycline system more applicable to liver-derived cells and in liver gene therapy in vivo.     

Numerical Taxonomy of Sarothamnus scoparius Rhizobia

Twenty nodule isolates from Sarothamnus scoparius (broom) growing in Poland and nine strains from plants growing in Japan were studied for phenotypic properties, plasmid presence, phage sensitivity, and host plant specificity. By numerical analysis of phenotypic properties, it was found that the studied nodule bacteria, originating from geographically different countries, constitute two separate groups affiliated to the bradyrhizobium cluster. The membership of S. scoparius rhizobia in the Bradyrhizobium genus was also supported by their long generation time, alkaline reaction in YEM medium with mannitol, lack of plasmids, and wide host plant range. Received: 12 June 2000 / Accepted: 17 July 2000     

Normal lymphoid homeostasis and lack of lethal autoimmunity in mice containing mature T cells with severely impaired IL-2 receptors

The importance of IL-2Rbeta function for immune regulation is highlighted by the severe impairment in lymphoid cell function in IL-2Rbeta-deficient mice. It has been speculated that failed IL-2/IL-2R signaling in peripheral T cells causes the associated autoimmunity, imbalanced peripheral lymphoid homeostasis, and defective T cell function. This study explored the requirement for IL-2Rbeta function in mature T lymphocytes. We show that transgenic thymic expression of the IL-2R beta-chain in IL-2Rbeta-deficient mice prevents lethal autoimmunity, restores normal production of B lymphocytes, and results in a peripheral T cell compartment that is responsive to triggering through the TCR, but not the IL-2R. The dysfunction of the IL-2R is illustrated by the near complete failure of mature T cells to proliferate to IL-2 in vitro and in vivo, to differentiate into CTL, and to up-regulate IL-2Ralpha expression. These data indicate that lymphoid homeostasis is largely maintained despite a nonfunctional IL-2R in mature T lymphocytes and suggest that IL-2Rbeta provides an essential signal during thymic development to regulate self-reactivity.     

The B-cell-specific Src-family kinase Blk is dispensable for B-cell development and activation

The B-cell lymphocyte kinase (Blk) is a src-family protein tyrosine kinase specifically expressed in B-lineage cells of mice. The early onset of Blk expression during B-cell development in the bone marrow and the high expression levels of Blk in mature B cells suggest a possible important role of Blk in B-cell physiology. To study the in vivo function of Blk, mice homozygous for the targeted disruption of the blk gene were generated. In homozygous mutant mice, neither blk mRNA nor Blk protein is expressed. Despite the absence of Blk, the development, in vitro activation, and humoral immune responses of B cells to T-cell-dependent and -independent antigens are unaltered. These data are consistent with functional redundancy of Blk in B-cell development and immune responses.