Brauselimonaden und reine Fruchtsaftlimonaden

Ohne ZusammenfassungMitteilung aus den vereinigten Laboratorien.     

Site-Specific Small Molecule Labeling of an Internal Loop in JC Polyomavirus Pentamers Using the π-Clamp-Mediated Cysteine Conjugation

The major capsid protein VP1 of JC Polyomavirus assembles into pentamers that serve as a model for studying viral entry of this potentially severe human pathogen. Previously, labeling of viral proteins utilized large fusion proteins or non-specific amine- or cysteine-functionalization with fluorescent dyes. Imaging of these sterically hindered fusion proteins or heterogeneously labeled virions limits reproducibility and could prevent the detection of subtle trafficking phenomena. Here we advance the π-clamp-mediated cysteine conjugation for site-selective fluorescent labeling of VP1-pentamers. We demonstrate a one-step synthesis of a probe consisting of a bio-orthogonal click chemistry handle bridged to a perfluoro-biphenyl π-clamp reactive electrophile by a polyethylene glycol linker. We expand the scope of the π-clamp conjugation by demonstrating selective labeling of an internal, surface exposed loop in VP1. Thus, the π-clamp conjugation offers a general method to selectively bioconjugate tags-of-interest to viral proteins without impeding their ability to bind and enter cells.     

Illness severity and self-efficacy as course predictors of DSM-IV alcohol dependence in a multisite clinical sample

OBJECTIVE: To measure cardiac and other effects of thioridazine and relate these to the plasma concentration of the parent drug and its principal metabolites. METHODS: A double-blind, randomized-order crossover study involving nine healthy male subjects compared the effects of single doses of thioridazine (10 mg and 50 mg) with placebo. Plasma concentrations of thioridazine and its ring sulfoxide, side-chain sulfoxide, and side-chain sulfone metabolites were measured, together with effects on the ECG, blood pressure, salivary flow, and a batch of psychomotor tests for 72 hours after administration. RESULTS: Thioridazine, 50 mg, reduced standing systolic blood pressure (mean peak changes from baseline [95% CI] -32 mm Hg [-55, 10 mm Hg]; p < 0.01 versus placebo) and diastolic blood pressure (-14 mm Hg [-26, -2 mm Hg]; p < 0.05), increased standing heart rate (7 beats/min [-1, 16 beats/min]; p < 0.05), impaired psychomotor function, and prolonged the JT (20 ms1/2 [7, 34 ms1/2]; p < 0.05), QTa (22 ms1/2 [8, 36 ms1/2]; p < 0.05), and QTc (22 ms1/2 [11, 33 ms1/2]; p < 0.01) intervals, but had no effect on QT dispersion (-12 ms1/2 [-31, 6 ms1/2]). Thioridazine, 1.0 mg, also significantly increased QTc, but the effect was less marked (9 ms1/2 [-1, 19 ms1/2]; p < 0.05). Plasma thioridazine and metabolite concentrations did not correlate significantly with these effects. Maximum effects on QTc occurred after peak concentrations of thioridazine but before peak concentrations of the ring sulfoxide and side-chain sulfone metabolites. CONCLUSIONS: These data suggest that thioridazine has dose-related effects on ventricular repolarization and that the parent drug causes an important proportion of these effects, although its metabolites may also contribute.     

Isolation and characterization of an intracellular serine proteinase inhibitor from a monkey kidney epithelial cell line

A recent report described a thrombin inhibitory activity in the soluble fraction of human placenta and the cytosolic fraction of K562 cells. Isolation and characterization of the functionally inactive 35-38-kDa placental form of this protein revealed that it was a novel serine proteinase inhibitor (Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993) J. Biol. Chem. 268, 9541-9547). In the present study, we observed a 67-kDa sodium dodecyl sulfate (SDS)-stable complex when 125I-thrombin was incubated with the cytosolic fraction of a monkey kidney epithelial cell line, BSC-1. This complex was not observed in either the particulate cell fraction extracted with 0.2% Triton X-100 or medium conditioned by cells, suggesting that the thrombin-complexing factor is confined to the cytoplasm. The cytoplasmic antithrombin activity was purified to apparent homogeneity from the cytosol of BSC-1 cells previously pulsed with [35S]methionine by a combination of heparin-agarose chromatography, Mono Q fast protein liquid chromatography, and anhydrotrypsin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-purified preparation by SDS-polyacrylamide gel electrophoresis and fluorography revealed a single protein with an apparent molecular mass of 38 kDa. The purified 38-kDa protein inhibited the amidolytic activities of thrombin, trypsin, urokinase, and factor Xa but not that of elastase. Incubation of the 38-kDa protein with excess thrombin identified approximately 60% of the labeled 38-kDa protein in an SDS-stable 67-kDa complex. The purified 38-kDa inhibitor was cleaved with cyanogen bromide and the isolated peptides subjected to microsequencing. Amino acid sequence obtained for a region within this protein exhibited significant homology with human antithrombin III and plasminogen activator inhibitors 1 and 2. This homologous peptide contained the full complement of residues designated as highly conserved in helix F of the greater serine proteinase inhibitor superfamily. In addition, an internal sequence of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported for an internal sequence of the human placental inhibitor. These findings confirm the existence of a novel cytoplasmic serine proteinase inhibitor in mammalian cells and provide additional details of its molecular properties. The physiological function of this novel serine proteinase inhibitor in cytoplasm is unknown.     

Surround repulsion of spinal sensory axons in higher vertebrate embryos

We have tested whether the orientation of axons sprouting from bipolar dorsal root ganglion neurons is influenced by diffusible cues from surrounding tissues. Surface ectoderm, dermomyotome, and notochord exert strong chemorepulsion on axons growing in collagen gels, operating at separations beyond those found in vivo and active in cocultures of chick and mouse tissues. Basal and alar plates of the neural tube are devoid of activity, as is the posterior-half-sclerotome, which repels in a contact-dependent manner. When ganglia are sandwiched between dermomyotome and notochord placed at a distance, axon growth is channeled in a bipolar trajectory. These results show that gradients of diffusible repulsion molecules flanking axon pathways can generate linear patterns of axon growth. We suggest that such "surround repulsion" may function generally, in concert with contact-dependent guidance mechanisms, to guide axons in the developing nervous system.