RhoA Interacts with the Fusion Glycoprotein of Respiratory Syncytial Virus and Facilitates Virus-Induced Syncytium Formation

The fusion glycoprotein (F) of respiratory syncytial virus (RSV), which mediates membrane fusion and virus entry, was shown to bind RhoA, a small GTPase, in yeast two-hybrid interaction studies. The interaction was confirmed in vivo by mammalian two-hybrid assay and in RSV-infected HEp-2 cells by coimmunoprecipitation. Furthermore, the interaction of F with RhoA was confirmed in vitro by enzyme-linked immunosorbent assay and biomolecular interaction analysis. Yeast two-hybrid interaction studies with various deletion mutants of F and with RhoA indicate that the key binding domains of these proteins are contained within, or overlap, amino acids 146 to 155 and 67 to 110, respectively. The biological significance of this interaction was studied in RSV-infected HEp-2 cells that were stably transfected to overexpress RhoA. There was a positive correlation between RhoA expression and RSV syncytium formation, indicating that RhoA can facilitate RSV-induced syncytium formation.     

Mechanochemical coupling in spin-labeled, active, isometric muscle

Observed effects of inorganic phosphate (P(i)) on active isometric muscle may provide the answer to one of the fundamental questions in muscle biophysics: how are the free energies of the chemical species in the myosin-catalyzed ATP hydrolysis (ATPase) reaction coupled to muscle force?. Pflugers Arch. 414:73-81) showed that active, isometric muscle force varies logarithmically with [P(i)]. Here, by simultaneously measuring electron paramagnetic resonance and the force of spin-labeled muscle fibers, we show that, in active, isometric muscle, the fraction of myosin heads in any given biochemical state is independent of both [P(i)] and force. These direct observations of mechanochemical coupling in muscle are immediately described by a muscle equation of state containing muscle force as a state variable. These results challenge the conventional assumption mechanochemical coupling is localized to individual myosin heads in muscle.     

Real-time imaging of the dynamics of secretory granules in growth cones

Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer.     

Stabilization of dry Mammalian cells: lessons from nature

The Center for Biostabilization at UC Davis is attempting tostabilize mammalian cells in the dry state. We review here someof the lessons from nature that we have been applying to thisenterprise, including the use of trehalose, a disaccharide foundat high concentrations in many anhydrobiotic organisms, to stabilizebiological structures, both in vitro and in vivo. Trehalosehas useful properties for this purpose and in at least in onecase—human blood platelets—introducing this sugarmay be sufficient to achieve useful stabilization. Nucleatedcells, however, are stabilized by trehalose only during theinitial stages of dehydration. Introduction of a stress proteinobtained from an anhydrobiotic organism, Artemia, improves thestability markedly, both during the dehydration event and followingrehydration. Thus, it appears that the stabilization will requiremultiple adaptations, many of which we propose to apply fromstudies on anhydrobiosis.     

Effects of temperature on calcium-sensitive fluorescent probes

The effect of temperature on the binding equilibria of calcium-sensing dyes has been extensively studied, but there are also important temperature-related changes in the photophysics of the dyes that have been largely ignored. We conducted a systematic study of thermal effects on five calcium-sensing dyes under calcium-saturated and calcium-free conditions. Quin-2, chlortetracycline, calcium green dextran, Indo-1, and Fura-2 all show temperature-dependent effects on fluorescence in all or part of the range tested (5-40 degrees C). Specifically, the intensity of the single-wavelength dyes increased at low temperature. The ratiometric dyes, because of variable effects at the two wavelengths, showed, in general, a reduction in the fluorescence ratio as temperature decreased. Changes in viscosity, pH, oxygen quenching, or fluorescence maxima could not fully explain the effects of temperature on fluorescence. The excited-state lifetimes of the dyes were determined, in both the presence and absence of calcium, using multifrequency phase-modulation fluorimetry. In most cases, low temperature led to prolonged fluorescence lifetimes. The increase in lifetimes at reduced temperature is probably largely responsible for the effects of temperature on the physical properties of the calcium-sensing dyes. Clearly, these temperature effects can influence reported calcium concentrations and must therefore be taken into consideration during any investigation involving variable temperatures.     

Helicobacter pylori urease binds to class II MHC on gastric epithelial cells and induces their apoptosis

Infection by Helicobacter pylori leads to injury of the gastric epithelium and a cellular infiltrate that includes CD4+ T cells. H. pylori binds to class II MHC molecules on gastric epithelial cells and induces their apoptosis. Because urease is an abundant protein expressed by H. pylori, we examined whether it had the ability to bind class II MHC and induce apoptosis in class II MHC-bearing cells. Flow cytometry revealed the binding of PE-conjugated urease to class II MHC+ gastric epithelial cell lines. The binding of urease to human gastric epithelial cells was reduced by anti-class II MHC Abs and by staphylococcal enterotoxin B. The binding of urease to class II MHC was confirmed when urease bound to HLA-DR1-transfected COS-1 (1D12) cells but not to untransfected COS-1 cells. Urease also bound to a panel of B cell lines expressing various class II MHC alleles. Recombinant urease induced apoptosis in gastric epithelial cells that express class II MHC molecules, but not in class II MHC- cells. Also, Fab from anti-class II MHC and not from isotype control Abs blocked the induction of apoptosis by urease in a concentration-dependent manner. The adhesin properties of urease might point to a novel and important role of H. pylori urease in the pathogenesis of H. pylori infection.     

Golf complements a GPA1 null mutation in Saccharomyces cerevisiae and functionally couples to the STE2 pheromone receptor

We have produced a plasmid designed for the expression of heterologous G protein alpha subunits in the yeast Saccharomyces cerevisiae. Introduction of these genes is by simple cassette replacement using unique restriction sites, and their expression is controlled by the regulatory sequences of the S. cerevisiae GPA1 gene. Levels of expression are therefore suitable for interaction of these heterologous proteins with elements of the yeast pheromone response pathway. We believe that this plasmid will facilitate the coupling of more members of the seven transmembrane domain superfamily of receptors, through their native G protein alpha subunit, to the yeast pheromone response pathway. The plasmid pRGP, is a stable centromeric shuttle vector with a HIS3-selectable marker. We have demonstrated that production of GPA1 from this plasmid functionally complements a gpal1- null mutation. A similar response is obtained when an alternative G protein alpha subunit, G(olf), is introduced using pRGP. We believe that this is the first example of a heterologous G protein shown to couple to a yeast pheromone receptor.     

Dynamics of parietal neural activity during spatial cognitive processing

Dynamic neural processing unrelated to changes in sensory input or motor output is likely to be a hallmark of cognitive operations. Here we show that neural representations of space in parietal cortex are dynamic while monkeys perform a spatial cognitive operation on a static visual stimulus. We recorded neural activity in area 7a during a visual maze task in which monkeys mentally followed a path without moving their eyes. We found that the direction of the followed path could be recovered from neuronal population activity. When the monkeys covertly processed a path that turned, the population representation of path direction shifted in the direction of the turn. This neural population dynamic took place during a period of unchanging visual input and showed characteristics of both serial and parallel processing. The data suggest that the dynamic evolution of parietal neuronal activity is associated with the progression of spatial cognitive operations.