A protein that is highly related to GTPase-activating protein-associated p62 complexes with phospholipase C gamma.

p62 is a highly tyrosyl phosphorylated protein that was first identified in immunoprecipitates of the GTPase-activating protein (GAP) of p21ras from cells transformed by oncogenic nonreceptor tyrosine kinases or stimulated through tyrosine kinase receptors (C. Ellis, M. Moran, F. McCormick, and T. Pawson, Nature 343:377-381, 1991). In this article we describe a highly related 62-kDa protein that becomes tyrosyl phosphorylated and associated with phospholipase C gamma (PLC gamma) in C3H10T1/2 cells stimulated with epidermal growth factor (EGF) or transformed by v-src. GAP-associated and PLC gamma-associated p62 comigrated in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited nearly identical phosphotryptic peptide patterns. That the association of p62 with PLC gamma was direct and not mediated through binding of GAP-p62 to PLC gamma or to the EGF receptor (and coprecipitation of the receptor with PLC gamma) was demonstrated by (i) the inability to detect GAP in PLC gamma immunocomplexes or PLC gamma in GAP immunocomplexes, (ii) the association of p62 with PLC gamma in v-src-transformed cells in the absence of EGF stimulation, and (iii) in vitro solution binding and direct blotting of p62 with a glutathione S-transferase fusion protein containing the Src homology 2 (SH2) domains of PLC gamma. Unlike GAP, whose N-terminal SH2 mediates the interaction between GAP and p62, PLC gamma was found to require both its N- and C-terminal SH2 regions for p62 binding. These studies demonstrate that a protein identical to or highly related to GAP-associated p62 binds PLC gamma and suggest a means by which "cross-talk" between PLC gamma- and GAP-mediated signalling may occur.     

Evaluation of counting error due to colony masking in bioaerosol sampling.

Colony counting error due to indistinguishable colony overlap (i.e., masking) was evaluated theoretically and experimentally. A theoretical model to predict colony masking was used to determine colony counting efficiency by Monte Carlo computer simulation of microorganism collection and development into CFU. The computer simulation was verified experimentally by collecting aerosolized Bacillus subtilis spores and examining micro- and macroscopic colonies. Colony counting efficiency decreased (i) with increasing density of collected culturable microorganisms, (ii) with increasing colony size, and (iii) with decreasing ability of an observation system to distinguish adjacent colonies as separate units. Counting efficiency for 2-mm colonies, at optimal resolution, decreased from 98 to 85% when colony density increased from 1 to 10 microorganisms cm-2, in contrast to an efficiency decrease from 90 to 45% for 5-mm colonies. No statistically significant difference (alpha = 0.05) between experimental and theoretical results was found when colony shape was used to estimate the number of individual colonies in a CFU. Experimental colony counts were 1.2 times simulation estimates when colony shape was not considered, because of nonuniformity of actual colony size and the better discrimination ability of the human eye relative to the model. Colony surface densities associated with high counting accuracy were compared with recommended upper plate count limits and found to depend on colony size and an observation system's ability to identify overlapped colonies. Correction factors were developed to estimate the actual number of collected microorganisms from observed colony counts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of cell pellets: I. Electrical properties and porosity.

Cell pellets formed by centrifugation provided a good system to study the osmotic behavior, electroporation, and interaction between cells. Rabbit erythrocyte pellets were used in this study because they were simpler than nucleated cells to model analytically. Structurally, cell pellets possessed properties of porous solid bodies and gels. Electrically, cell pellets were shown to behave as a parallel set of resistance, Rp, and capacitance, Cp. Information on pellet structures was obtained from electric measurements. The pellet resistance reflected the intercellular conductivity (porosity and gap conductivity), whereas the pellet capacitance depended mostly on membrane capacitance. The pellet resistance was more sensitive to experimental conditions. The intercellular gap distance can be derived from pellet porosity measurements, providing the cell volume and surface area were known. Rp increased and relaxed exponentially with time when centrifugation started and stopped; the cycles were reversible. When supernatants were exchanged with solutions containing hypotonic electrolytes or macromolecules (such as PEG) after the pellets were formed, complicated responses to different colloidal osmotic effects were observed. A transient decrease followed by a large increase of Rp was observed after the application of a porating electric pulse, as expected from a momentary membrane breakdown, followed by a limited colloidal-osmotic swelling of pelleted cells. The equilibrium values of Rp, Cp, pellet porosity, and intercellular distances were measured and calculated as functions of cell number, centrifugation force, and ionic strength of the exchanged supernatant. Thus, the structure and properties of cell pellets can be completely characterized by electrical measurements.     

Disparate modulation of plasma membrane protein lateral mobility by various cell permeabilizing agents

The mobility of a cell surface protein on cells osmotically swollen by treatment with several different cell permeabilizing agents retains specific restraints despite detachment of the plasma membrane from the cortical cytoskeleton. Fluorescence photobleaching recovery experiments indicate that the lateral diffusion constants of immunoglobulin E (IgE)-receptor complexes on the surface of rat basophilic leukemia cells increase 2–5 × following permeabilization with streptolysin O or digitonin, with little change in their mobile fractions. Swelling by hypo-osmotic treatment in water enhances lateral diffusion of IgE-receptor complexes and raises the mobile fractions to near 100%. In contrast, swelling by treatment with filipin arrests lateral diffusion, although rotational mobility remains unhindered. Lateral mobility of a fluorescent lipid analogue remains unchanged under these conditions. Crosslinking by anti-IgE antibodies redistributes the IgE-receptor complexes into large patches on untreated cells and on cells swollen by permeabilization with streptolysin O or digitonin, but rot on cells swollen by treatment with filipin. The results indicate a diversity of effects of the various permeabilizing agents on the mobility of membrane proteins. In particular, treatment with filipin appears to reorganize the plasma membrane into a network of fluid domains on a scale smaller than the bleaching spot size used (～1.5 μm). © 1994 Wiley-Liss, Inc.     

Stochastic versus deterministic variability in simple neuronal circuits: II. Hippocampal slice.

Long time series of Schaffer collateral to CA1 pyramidal cell presynaptic volleys (stratum radiatum) and population spikes (stratum pyramidale) were evoked (driven) in rat hippocampal slices. From the driven CA1 region in normal [K+] perfusate, both population spike amplitude and an input-output function consisting of population spike amplitude divided by the presynaptic volley amplitude were analyzed. Raising [K+] in the perfusion medium to 8.5 mM, slices were induced to spontaneously burst fire in CA3 and long time series of inter-burst intervals were recorded. Three tests for determinism were applied to these series: a discrete adaptation of a local flow approach, a local dispersion approach, and nonlinear prediction. Surrogate data were generated to serve as mathematical and statistical controls. All of the population spike (6/6) and input-output (6/6) time series from the normal [K+] driven circuitry were stochastic by all three methods. Although most of the time series (5/6) from the autonomously bursting high [K+] state failed to demonstrate evidence of determinism, one (1/6) of these time series did demonstrate significant determinism. This single instance of predictability could not be accounted for by the linear correlation in these data.     

Contrasting rates of nucleotide substitution in the X-Linked and Y-Linked zinc finger genes

We have sequenced the entire exon (1,180 bp) encoding the zinc finger domain of the X-linked and Y-linked zinc finger genes (ZFX and ZFY, respectively) in the orangutan, the baboon, the squirrel monkey, and the rat; a total of 9,442 by were sequenced. The ratio of the rates of synonymous substitution in the ZFY and ZFX genes is estimated to be 2.1 in primates. This is close to the ratio of 2.3 estimated from primate ZFY and ZFX intron sequences and supports the view that the male-to-female ratio of mutation rate in humans is considerably higher than 1 but not extremely large. The ratio of synonymous substitution rates in ZFY and ZFX is estimated to be 1.3 in the rat lineage but 4.2 in the mouse lineage. The former is close to the estimate (1.4) from introns. The much higher ratio in the mouse lineage (not statistically significant) might have arisen from relaxation of selective constraints. The synonymous divergence between mouse and rat ZFX is considerably lower than that between mouse and rat autosomal genes, agreeing with previous observations and providing some evidence for stronger selective constraints on synonymous changes in X-linked genes than in autosomal genes. At the protein level ZFX has been highly conserved in all placental mammals studied while ZFY has been well conserved in primates and foxes but has evolved rapidly in mice and rats, possibly due to relaxation of functional constraints as a result of the development of X-inactivation of ZFX in rodents. The long persistence of the ZFY-ZFX gene pair in mammals provides some insight into the process of degeneration of Y-linked genes.Correspondence to: W.-H. Li     

Electrically induced fusion of mammalian cells in the presence of polyethylene glycol

Chinese Hamster Ovary (CHO) cells were fused by subjecting cell suspensions to an exponentially decaying electric pulse in the presence of polyethylene glycol (PEG), Dextran or Ficoll. PEG (MW 1,000, 3,350, 8,000, 10,000 and 18,500), Dextran (MW 71,200) and Ficoll (MW 400,000) were added to the pulsing medium. A single exponential electric pulse with peak field strength of 4 kV/cm, and a half-time of 0.72 msec was used. The combination of two techniques, PEG-induced fusion and electrofusion, resulted in highly efficient fusion of CHO cells. Fusion yields (FY) at different concentrations of these polymers were measured using phase-contrast microscopy. FY was highly dependent on the concentration of PEG in media, while the presence of Dextran and Ficoll had no influence on fusion yield. PEG with MW 8,000 was found to be the most effective in causing cell aggregation, and to give the highest FY (40%). An optimal concentration for fusion was found for PEG of each molecular weight. Diluting cells suspended in higher concentrations of PEG to these optimal concentrations after the pulse application regained the optimal FY. It was concluded that PEG-induced prepulse aggregation and moderate cell swelling immediately after the pulse were important factors in achieving high fusion yields.This work is supported by a grant GM-30969 from the National Institutes of Health. Traveling fellowship to N.G.S. was supported from Foundation Cyrill and Methodius and grant N-189 from MCES of Bulgaria.     

The effect of methyl palmoxirate on incorporation of [U-14C]palmitate into rat brain

We examined the dose response, time course and reversibility of the effect of methyl 2-tetradecylglycidate (McN-3716, methyl palmoxirate or MEP), an inhibitor of -oxidation of fatty acids, on incorporation of radiolabeled palmitic acid ([U-¹⁴C]PA) from plasma into brain lipids of awake rats. MEP (0.1, 1 and 10 mg/kg) or vehicle was administered intravenously from 10 min to 72 hr prior to infusion of [U-¹⁴C]PA. Two hr pretreatment with MEP (0.1 to 10 mg/kg) increased brain organic radioactivity 1.2 to 1.8 fold and decreased brain aqueous radioactivity by 1.2 to 3.0 fold when compared to control values. At 10 mg/kg, MEP significantly increased brain organic fraction from 40% in controls to 85%, 30 min to 6 hr pretreatment, and resulted in a redistribution of the radiolabeled fatty acid toward triacylglycerol. MEP changed the lipid/aqueous brain ratio of incorporated [U-¹⁴C]PA from 0.67 to 5.7. The incorporation rate coefficient, k^*, was significantly increased by MEP (10 mg/kg) at 2 hr (31%), 4 hr (59%) and 6 hr (34%). All effects were reversed by 72 hr, consistent with a half-life of 2 days for carnitine palmitoyl transferase I. These results indicate that intravenous MEP may be used with [1-¹¹C]palmitic acid for studying brain lipid metabolism in vivo by positron emission tomography, as it significantly reduces the large unincorporated aqueous fraction that would result in high background radioactivity.