Phasing beams with different dispersions and application to the petawatt-class beamline at the National Ignition Facility.

In order to achieve the highest intensities possible with the short-pulse Advanced Radiographic Capability beamline at the National Ignition Facility (NIF), it will be necessary to phase the individual ARC apertures. This is made especially challenging because the design of ARC results in two laser beams with different dispersions sharing the same NIF aperture. The extent to which two beams with different dispersions can be phased with each other has been an open question. This paper presents results of an analysis showing that the different dispersion values that will be encountered by the shared-aperture beams will not preclude the phasing of the two beams. We also highlight a situation in which dispersion mismatch will prevent good phasing between apertures, and discuss the limits to which higher-order dispersion values may differ before the beams begin to dephase.

Externalization of host cell protein kinase C during enteropathogenic Escherichia coli infection.

Enteropathogenic Escherichia coli (EPEC) is a common cause of diarrhea in children in developing countries. Protein kinase C (PKC), a serine- and threonine-directed protein kinase, is rapidly activated following EPEC infection and this is accompanied by its translocation to a membrane-bound location where it is tightly bound to phosphatidylserine (PS). EPEC infection causes host cell death, one of whose features is externalization of PS. We hypothesized that externalization of PS would be accompanied by externalization of PKC as well. We report that EPEC infection triggers the externalization of PKC to the outer surface of the host cell. Ecto-PKC remains firmly tethered to the cell but can be released by incubation with peptide or protein substrates for the enzyme. Ecto-PKC is intact and biologically active and able to phosphorylate protein substrates on the surface of the host cell. Phosphorylation of whole EPEC bacteria or EPEC-secreted proteins could not be detected. Externalization of PKC could be reproduced by the combination of an apoptotic stimulus (ultraviolet (UV) irradiation) and phorbol myristate acetate (PMA), a procedure which resulted in externalization of >25% of the total cellular content of PKC-alpha. In the presence of ATP, ecto-PKC inhibited UV-induced cell shrinkage, membrane blebbing, and propidium iodide uptake but not the activation of caspases 3 and 7. This is the first report that expression of an ecto-protein kinase is altered by a microbial pathogen and the first to note that externalization of PKC can accompany apoptosis.

Role of EspF in host cell death induced by enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) causes diarrhoea in children in developing countries. Many EPEC genes involved in virulence are contained within the locus of enterocyte effacement (LEE), a large pathogenicity island. One of the genes at the far righthand end of the LEE encodes EspF, an EPEC secreted protein of unknown function. EspF, like the other Esps, is a substrate for secretion by the type III secretory system. Previous studies found that an espF mutant behaved as wild type in assays of adherence, invasion, actin condensation and tyrosine phosphorylation. As EPEC can kill host cells, we tested esp gene mutants for host cell killing ability. The espF mutant was deficient in host cell killing despite having normal adherence. The addition of purified EspF to tissue culture medium did not cause any damage to host cells, but expression of espF in COS or HeLa cells caused cell death. The mode of cell death in cells transfected with espF appeared to be pure apoptosis. EspF appears to be an effector of host cell death in epithelial cells; its proline-rich structure suggests that it may act by binding to SH3 domains or EVH1 domains of host cell signalling proteins.

Redistribution of cyclic GMP in response to sodium butyrate in colon cells.

The effect of butyrate on the response to guanylin and Escherichia coli heat-stable enterotoxin, STa, was assessed in T84 cells and Caco-2 cells, cultured colon cell lines possessing the guanylyl cyclase C which is the receptor for these peptides. Butyrate treatment of these cells resulted in an apparent increase in cyclic GMP (cGMP) accumulation when the cGMP content of cells and the supernatant medium was measured. Butyrate treatment did not change the guanylyl cyclase activity or (125)I-STa binding parameters in T84 cells, but the butyrate effect was completely blocked by cycloheximide. Butyrate did not have any effect on STa-stimulated cGMP accumulation in COS cells transfected with the human or porcine GC-C. Further experiments showed that butyrate treatment caused a large increase in the cGMP released into the culture medium, and in cells grown in polarized fashion in Transwell inserts, cGMP efflux was predominantly from the basolateral surface of the cell; intracellular cGMP was actually lowered by butyrate treatment. Exposure of T84 cells to butyrate had no effect on the disposition of cyclic AMP generated in response to forskolin. The effects of butyrate on cGMP were reversible within 24 h of butyrate withdrawal. In colon cells, butyrate treatment induced a previously undescribed, cGMP-specific efflux mechanism which lowered intracellular cGMP and elevated extracellular cGMP in response to peptide agonists such as guanylin and STa.

Preformed bacterial toxins.

Food poisoning syndromes caused by four different bacteria are described. For all types, food kept at a permissive temperature allows growth of the vegetative forms of the bacteria and production of a toxin or toxins. The key features of these syndromes, as well as possible new trends of concern, are summarized in Table 1.

Host cell death due to enteropathogenic Escherichia coli has features of apoptosis.

Enteropathogenic Escherichia coli (EPEC) is a cause of prolonged watery diarrhea in children in developing countries. The ability of EPEC to kill host cells was investigated in vitro in assays using two human cultured cell lines, HeLa (cervical) and T84 (colonic). EPEC killed epithelial cells as assessed by permeability to the vital dyes trypan blue and propidium iodide. In addition, EPEC triggered changes in the host cell, suggesting apoptosis as the mode of death; such changes included early expression of phosphatidylserine on the host cell surface and internucleosomal cleavage of host cell DNA. Genistein, an inhibitor of tyrosine kinases, and wortmannin, an inhibitor of host phosphatidylinositol 3-kinase, markedly increased EPEC-induced cell death and enhanced the features of apoptosis. EPEC-induced cell death was contact dependent and required adherence of live bacteria to the host cell. A quantitative assay for EPEC-induced cell death was developed by using the propidium iodide uptake method adapted to a fluorescence plate reader. With EPEC, the rate and extent of host cell death were less that what has been reported for Salmonella, Shigella, and Yersinia, three other genera of enteric bacteria known to cause apoptosis. However, rapid apoptosis of the host cell may not favor the pathogenic strategy of EPEC, a mucosa-adhering, noninvasive pathogen.

Mycobacterium bovis BCG causing vertebral osteomyelitis (Pott's disease) following intravesical BCG therapy.

We report a case of Mycobacterium bovis BCG vertebral osteomyelitis in a 79-year-old man 2.5 years after intravesical BCG therapy for bladder cancer. The recovered isolate resembled M. tuberculosis biochemically, but resistance to pyrazinamide (PZA) rendered that diagnosis suspect. High-pressure liquid chromatographic studies confirmed the diagnosis of M. bovis BCG infection. The patient was originally started on a four-drug antituberculous regimen of isoniazid, rifampin, ethambutol, and PZA. When susceptibility studies were reported, the regimen was changed to isoniazid and rifampin for 12 months. Subsequently, the patient was transferred to a skilled nursing facility for 3 months, where he underwent intensive physical therapy. Although extravesical adverse reactions are rare, clinicians and clinical microbiologists need to be aware of the possibility of disseminated infection by M. bovis BCG in the appropriate setting of clinical history, physical examination, and laboratory investigation.

Activation of host cell protein kinase C by enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) consists of a group of diarrhea-producing E. coli strains, common in developing countries, which do not produce classical toxins and are not truly invasive. EPEC strains adhere to mammalian cells in an intimate fashion, trigger a localized increase in intracellular calcium levels, and elevate inositol phosphate production. We hypothesized that these mediators could activate host cell protein kinase C (PKC) and tested this idea in vitro with two cultured human cell lines, HeLa cells and T84 cells. Using a recently described subculturing protocol to "induce" or accelerate EPEC adherence, we infected the cells with EPEC at a multiplicity of infection of approximately 100:1 for 30 to 60 min. Under these conditions, EPEC E2348 increased membrane-bound PKC activity 1.5- to 2.3-fold in HeLa cells and T84 cells, respectively. The increase in membrane-bound PKC activity was accompanied by a decrease in cytosolic PKC activity in EPEC-infected HeLa cells. Nonadherent laboratory E. coli strains such as HB101 and H.S. failed to trigger any consistent change in PKC production, similar to the nonadherent mutant strains derived from E2348, JPN15 (plasmid cured) and CVD206 (eaeA). In addition, immunoblots performed on extracts of T84 cells with a monoclonal antibody against PKC-alpha showed an increased PKC content in membranes of EPEC-infected cells. Finally, EPEC-infected T84 cells showed a 60% increase in responsiveness to the E. coli heat-stable toxin. We conclude that mediators produced in response to EPEC adherence activate PKC in intestinal and nonintestinal cells.

Phosphorylation and activation of the intestinal guanylyl cyclase receptor for Escherichia coli heat-stable toxin by protein kinase C.

The heat-stable enterotoxin STa of E. coli causes diarrhea by binding to and stimulating intestinal membrane-bound guanylyl cyclase, triggering production of cyclic GMP. Agents which stimulate protein kinase C (PKC), including phorbol esters, synergistically enhance STa effects on cGMP and secretion. We investigated whether PKC causes phosphorylation of the STa receptor in vivo and in vitro. Immunoprecipitation of the STa receptor-guanylyl cyclase was carried out from extracts of T84 colon cells metabolically labelled with [32P]-phosphate using polyclonal anti-STa receptor antibody. The STa receptor was phosphorylated in its basal state, and 32P content in the 150 kDa holoreceptor band increased 2-fold in cells exposed to phorbol ester for 1 h. In vitro, immunopurified STa receptor was readily phosphorylated by purified rat brain PKC. Phosphorylation was inhibited 40% by 5 microM of a synthetic peptide corresponding to the sequence around Ser1029 of the STa receptor, a site previously proposed as a potential PKC phosphorylation site. Treatment of the immunopurified STaR/GC with purified PKC increased STa-stimulated guanylyl cyclase activity 2-fold. We conclude that PKC phosphorylates and activates the STa receptor/guanylyl cyclase in vitro and in vivo; Ser1029 of the STaR/GC remains a candidate phosphorylation site by PKC.

Oligosaccharides from human milk block binding and activity of the Escherichia coli heat-stable enterotoxin (STa) in T84 intestinal cells.

Enterotoxin-producing Escherichia coli are major causes of pediatric diarrhea in developing countries. The heat-stable enterotoxin of Escherichia coli (STa) causes diarrhea by virtue of its ability to bind to and stimulate intestinal membrane-bound guanylate cyclase, generating cyclic GMP (cGMP). Previous work showed that a fucosylated oligosaccharide fraction of human milk was able to protect suckling mice from the secretory effects of STa, but the mechanism of the protection could not be determined. Oligosaccharide fractions from human milk were tested for their ability to block the biochemical effects of STa in T84 cells, a human colon carcinoma line responsive to the toxin. Total and fucosylated oligosaccharide fractions were found to inhibit STa-stimulated guanylate cyclase activity in T84 cell membranes and cGMP production in intact T84 cells by 60-80%. In addition, the total oligosaccharide fraction and the fucosylated oligosaccharide fraction inhibited 125I-STa binding significantly (17% and 27% inhibition, respectively). These findings demonstrate the protective activity of human milk oligosaccharides against STa in a human-derived cell line and show that the biochemical step blocked by oligosaccharides is STa-mediated stimulation of guanylate cyclase. This represents a novel mechanism by which human milk oligosaccharides protect against diarrhea.

Regulation of intestinal guanylate cyclase by the heat-stable enterotoxin of Escherichia coli (STa) and protein kinase C.

The heat-stable enterotoxin of Escherichia coli (STa) stimulates membrane-bound guanylate cyclase in intestinal epithelium and induces fluid and ion secretion. Using the T84 human colon carcinoma cell line as a model, we observed that phorbol esters markedly enhanced STa-stimulated cyclic GMP accumulation in T84 cells (C. S. Weikel, C. L. Spann, C. P. Chambers, J. K. Crane, J. Linden, and E. L. Hewlett, Infect. Immun. 58:1402-1407, 1990). In this study we document that the phorbol ester treatment increases 125I-STa-binding sites as well as membrane-bound guanylate cyclase activity in T84 cells and provide evidence that both effects are mediated by phosphorylation. Guanylate cyclase activity was increased approximately 50% in membranes prepared from intact T84 cells treated with phorbol-12,13-dibutyrate (beta-PDB) and after treatment of homogenates with beta-PDB in a manner dependent on ATP, MgCl2, and cytosol. Similarly, treatment of membranes with purified bovine brain protein kinase C in the presence of appropriate cofactors and beta-PDB resulted in an increase in STa-stimulated guanylate cyclase activity of about 70%. Likewise, the number of 125I-STa-binding sites was increased by about 25 to 40% in membranes prepared from intact cells or homogenates treated with beta-PDB; no effect on binding affinity (Kd = 0.15 nM) was noted. These experiments suggest that protein kinase C may phosphorylate the STa receptor-guanylate cyclase or a closely related protein and increase guanylate cyclase activity. The stimulatory effects of protein kinase C on STa-sensitive guanylate cyclase are opposite in direction to the profound inhibitory effects of the kinase on atrial natriuretic peptide-stimulated guanylate cyclase, demonstrating differential regulation by protein kinases within the guanylate cyclase-receptor family.

Measurement of the local electron density by means of stimulated Raman scattering in a laser-produced gas jet plasma.

By measuring the spectrum of the backscattered light from a short-pulse laser-produced plasma in a gas jet, a direct and highly accurate measurement of the local electron density can be obtained. The measurement is based on the density-dependent spectral shift of the backscattered Raman wave.

High-field harmonic generation in helium.

We observe harmonics of 526-nm laser light up to the 45th order, 11.7 nm, in helium. We discuss the extension of the harmonic plateau with increasing laser intensity. The data suggest that the highest harmonic order produced depends on the highest intensity seen by the atom before photoionization. Harmonics are generated predominantly from neutrals. Harmonic generation from ions is weak owing to poor phase matching between the fundamental and harmonic fields at high electron densities.

Characterization of the synergistic interaction of Escherichia coli heat-stable toxin and carbachol.

STa, the heat-stable enterotoxin of Escherichia coli, is a specific activator of membrane-bound guanylyl cyclase and stimulates secretion of Cl- in a human colonic carcinoma cell line (T84). We investigated the effect of the cholinergic agent carbachol on the secretory response to STa. T84 cell monolayers were studied under voltage-clamped conditions in modified Ussing chambers. Simultaneous addition of STa and carbachol resulted in a biphasic synergistic response characterized by a brief peak in short-circuit current (Isc) followed by a prolonged plateau phase lasting up to 90 min. A synergistic response was also seen with sequential addition of the agonists, and was altered by the order and timing of agonist addition. Pretreatment with STa enhanced the synergistic response to carbachol, while the reverse order of additions produced synergy only when STa was added during or immediately after the Isc response to carbachol. Synergy occurred only with a concentration of STa sufficient to produce an Isc response alone. However, a concentration of carbachol that caused neither an increase in Isc nor intracellular Ca2+ mobilization was sufficient to evoke a synergistic response. Addition of 8-bromoguanosine 3',5'-cyclic monophosphate also produced a synergistic Isc response with carbachol, although maximal synergism was seen with simultaneous addition. Augmentation of the intracellular Ca2+ response to carbachol by STa is not the mechanism of synergy. Although the mechanism of synergy is not understood, these studies suggest that STa-induced cGMP interacts with other second messengers to produce the synergistic response, and that multiple intracellular mediators may influence the ability of STa to cause disease.

Group-delay diagnostic for measuring vapor column density.

We describe a technique for determining Nfl by measuring the group-velocity delay of a probe laser beam propagating through a vapor. This diagnostic has wide dynamic range, is simple to implement, and can be used as a high-bandwidth vapor rate monitor. In addition, it can be used to measure column density, Nl, number density, N, oscillator strengths, f, or absorption cross sections, collisional line broadening, and vapor group-velocity delay.

Carbachol mimics phorbol esters in its ability to enhance cyclic GMP production by STa, the heat-stable toxin of Escherichia coli.

STa, the heat-stable enterotoxin of Escherichia coli, stimulates membrane-bound guanylate cyclase in enterocytes, elevates cyclic GMP, and results in intestinal secretion of ions and fluid. Using the T84 colon carcinoma cell line as a model. Weikel et al. reported that phorbol esters enhance STa-stimulated cyclic GMP production by 60-140% [(1990) Infect. Immun. 58, 1402-1407]. In the present report we demonstrate that the acetylcholine analog carbachol enhanced toxin-stimulated cyclic GMP accumulation in intact T84 cells by 50-100% and that this effect was blocked by 10 microM atropine and 10 microM sphingosine. Pertussis toxin treatment of the T84 cells did not affect the subsequent response to carbachol. Carbachol, which elevates intracellular calcium in these cells, may act through protein kinase C to enhance cyclic GMP production.