Calcium-dependent protection from complement lysis in Naegleria fowleri amebae.

Pathogenic Naegleria fowleri amebae are resistant to the lytic effects of serum complement. The presence of surface glycoproteins or removal of the membrane attack complex (MAC) of complement from the cell surface by vesiculation serve to protect the amebae from complement lysis. The specific mediators important in stimulating complement resistance are not defined. These studies were undertaken to examine the effect of Ca(2+) ions in initiating complement resistance of N. fowleri in contrast to non-pathogenic complement-sensitive N. gruberi. Chelation of extracellular calcium with ethylene glycol tetraacetic acid (EGTA) or chelation of intracellular calcium with 1,2-bis-(O-Aminophenoxy) ethane-N,N,N,N tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM) increased complement lysis of N. fowleri. Chelation of calcium ions did not affect complement sensitivity of N. gruberi. Increased lysis of ionomycin-treated N. fowleri was detected after exposure to serum complement, suggesting that a threshold level of Ca(2+) mediates complement resistance before survival mechanisms are overwhelmed and lysis occurs. A differential influx of Ca(2+) ions occurred in fura-2 labeled N. fowleri after deposition of complement component C9 to form the MAC complex on the cell surface in comparison to N. gruberi. These studies suggest that Ca(2+) ions influence complement resistance in N. fowleri but do not play a role in altering the sensitivity of N. gruberi to complement.

Hypokalemia and salbutamol therapy in asthma.

Hypokalemia is a common side effect in adult asthmatic patients on beta 2 adrenergic therapy. There is limited information in regard to hypokalemia and its relation to the clinical responses following administration of beta 2 agonist therapy in children with asthma. We observed that salbutamol inhalation significantly improved asthmatic symptoms as demonstrated by increases in peak expiratory flow (PEF: 122.37+/-75.38 vs. 152.59+/-80.29; P < 0.001) and venous oxygen tension (Pv,O2: 33.24+/-4.95 vs. 58.16+/-2.31; P < 0.001), and decreases in respiratory rate (RR: 36.39+/-3.78 vs. 28.62+/-3.12; P< 0.01), clinical scores (CS: 3.59+/-1.28 vs. 1.59+/-0.71), and venous PCO2 tensions (Pv,CO2: 40.84+/-2.67 vs. 34.75+/-2.31; P < 0.001). Salbutamol-induced hypokalemia was correlated with a decrease in RR, and an increase of Pv,O2 and PEF. These findings suggest that the same mechanism is involved in eliciting hypokalemia and bronchodilatation.

Primary staphylococcal infection and toxic shock syndrome diagnosed by polymerase chain reaction.

Primary staphylococcal pneumonia complicated with toxic shock syndrome (TSS) is relatively uncommon in children. Staphylococcus aureus exotoxins are thought to function as superantigens, and seem to promote disease manifestations. The identification of staphylococcal toxin genes by polymerase chain reaction (PCR) offers a specific and rapid diagnostic method for TSS. We describe a 7-year-old child with TSS resulting from staphylococcal pneumonia. S. aureus enterotoxins A and B were detected in the sputum of this patient by PCR.

Protein kinase activation and protein phosphorylation in Naegleria fowleri amebae in response to normal human serum.

Activation of signal transduction pathways in response to serum complement in Naegleria fowleri amebae was investigated. We examined the activation of protein kinases and changes in the phosphorylation state of proteins in N. fowleri stimulated by normal human serum (NHS). To determine differences in phosphorylation of proteins when amebae were exposed to NHS or heat inactivated serum (HIS) lacking complement, amebae were labeled with [32P] orthophosphate. An increase in phosphorylation of relatively low molecular weight proteins was noted in N. fowleri incubated in NHS with a concomitant decrease in phosphorylation of high molecular mass polypeptides. To investigate whether serine/threonine or tyrosine kinases were stimulated by NHS, amebae were treated with protein kinase inhibitors H7, staurosporine or genistein, prior to serum exposure and examined for susceptibility to complement. Treatment with each of these inhibitors resulted in increased complement lysis. Incubation of N. fowleri with genistein specifically inhibited tyrosine phosphorylation of proteins stimulated by NHS. A tyrosine kinase activity assay using exogenous polyGlu-Tyr substrate demonstrated differential activation of tyrosine kinases in amebae treated with NHS when compared to treatment with HIS. The results suggest that activation of protein kinases and subsequent protein phosphorylation are important in mediating complement resistance in N. fowleri.

Activation by cyclic GMP binding causes an apparent conformational change in cGMP-dependent protein kinase.

Cyclic nucleotide binding activates cyclic nucleotide-dependent protein kinases, but the molecular mechanism is unknown. In the present studies, cGMP binding to type Ialpha or type Ibeta cGMP-dependent protein kinase (PKG) caused (i) a large electronegative charge shift of each enzyme on ion exchange chromatography, (ii) an increase in the Stokes radius (>3 A) of each enzyme, and (iii) a decreased mobility of type Ibeta PKG on native gel electrophoresis. These physical changes were not detected in the monomeric form of type Ibeta PKG upon activation by cGMP. However, the results of partial proteolysis of type Ialpha PKG revealed some degree of cGMP-induced conformational change within the PKG-monomer, since cGMP binding protects the PKG-monomer against chymotryptic cleavage. The altered sensitivity to proteolysis occurs at Met-200, which is located between the B and C alpha-helices in the high affinity site (site A), and implies that the cGMP-induced structural perturbations in this region may participate in activation of dimeric PKG. The cGMP-induced conformational effects observed using the physical separation methods are likely to reflect altered interactions within the dimeric PKG that are caused by structural alterations within the subunits.

Amebicidal activity of plant extracts from Southeast Asia on Acanthamoeba spp.

The effect of 100 polar and 100 nonpolar plant extract materials obtained from Southeast Asia were evaluated for amebicidal activity in vitro against three species of Acanthamoeba. A. culbertsoni, A. castellanii, and A. polyphaga, the causative agents of granulomatous amebic encephalitis and amebic keratitis, were studied in vitro to determine whether the plant extracts exhibited amebicidal activity or induced encystment of the amebae. Of the 200 plant extracts tested, extracts obtained from three plants (Ipomoea sp., Kaempferia galanga, and Cananga odorata) were amebicidal for all three species of Acanthamoeba and a fourth extract prepared from Gastrochilus panduratum was lytic for A. polyphaga and growth-inhibitory for A. castellanii and A. culbertsoni. Three plant extracts induced encystment of all three species of Acanthamoeba. Select plant extracts were tested as well for tumoricidal activity against B103 neuroblastoma cells. Some plant extracts that exhibited tumoricidal activity for B103 cells were not amebicidal for Acanthamoeba spp. Additionally, the polar and nonpolar extracts that exhibited amebicidal activity were also tested for activity against primary murine peritoneal macrophage cultures. Plant extracts that demonstrated tumoricidal or amebicidal activity were not lytic for normal macrophage cultures.

Activation by autophosphorylation or cGMP binding produces a similar apparent conformational change in cGMP-dependent protein kinase.

Binding of cyclic nucleotide to or autophosphorylation of cGMP-dependent protein kinase (PKG) activates this kinase, but the molecular mechanism of activation for either process is unknown. Activation of PKG by cGMP binding produces a conformational change in the enzyme (Chu, D.-M., Corbin, J. D., Grimes, K. A., and Francis, S. H. (1997) J. Biol. Chem. 272, 31922-31928; Zhao, J., Trewhella, J., Corbin, J., Francis, S., Mitchell, R., Brushia, R., and Walsh, D. (1997) J. Biol. Chem. 272, 39129-31936). In the present studies, activation of type Ibeta PKG by either autophosphorylation or cGMP-binding alone causes (i) an electronegative charge shift on ion exchange chromatography, (ii) a similar increase ( approximately 3.5 A) in the Stokes radius as determined by gel filtration chromatography, and (iii) a similar decrease in the mobility of the enzyme on native gel electrophoresis. Consistent with these results, cGMP binding increases the rate of phosphoprotein phosphatase-1 catalyzed dephosphorylation of PKG which is autophosphorylated only at Ser-63 (not activated); however, dephosphorylation of PKG that is highly autophosphorylated (activated) is not stimulated by cGMP. The combined results suggest that activation of PKG by either autophosphorylation or cGMP binding alone produces a similar apparent elongation of the enzyme, implying that either process activates the enzyme by a similar molecular mechanism.

[Neonatal chylous ascites: report of two cases].

Chylous ascites in neonates is an unusual and etiologically poor understood entity. Our first case was a female newborn who suffered from abdominal distension and recurrent vomiting after birth. The history, physical, laboratory, and radiologic evaluations were not diagnostic except the evidence of obvious ascites. Paracentesis was performed and ascitic fluid was obtained. She was later discharged on a strict low-fat medium-chain triglycerides formula. She was found to have continue increase in abdominal girth, poor growth and development, and respiratory distress in which led her to readmission at 8 months of age. Exploratory laparotomy was done in order to rule out an anatomical lesion in which may be obstructing the lymphatic flow; but no such lesion could be found. She expired at 1 year of age with chylothorax, chylopericardium and lobar pneumonia. The second case, a 37-day-old male baby, who was admitted because of right inguinal hernia. Milky ascitic fluid in the abdomen was incidentally found during herniorrhaphy. Analysis of the fluid revealed protein 1,616 mg/dl, glucose 487 mg/dl, and triglyceride 796 mg/dl. Culture of peritoneal fluid grew no bacteria. Other laboratory findings were: serum protein 4.8 mg/dl, and BUN 14 mg/dl. A plain film of abdomen and sonogram showed massive ascites. The infant was then put on Pregestimil with the hope that the medium-chain triglyceride formula would improve his condition. Since then the child's abdominal girth did not increase and he continued to growth and develop normally at 4 months follow up.

A single dose of nebulized budesonide decreases exhaled nitric oxide in children with acute asthma.

This study was conducted to investigate whether a single dose of nebulized budesonide effectively decreased airway inflammation as demonstrated by exhaled nitric oxide (eNO) levels. A single dose of nebulized budesonide, but not nebulized terbutaline, rapidly decreased eNO levels in 6 hours. The decrease in eNO levels induced by nebulized budesonide was correlated to an increase in peak expiratory flow rate.

Ligand-induced conformational changes in cyclic nucleotide phosphodiesterases and cyclic nucleotide-dependent protein kinases.

Three methods have been used to assess the conformational effects associated with ligand binding to two unrelated cyclic nucleotide receptor proteins: the cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE or PDE5A) and the cGMP-dependent protein kinase (PKG). The methods should be applicable to other proteins and to other types of modification such as phosphorylation. The procedures use either ion-exchange chromatography, size-exclusion chromatography, or native gel electrophoresis of these proteins in the absence and presence of regulatory ligands. Measurements from these respective approaches allow documentation of changes in the quaternary structure, surface electronegativity, and relative compactness (Stokes radius) of the protein molecule. The combined data allow the changes in protein conformation to be quantitated in terms of alterations in the axial ratio or length/width dimension of the molecule. The methods can be applied to partially purified proteins and to proteins that are available in limited quantities. Conformational changes due to stable modifications of proteins can be potentially examined in crude extracts of intact cells. Each of the methods can be tailored to optimize resolution of a particular protein under a variety of conditions. Activity measurements, Coomassie brilliant blue or silver staining of gels, radioautography, or Western blot analysis can be used for detection of the protein.

Characterization of Vibrio fluvialis-like strains implicated in limp lobster disease.

Studies were undertaken to characterize and determine the pathogenic mechanisms involved in a newly described systemic disease in Homarus americanus (American lobster) caused by a Vibrio fluvialis-like microorganism. Nineteen isolates were obtained from eight of nine lobsters sampled. Biochemically, the isolates resembled V. fluvialis, and the isolates grew optimally at 20 degrees C; none could grow at temperatures above 23 degrees C. The type strain (1AMA) displayed a thermal reduction time (D value) of 5.77 min at 37 degrees C. All of the isolates required at least 1% NaCl for growth. Collectively, the data suggest that these isolates may embody a new biotype. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates revealed five closely related subgroups. Some isolates produced a sheep hemagglutinin that was neither an outer membrane protein nor a metalloprotease. Several isolates possessed capsules. The isolates were highly susceptible to a variety of antibiotics tested. However, six isolates were resistant to erythromycin. Seventeen isolates harbored plasmids. Lobster challenge studies revealed that the 50% lethal dose of a plasmid-positive strain was 100-fold lower than that of a plasmid-negative strain, suggesting that the plasmid may enhance the pathogenicity of these microorganisms in lobsters. Microorganisms that were recovered from experimentally infected lobsters exhibited biochemical and PFGE profiles that were indistinguishable from those of the challenge strain. Tissue affinity studies demonstrated that the challenge microorganisms accumulated in heart and midgut tissues as well as in the hemolymph. Culture supernatants and polymyxin B lysates of the strains caused elongation of CHO cells in tissue culture, suggesting the presence of a hitherto unknown enterotoxin. Both plasmid-positive and plasmid-negative strains caused significant dose-related intestinal fluid accumulations in suckling mice. Absence of viable organisms in the intestinal contents of mice suggests that these microorganisms cause diarrhea in mice by intoxication rather than by an infectious process. Further, these results support the thermal reduction data at 37 degrees C and suggest that the mechanism(s) that led to fluid accumulation in mice differs from the disease process observed in lobsters by requiring neither the persistence of viable microorganisms nor the presence of plasmids. In summary, results of lobster studies satisfy Koch's postulates at the organismal and molecular levels; the findings support the hypothesis that these V. fluvialis-like organisms were responsible for the originally described systemic disease, which is now called limp lobster disease.