Metabolic activation of A549 human airway epithelial cells by organic dust: a study based on microphysiometry

A Cytosensor microphysiometer, which measures extracellular acidification rate (ECAR), was used to study the early metabolic activation by organic dust from a swine confinement building in a human airway epithelial cell line, A549. The dust is known to cause an intense airway inflammatory reaction following inhalation in vivo and cytokine release in vitro. Dimethyl amiloride (DMA) was used to study sodium/proton exchanger (NHE) activity in cells growing at different cell densities. Exposing cells at low density to dust induced an initial release of acid not involving NHE, followed by a sustained DMA-sensitive NHE activation. In cells near high density, NHE was not activated during exposure resulting in a modest increase in ECAR. Exposing cells at high density resulted in a bi-phasic ECAR pattern; an initial increase in proton release followed by an inhibition of ECAR below baseline. Pretreatment with pertussis toxin (PTX), an inhibitor of receptor/G(i alpha)-coupled signal transductions did not affect ECAR in low and medium density cells, but abolished the inhibition of ECAR in high-density cells. The dust did not prevent forskolin-induced cAMP accumulation and PTX did not affect cAMP in near-confluent cells suggesting the PTX-effect to be cAMP-independent. The ECAR response to organic dust was similar to that of lipopolysaccharide (LPS) except for high-density cells where PTX did not influence the LPS-induced decrease in ECAR below baseline. In summary, the organic dust induces PTX-sensitive (cAMP independent) signalling in near-confluent A549 epithelial cells and, depending on cell density opposing effects on NHE activity during exposure.     

Acid-induced movements in the glycoprotein shell of an alphavirus turn the spikes into membrane fusion mode

In the icosahedral (T = 4) Semliki Forest virus, the envelope protomers, i.e. E1-E2 heterodimers, make one-to-one interactions with capsid proteins below the viral lipid bilayer, transverse the membrane and form an external glycoprotein shell with projections. The shell is organized by protomer domains interacting as hexamers and pentamers around shell openings at icosahedral 2- and 5-fold axes, respectively, and the projections by other domains associating as trimers at 3- and quasi 3-fold axes. We show here, using cryo- electron microscopy, that low pH, as occurs in the endosomes during virus uptake, results in the relaxation of protomer interactions around the 2- and the 5-fold axes in the shell, and movement of protomers towards 3- and quasi 3-fold axes in a way that reciprocally relocates their putative E1 and E2 domains. This seemed to be facilitated by a trimerization of transmembrane segments at the same axes. The alterations observed help to explain several key features of the spike-mediated membrane fusion reaction, including shell dissolution, heterodimer dissociation, fusion peptide exposure and E1 homotrimerization.     

NMR solution structure and dynamics of motilin in isotropic phospholipid bicellar solution

The structure and dynamics of the gastrointestinal peptide hormone motilin, consisting of 22 amino acid residues, have been studied in the presence of isotropic q=0.5 phospholipid bicelles. The NMR solution structure of the peptide in acidic bicelle solution was determined from 203 NOE-derived distance constraints and six backbone torsion angle constraints. Dynamic properties for the ¹³C-¹H vector in Leu10 were determined for motilin specifically labeled with ¹³C at this position by analysis of multiple-field relaxation data. The structure reveals an ordered -helical conformation between Glu9 and Lys20. The N-terminus is also well structured with a turn resembling that of a classical -turn. The ¹³C dynamics clearly show that motilin tumbles slowly in solution, with a correlation time characteristic of a large object. It was also found that motilin has a large degree of local flexibility as compared with what has previously been reported in SDS micelles. The results show that motilin interacts with the bicelle, displaying motional properties of a peptide bound to a membrane. In comparison, motilin in neutral bicelles seems less structured and more flexible. This study shows that the small isotropic bicelles are well suited for use as membrane-mimetic for structural as well as dynamical investigations of membrane-bound peptides by high-resolution NMR.     

Effects of estradiol and estradiol sulfamate on the liver of ovariectomized or ovariectomized and hypophysectomized rats

This study was performed to evaluate and compare the effects of estradiol sulfamate (J995) and estradiol (E2) on the hepatic levels of the estrogen receptor (ER) and its mRNA, in ovariectomized (OVX) and OVX+hypophysectomized (OVXHX) female rats and to study the effects on the liver-derived serum compounds angiotensin I, triglycerides, high-density lipoprotein (HDL) and cholesterol. ER concentrations were determined using ligand-binding assay (LBA) and enzyme immuno assay (EIA), and the mRNA levels using solution hybridization.

The rats were treated orally (p.o.) or subcutaneously (s.c.) for 7 days, with treatments initiated 14 days after surgery.

No differences were found in ER mRNA levels between J995 and E2 treated rats.

The s.c. administered estrogens increased ER levels in OVX rats. Addition of GH+DEX to OVXHX rats restored the ER to levels above those seen in intact rats, whereas simultaneous oral treatment with E2 significantly decreased ER levels again. The s.c. treatment with either J995 or E2 limited the increase caused by addition of GH+DEX.

After oral treatment angiotensin I levels were increased by E2, but not by J995, while triglycerides, HDL and cholesterol levels were decreased by oral E2, J995 showing a similar pattern but was less effective.

In summary, these results on hepatic ER levels and estrogen dependent compounds produced by the liver showed that J995 has a lower impact on the normal liver functions after oral treatment than E2. Thus, J995 is a very promising substance for development of oral estrogen treatment with reduced hepatic side effects.     

Development and validation of a liquid chromatographic method for Casiopeina IIIi in rat plasma

In order to measure changes in physiological CO concentrations in blood with good accuracy, a method was developed using gas chromatography with flame ionisation detection (250 degrees C). A nickel catalyst system was fitted to convert CO to methane at 375 degrees C after separation with a molecular sieve column at 35 degrees C. Helium was used as carrier at 30 ml/min. Porcine or human blood (400 microl) was sampled in gastight tubes and treated with sulfuric acid and saponin (800 microl). Accuracy was 1.4% and 1.5% (RSD), respectively. Precision was 2.8% (porcine blood). Limit of detection was 0.01 nmol/ml gas and limit of quantification 12 nmol/ml blood. Calibration was made in the interval 12-514 nmol/ml blood (corresponding to 0.1-6% COHb). Samples were stable for at least a month at +4 degrees C. This paper describes a method with high sensitivity and good accuracy, suitable for analysis of low CO concentrations.     

Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice

The Lyme disease vaccine is based on the outer-surface lipoprotein (OspA) of the pathogen Borrelia burgdorferi, and 95% of vaccine recipients develop substantial titers of antibodies against OspA. Here, we identified seven individuals with very low antibody titers after vaccination (low responders). The macrophages of low responders produced less tumor necrosis factor-alpha and interleukin-6 after OspA stimulation and had lower cell-surface expression of Toll-like receptor (TLR) 1 as compared to normal cells, but normal expression of TLR2. TLRs activate innate responses to pathogens, and TLR2 recognizes lipoproteins and peptidoglycan (PGN). After OspA immunization, mice genetically deficient in either TLR2 (TLR2(-/-)) or TLR1 (TLR1(-/-)) produced low titers of antibodies against OspA. Notably, macrophages from TLR2(-/-) mice were unresponsive to OspA and PGN, whereas those from TLR1(-/-) mice responded normally to PGN but not to OspA. These data indicate that TLR1 and TLR2 are required for lipoprotein recognition and that defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination.     

Effects after inhalation of (1-->3)-beta-D-glucan and relation to mould exposure in the home

BACKGROUND: Damp conditions indoors favour the growth of microorganisms, and these contain several agents that may cause inflammation when inhaled. Moulds contain a polyglucose in their cell wall, defined as (1-->3)-beta-D-glucan, exhibiting effects on inflammatory cells. AIM: The aim of the present study was to evaluate whether an inhalation challenge to purified (1-->3)-beta-D-glucan (grifolan) in humans could induce effects on inflammatory markers in blood, and to evaluate whether the reactions were related to the home exposure to (1-->3)-beta-D-glucan. METHODS: Seventeen subjects in homes with high levels of airborne (1-->3)-beta-D-glucan (G-high) and 18 subjects in homes with low levels of (1-->3)-beta-D-glucan (G-low) underwent two randomised, double-blind inhalation challenges, one to (1-->3)-beta-D-glucan suspended in saline and one to saline alone. A blood sample was taken before and after the challenges, and differential cell count, granulocyte enzymes in serum and the secretion of cytokines from peripheral blood mononuclear cells (PBMC) were measured. RESULTS: Inhalation challenge with (1-->3)-beta-D-glucan induced a decrease in the secretion of tumour necrosis factor alpha from endotoxin-stimulated PBMC in the G-high group as well as in the G-low group. In the G-high group, the inhalation of (1-->3)-beta-D-glucan induced an increase in blood lymphocytes that was significantly different from the saline-induced effect. CONCLUSIONS: The results suggest that an inhalation challenge to (1-->3)-beta-D-glucan has an effect on inflammatory cells and this effect may be related to a chronic exposure to moulds at home.     

Expression in insect cells of active mature cruzipain from Trypanosoma cruzi,containing its C-terminal domain

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, is a member of the papain family that contains a C-terminal domain in the mature enzyme, in addition to a catalytic moiety homologous to papain and some mammalian cathepsins. The native enzyme is expressed as a complex mixture of isoforms and has not been crystallized. Previous attempts to express recombinant mature cruzipain containing the C-terminal domain have failed. For this reason, the three-dimensional structure of the complete mature enzyme is not known, although the structure of a recombinant truncated molecule lacking the C-terminal domain (cruzaindeltac) has been determined. We report here the expression of active, N-glycosylated, complete mature cruzipain in an insect cell/baculovirus system. The purified recombinant enzyme, obtained with a yield of about 0.2 mg/100 ml of culture supernatant, has an apparent molecular mass similar, and an identical N-terminal sequence, compared with the native enzyme. The expressed protein is able to process itself by self-proteolysis, leaving the isolated C-terminal domain, and has kinetic properties similar to those of native cruzipain, although some differences in substrate specificity were found. These results open up the possibility of obtaining recombinant intact mature cruzipain of a quality and in quantity suitable for X-ray crystallography.     

White matter lesions and soluble interleukin-1 receptor type II in CSF from demented and non-demented subjects

White matter lesions (WMLs) in the brain is a common, unspecific finding on magnetic resonance imaging appearing both in the healthy elderly as well as in a number of different diseases including dementia disorders. However, the pathophysiological and clinical significance of WMLs in dementia disorders is still unknown. In this study, we investigated the possibility of their origin being inflammatory by studying the correlation between WMLs and cerebrospinal fluid (CSF) levels of the proinflammatory cytokine soluble interleukin-1 receptor type II (sIL-1RII). The sIL-1RII is a member of the IL-1 family, and has been found to be elevated in CSF from Alzheimer's disease (AD) patients. In the present study, two groups of patients complaining of memory disturbances with little or extensive WMLs respectively, were examined, as well as healthy subjects. In accordance with other reports, WML scores (total, periventricular as well as deep lesions) were positively correlated with age but not mini mental state examination (MMSE) scores, and were significantly higher in patients with a dementia diagnosis as compared to non-demented subjects. There were no differences in sIL-1RII levels in CSF regardless of amount of total, periventricular or deep WMLs, nor were there any differences between demented and non-demented subjects. In conclusion, sIL-1RII levels in CSF are not correlated to magnetic resonance imaging WMLs in patients with dementia disorders or in healthy subjects.     

The tyrosine kinase inhibitor genistein increases basal cAMP and potentiates forskolin-induced cAMP accumulation in A549 human airway epithelial cells

Genistein is often used as an inhibitor of tyrosine kinases. A less studied side effect of genistein is an inhibition of cyclic AMP-phosphodiesterase (cAMP-PDE) activity resulting in increased cAMP accumulation. The effect of genistein on intracellular cAMP-levels, basal and forskolin-induced, was studied in A549 human airway epithelial cells and compared with the unspecific PDE inhibitor, isobutylmethylxanthine (IBMX). It was shown that genistein (50 M) increased basal cAMP and potentiated forskolin-induced cAMP accumulation to the same extent as IBMX (100 M). Thus, the use of genistein in studies on signaling transductions may result in erroneous conclusions since increased cAMP may cause or contribute to the observed effects.