Unfolding and refolding of the glutamine-binding protein from Escherichia coli and its complex with glutamine induced by guanidine hydrochloride

The aim of this work was to study the conformational changes of the Escherichia coli glutamine-binding protein (GlnBP) induced by GdnHCl and the effect of the binding of glutamine (Gln) on these processes. To this end, GdnHCl-induced unfolding of GlnBP alone and its GlnBP-Gln complex was studied by protein intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy. The obtained spectroscopic data were interpreted taking into the account the peculiarities of protein three-dimensional structure. In particular, the fact that formation of a complex of GlnBP and Gln, which essentially changes the global structure of protein, affects only insignificantly the microenvironments of tryptophan residues elucidates the similarity of the emission spectra of GlnBP and the GlnBP-Gln complex, and the existence of quenching groups near tyrosine residues and an effective nonradiative Tyr --> Trp and/or Tyr --> Tyr --> Trp energy transfer provide an explanation for the negligibly small contribution of tyrosine to the bulk fluorescence of the native protein and for its increase in protein unfolding. The use of the parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP-Gln unfolding are three-step processes (N --> I(1) --> I(2) --> U), though in the case of the GlnBP-Gln complex these stages essentially overlap. Despite the complex character, GlnBP unfolding is completely reversible. The dramatic shift of the N --> I(1) process to higher GdnHCl concentrations for the GlnBP-Gln complex in comparison with GlnBP was shown.     

Evaluation of chromogenic medium (ORSAB) for routine identification of methicillin resistant Staphylococcus aureus (MRSA)

Performance of chromogenic medium (ORSAB) for routine detection of methicillin resistant S. aureus (MRSA) was evaluated on 510 specimens collected from patients suspected of MRSA infection or colonization. Addition of ORSAB plates to the routine protocol allowed MRSA identification in 24 hours from samples plating. In 18 samples MRSA colonies were identified only on ORSAB plates, those cases would have been missed by routine protocol alone.     

Type 2 cytotoxic T lymphocytes modulate the activity of dendritic cells toward type 2 immune responses

Activated CD8+ T cells can differentiate into type 1 (Tc1) cells, producing mainly IFN-gamma, and type 2 (Tc2) cells, producing mostly IL-4, IL-5, and IL-10. Tc1 cells are potent CTL involved in the defense against intracellular pathogens and cancer cells. The role of Tc2 cells in the immune response is largely unknown, although their presence in chronic infections, cancer, and autoimmune diseases is associated with disease severity and progression. Here, we show that mouse Tc2 cells modify, through a cell-to-cell contact mechanism, the function of bone marrow-derived dendritic cells (DC). Indeed, Tc2-conditioned DC displayed a reduced expression of MHC class II and costimulatory molecules, produced IL-10 instead of IL-12, and favored the differentiation of both naive CD4+ and CD8+ T cells toward type 2 cells in the absence of added polarizing cytokines. The novel function for Tc2 cells suggests a type 2 loop in which Tc2 cells modify DC function and favor differentiation of naive T cells to type 2 cells. The type 2 loop may at least in part explain the unexpected high frequency of type 2 cells during a chronic exposure to the Ag.     

Folding of Aplysia limacina apomyoglobin involves an intermediate in common with other evolutionarily distant globins

In the globin family, similarities in the folding mechanism have been found among different mammalian apomyoglobins (apoMb). The best-characterized intermediate of sperm whale apoMb, called I(AGH), is mainly stabilized by nativelike contacts among the A, G, and H helices involving a cluster of hydrophobic residues that includes two conserved tryptophans. To verify the hypothesis of a common intermediate in the folding of all members of the globin family, we have extensively studied a site-directed mutant of the myoglobin from Aplysia limacina, distantly related to the mammalian counterpart, in which one of the two tryptophans in the A-G-H cluster [i.e., Trp(H8)130] has been mutated to tyrosine. The results presented here show that this mutation destabilizes both the native state and the acid intermediate I(A) but exerts little or no effect on the thermally stable core of an intermediate species (called I(T)) peculiar to Aplysia apomyoglobin. Dynamic quenching of Trp emission by acrylamide provides information on the accessibility of the chromophores at the native and the intermediate states of wild-type and mutant Aplysia apomyoglobin, consistent with the thermodynamics. Our results agree well with those obtained for the corresponding topological position of apomyoglobin from sperm whale and clearly show that the H8 position is involved in the stabilization of the main intermediate in both apoproteins. This residue thus plays a role which is evolutionarily conserved in the globin family from invertebrates to mammals; our results support the contention that the A-G-H cluster is important in the folding pathway of different globins.     

Coumarin inhibits the growth of carrot (Daucus carota L. cv. Saint Valery) cells in suspension culture

We used a carrot (Daucus carota L. cv. Saint Valery) cell suspension culture as a simplified model system to study the effects of the allelochemical compound coumarin (1,2 benzopyrone) on cell growth and utilisation of exogenous nitrate, ammonium and carbohydrates. Exposure to micromolar levels of coumarin caused severe inhibition of cell growth starting from the second day of culture onwards. At the same time, the presence of 50 mumol/L coumarin caused accumulation of free amino acids and of ammonium in the cultured cells, and stimulated their glutamine synthetase, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase and phosphoenolpyruvate carboxylase activities. Malate dehydrogenase, on the other hand, was inhibited under the same conditions. These effects were interpreted in terms of the stimulation of protein catabolism and/or interference with protein biosynthesis induced by coumarin. This could have led to a series of compensatory changes in the activities of enzymes linking nitrogen and carbon metabolism. Because coumarin seemed to abolish the exponential phase and to accelerate the onset of the stationary phase of cell growth, we hypothesise that such allelochemical compounds may act in nature as an inhibitor of the cell cycle and/or as a senescence-promoting substance.     

Quantitative determination of the enantiomers of methadone and its metabolite (EDDP) in human saliva by enantioselective liquid chromatography with mass spectrometric detection

A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection (LC-MS) has been developed and validated for the simultaneous determination of saliva concentrations of (R)- and (S)-methadone (Met) and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (EDDP, a primary metabolite of Met). Saliva specimens were collected using Salivette devices (Sarsedt), and centrifuged; collected saliva was then spiked with deuterated internal standards, D3-Met and D3-EDDP, and directly injected into the LC-MS. Enantioselective separations were achieved on a liquid chromatographic chiral stationary phase (CSP) based upon immobilized alpha(1)-acid glycoprotein (AGP) using a mobile phase composed of acetonitrile: ammonium acetate buffer (10mM, pH 7.0) in a ratio of 18:82 (v/v), a flow rate of 0.9 ml/min and a temperature of 25 degrees C. Under these conditions, enantioselective separations were observed for methadone (alpha=1.30) and EDDP (alpha=1.17) within 15 min. Met, EDDP, D3-Met and D3-EDDP were detected using selected ion monitoring at m/z 310.20, 278.20, 313.20 and 281.20, respectively. Linear relationships between peak height ratio and drug-enantiomer concentrations were obtained for methadone in the range of 5.0-600.0 ng/ml, and for EDDP from 0.5 to 15.0 ng/ml per enantiomer with correlation coefficients better than 0.9994, where lower limit of quantification (LLOQ) for Met was 5 ng/ml and for EDDP 0.5 ng/ml. Acceptable intra- and inter-day precision of the method (CVs<4.0%) and accuracy (CVs<4.0%) were obtained. These findings demonstrate the accuracy and precision of the method used to successfully analyze saliva obtained from patients enrolled in a methadone-maintenance program.     

Selective spatiotemporal induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 transcription after myocardial infarction

Myocardial remodeling after myocardial infarction (MI) is associated with increased levels of the matrix metalloproteinases (MMPs). Levels of two MMP species, MMP-2 and MMP-9, are increased after MI, and transgenic deletion of these MMPs attenuates post-MI left ventricular (LV) remodeling. This study characterized the spatiotemporal patterns of gene promoter induction for MMP-2 and MMP-9 after MI. MI was induced in transgenic mice in which the MMP-2 or MMP-9 promoter sequence was fused to the beta-galactosidase reporter, and reporter level was assayed up to 28 days after MI. Myocardial localization with respect to cellular sources of MMP-2 and MMP-9 promoter induction was examined. After MI, LV diameter increased by 70% (P < 0.05), consistent with LV remodeling. beta-Galactosidase staining in MMP-2 reporter mice was increased by 1 day after MI and increased further to 64 +/- 6% of LV epicardial area by 7 days after MI (P < 0.05). MMP-2 promoter activation occurred in fibroblasts and myofibroblasts in the MI region. In MMP-9 reporter mice, promoter induction was detected after 3 days and peaked at 7 days after MI (53 +/- 6%, P < 0.05) and was colocalized with inflammatory cells at the peri-infarct region. Although MMP-2 promoter activation was similarly distributed in the MI and border regions, activation of the MMP-9 promoter was highest at the border between the MI and remote regions. These unique findings visually demonstrated that activation of the MMP-2 and MMP-9 gene promoters occurs in a distinct spatial relation with reference to the MI region and changes in a characteristic time-dependent manner after MI.     

Ultrafine carbon particles induce apoptosis and proliferation in rat lung epithelial cells via specific signaling pathways both using EGF-R

Apoptosis and proliferation are important causes of adverse health effects induced by inhaled ultrafine particles. The molecular mechanisms of particle cell interactions mediating these end points are therefore a major topic of current particle toxicology and molecular preventive medicine. Initial studies revealed that ultrafine particles induce apoptosis and proliferation in parallel in rat lung epithelial cells, dependent on time and dosage. With these end points, two antagonistic reactions seem to be induced by the same extracellular stimulus. It was therefore investigated whether proliferation is induced directly by the particles or as a compensation of particle-caused cell death. Experimental conditions excluding compensatory proliferation demonstrated that both end points are induced independently by specific signaling pathways. Events eliciting signaling cascades leading to apoptosis and proliferation were studied with specific inhibitors of membrane receptors. Epidermal growth factor receptor (EGF-R) kinase activity was identified as essential for apoptosis as well as for proliferation. As ultrafine particle-induced proliferation alone was dependent on the activation of beta1-integrins, these membrane receptors are suggested to mediate the specificity of EGF-R signaling concerning the decision as to whether apoptosis or proliferation is triggered. Accordingly, MAP kinase signaling downstream of EGF-R showed comparable specificity with regard to receptor-dependent induction of apoptosis and proliferation. As key mediators of signaling cascades, the activation of extracellular signal-regulated kinases 1 and 2 proved to be specific for proliferation in a beta1-integrin-dependent manner, whereas phosphorylation of c-Jun NH2-terminal kinases 1 and 2 was correlated with the induction of apoptosis.     

Spermiogenesis and introsperm ultrastructure of Scoloplax distolothrix (Ostariophysi: Siluriformes: Scoloplacidae)

Spermiogenesis in scoloplacids is characterized by initial lateral development of the flagellum, nuclear rotation, medial nuclear fossa formation, complex centriolar migration, and cytoplasmic channel formation. The scoloplacid spermiogenesis is similar to those found in Diplomystidae, the most primitive siluriform family. The scoloplacid spermatozoa have all the main characteristics of introsperm. They exhibit a conic head, a symmetric midpiece, a medial flagellum, and no acrosome. The conic forward‐elongated nuclei contain homogeneous chromatin. The thin extremity of the nuclei is strongly curved and along its internal face there is a well‐developed membranous compartment. The centrioles are completely inside the medial nuclear fossa, perpendicular to each other and with an electron‐dense material between them. In a cross view of the midpiece, the mitochondria form a ring surrounding internally the cytoplasmic channel, and in a longitudinal view they are organized in a row along it. Several elongated vesicles are distributed peripherally, mainly concentrated in the mid‐piece basal region. The flagellum contains the classical axoneme (9 + 2) and has two lateral projections or fins. The spermatozoa of scoloplacids share several characteristics with those of Auchenipteridae. Since these two families are not phylogenetically related this similarity seems to be due to convergence once both families are, until now, the only known siluriform families with introsperm.