A novel alpha-Proteobacterium resides in the mitochondria of ovarian cells of the tick Ixodes ricinus

An intracellular bacterium from Ixodes ricinus ticks collected in Italy was characterized by electron microscopy (EM), PCR sequencing of the 16S rRNA gene, molecular phylogenetic analysis, and in situ hybridization (ISH). This bacterium was shown by EM to be present in the cytoplasm, as well as in the mitochondria of ovarian cells. When universal 16S rRNA bacterial primers were used, PCR amplification of ovarian DNA followed by cloning and sequencing resulted in the same sequence being found in each sample. Phylogenetic analysis of this sequence showed that the bacterium from which it was derived, tentatively designated IricES1, is part of a novel clade in the alpha subdivision of the Proteobacterium: ISH and PCR assays of various tissues performed with oligonucleotides specific for the IricES1 16S rRNA showed that IricES1 is restricted to ovarian cells. Based on the results obtained, we inferred that the bacteria seen by EM in ovarian cells are a single type of bacteria, corresponding to IricES1. PCR screening of 166 ticks from various parts of Italy and one site in England showed that IricES1 was present in 96% of adult females and 44% of nymphs (unsexed). No adult males were found to be infected. Despite the apparent parasitism of host mitochondria by IricES1, the available information suggests that the bacterium has an obligate relationship with its host, although this must be confirmed.     

Effect of maprotiline on Ca(2+) movement in human neuroblastoma cells

In human neuroblastoma IMR32 cells, the effect of the anti-depressant maprotiline on baseline intracellular Ca2+ concentrations ([Ca2+]i) was explored by using the Ca2+-sensitive probe fura-2. Maprotiline at concentrations greater than 100 microM caused a rapid rise in [Ca2+]i in a concentration-dependent manner (EC50 = 200 microM). Maprotiline-induced [Ca2+]i rise was reduced by 50% by removal of extracellular Ca2+. Maprotiline-induced [Ca2+]i rises were inhibited by half by nifedipine, but was unaffected by verapamil or diiltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of maprotiline on [Ca2+]i was abolished. U73122, an inhibitor of phospholipase C, did not affect maprotiline-induced [Ca2+]i rises. These findings suggest that in human neuroblastoma cells, maprotiline increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum via a phospholiase C-independent manner.     

Hydrophobic properties of gram-negative rods colonizing upper respiratory tract of healthy people

The cell surface hydrophobicity is one of the non specific factors of adhesion influencing the ability of microorganisms to colonize nasopharynx. The aim of this paper was to evaluate via salt aggregation test (SAT) the cell surface hydrophobicity of 150 strains of gram-negative rods isolated from the throat or/and nasal specimens of healthy people. It has been found that among the nonfermenting rods hydrophobic strains were predominant. In contrast, the isolates of Enterobacteriaceae family were characterized by the distinctive features of the cell surface within particular genera or even species. The obtained results show that, despite differences in cell surface hydrophobicity, numerous species of gram-negative rods have the ability to colonize the mucous membrane of upper respiratory tract. This suggests that the cell surface hydrophobicity is rather a feature of species or genus, but it is not related to the ecological niche of microorganisms in human body.     

A preliminary phylogeny of the Pterasteridae (Echinodermata,Asteroidea) and the first fossil record: Late Cretaceous of Germany and Belgium

The Pterasteridae comprises a diversified group of extant largely deep-sea starfishes. Generic diagnoses have been based classically on soft tissue characters and skeletal architecture. A preliminary phylogeny of sixteen extant species is here worked out by cladistic analysis. The resulting tree suggests monophyly of extant genera and the validity of dissociated plates for identification of genera. Fossil remains of Pterasteridae are here described for the first time. By comparison with extant species, all the skeletal remains from the lower Upper Campanian of Belgium and from the lower Maastrichtian of Germany are tentatively assigned to the genusPteraster. The fossil record of starfishes is poor, but the present Late Cretaceous pterasterids provide one more piece of evidence of the high diversity of starfishes during the Mesozoic. Known Late Cretaceous and Paleogene fossils are broadly similar, which suggests the end-Cretaceous extinction event did not cause major turnover in asteroid faunal composition. As suggested for other starfish groups, both the fossil record of deep-sea Pterasteridae in shelf settings and tree topology imply an onshore-offshore evolutionary trend.      

Determination of glutathionyl-hemoglobin in human erythrocytes by cation-exchange high-performance liquid chromatography

Since glutathionyl-hemoglobin has been suggested to be a clinical marker of oxidative stress in human blood and given the growing biological relevance of oxidative stress as a pathogenic factor in several diseases, we describe a method to measure glutathionyl-hemoglobin concentration in erythrocytes, by using cation-exchange high-pressure liquid chromatography with UV detection. The glutathionyl-hemoglobin peak has been identified on the basis of the following findings: (a) the peak increased when the sample was incubated with oxidized glutathione; (b) the peak disappeared when the sample was reduced with dithiothreitol, with the simultaneous increase of that corresponding to hemoglobin A(0); (c) the peak could be detected by incubating hemoglobin A(0) with reduced glutathione; (e) deconvoluted mass spectrum of the glutathionyl-hemoglobin peak showed a 16172.0-Da molecular mass, corresponding to hemoglobin beta bound to glutathione. Glutathionyl-hemoglobin concentration has been determined in erythrocytes of 40 healthy subjects, with a mean value of 2.58+/-0.7%, calculated as the percentage of its peak area ratio to that of total hemoglobin (HbA(0)+HbA(2)+HbA(1C)+glutathionyl-hemoglobin). The availability of a simple and reproducible method to detect glutathionyl-hemoglobin concentration in blood could be useful in monitoring oxidative stress, and for investigating the efficacy of antioxidant therapies in clinical trials.     

Functional characterization of Narc 1, a novel proteinase related to proteinase K

The NARC 1 gene encodes a novel proteinase K family proteinase. The domain structure of rat Narc 1 resembles that of the subtilisin-like proprotein convertases (SPCs), except that rNarc 1 lacks the canonical P-domain of SPCs, retaining only the RGD motif as part of what might be a cryptically functioning P-domain. Narc 1 undergoes autocatalytic intramolecular processing at the site LVFAQ/, resulting in the cleavage of its prosegment and the generation of an active proteinase with a broad alkaline pH optimum and no apparent calcium requirement for activity. Both primary and secondary structural determinants influence Narc 1 substrate recognition. Our functional characterization of Narc 1 reinforces the inference drawn from the analysis of its predicted structure that this enzyme is most closely related to representatives of the proteinase K family, but that it is also sufficiently different to warrant its possible classification in a separate sub-family.     

Biophysical characterization of the interactions of HTI-286 with tubulin heterodimer and microtubules

HTI-286 is a synthetic analogue of the natural product hemiasterlin and is a potent antimitotic agent. HTI-286 inhibits the proliferation of tumor cells during mitosis. The observed antimitotic activity is due to the binding of HTI-286 to tubulin. This report details the effects of HTI-286 on soluble tubulin and preassembled microtubules. HTI-286 binds tubulin monomer and oligomerizes it to an 18.5 S species corresponding to a discrete ring structure consisting of about 13 tubulin units as determined by sedimentation equilibrium analyses. The rate of formation of the oligomers is dependent on the concentration of HTI-286 and the time of incubation. Tubulin oligomers, specifically the 18.5 S species, form slowly. The interactions of HTI-286 with tubulin were studied by isothermal titration calorimetry. HTI-286 binds tubulin rapidly, and the initial association of HTI-286 with tubulin is enthalpically driven with a DeltaH value of -14 kcal/mol at 25 degrees C and a dissociation constant of ca. 100 nM. However, the accompanying tubulin oligomerization event does not produce measurable heats at 25 degrees C. The dissociation constant estimated from the changes in the intrinsic fluorescence of tubulin was found to be consistent with the calorimetric results. Both HTI-286 and hemiasterlin bind tubulin with nearly equal potency. However, the stability of the tubulin oligomers is not identical under size-exclusion column chromatographic conditions. The tubulin oligomers formed in the presence of HTI-286 dissociate on the column, while the corresponding oligomers formed in the presence of hemiasterlin are stable. Tubulin undergoes a change in the secondary structure in the presence of HTI-286, which is evidenced by changes in the circular dichroic absorption spectrum of tubulin. In contrast to the microtubule-stabilizing effects of paclitaxel, both HTI-286 and hemiasterlin depolymerize preassembled microtubules at micromolar concentrations.     

Thermodynamic description of the effect of the mutation Y49F on human glutathione transferase P1-1 in binding with glutathione and the inhibitor S-hexylglutathione

The thermodynamics of binding of both the substrate glutathione (GSH) and the competitive inhibitor S-hexylglutathione to the mutant Y49F of human glutathione S-transferase (hGST P1-1), a key residue at the dimer interface, has been investigated by isothermal titration calorimetry and fluorescence spectroscopy. Calorimetric measurements indicated that the binding of these ligands to both the Y49F mutant and wild-type enzyme is enthalpically favorable and entropically unfavorable over the temperature range studied. The affinity of these ligands for the Y49F mutant is lower than those for the wild-type enzyme due mainly to an entropy change. Therefore, the thermodynamic effect of this mutation is to decrease the entropy loss due to binding. Calorimetric titrations in several buffers with different ionization heat amounts indicate a release of protons when the mutant binds GSH, whereas protons are taken up in binding S-hexylglutathione at pH 6.5. This suggests that the thiol group of GSH releases protons to buffer media during binding and a group with low pKa (such as Asp98) is responsible for the uptake of protons. The temperature dependence of the free energy of binding, DeltaG0, is weak because of the enthalpy-entropy compensation caused by a large heat capacity change. The heat capacity change is -199.5 +/- 26.9 cal K-1 mol-1 for GSH binding and -333.6 +/- 28.8 cal K-1 mol-1 for S-hexylglutathione binding. The thermodynamic parameters are consistent with the mutation Tyr49 --> Phe, producing a slight conformational change in the active site.     

Differential effects of quercetin,a natural polyphenolic flavonoid,on L-type calcium current in pituitary tumor (GH3) cells and neuronal NG108-15 cells

The effects of quercetin, a natural polyphenolic compound, on voltage-dependent L-type Ca(2+) current (I(Ca,L)) in rat pituitary GH(3) cells were investigated with the aid of the whole-cell voltage-camp technique. Quercetin (0.5-200 microM) stimulated I(Ca,L) in a concentration-dependent manner. The current-voltage (I-V) relationship of I(Ca,L) was slightly shifted to more negative potentials in the presence of quercetin. The EC(50) value of the quercetin-induced stimulation of I(Ca,L) was about 7 microM. The presence of quercetin (5 microM) shifted the steady state inactivation curve of I(Ca,L) to a more negative potential by approximately -10 mV. Although quercetin might increase intracellular cyclic AMP, sp-cAMPS did not affect I(Ca,L). In addition, neither flavone nor wortmannin had any effect on the amplitude of I(Ca,L), while epicatechin and genistein slightly suppressed it. Quercetin (50 microM) decreased the amplitude of tetrodotoxin-sensitive Na(+) current in GH(3) cells. Under current-clamp configuration, quercetin could increase the firing frequency of actions potentials. Conversely, in NG108-15 neuronal cells, quercetin suppressed the amplitude of I(Ca,L). The quercetin-induced inhibition of I(Ca,L) was abolished in NG108-15 cells preincubated with t-butyl hydroperoxide (1 mM). Quercetin-mediated stimulation of I(Ca,L) in GH(3) cells was presumably not associated with the level of intracellular cyclic AMP, or with the activity of tyrosine or phosphoinositide 3-kinases. Therefore, the effects of quercetin on ion currents may, at least in part, contribute to the underlying mechanisms through which it affects neuronal or neuroendocrine function.