Impact of Protein Domains on PE_PGRS30 Polar Localization in Mycobacteria

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.     

Anthropogenic radionuclides bioaccumulation in Antarctic marine fauna and its ecological relevance

The paucity of investigations on the presence of artificial radionuclides and their bioaccumulation in Antarctic fauna is due to the erroneous belief that this area is pristine. We report evidence that significant levels of the artificial radionuclides Sr-90, Cs-137, Am-241 and plutonium isotopes can be found in sponges, bivalves, krill and demersal fish fauna of Terra Nova Bay (Ross Sea), sometimes with a seasonal pattern. Increasing concentrations of Cs-137 were detected in the bivalve Adamussium colbecki (Antarctic scallop) during austral summer months, as a result of major trophic activity and changes in metabolic rates. Bioconcentration factors for artificial radionuclides in different Antarctic species are presented and discussed in relation to their different trophic strategies. Unexpectedly high radiocesium bioconcentration factors determined in bivalves suggested the particular role played by filter feeding in bioaccumulation, particularly in summer when radionuclide bioavailability is enhanced. The feeding preference of the trematomiid fish Trematomus bernacchii for the scallop A. colbecki is confirmed, not only by fish gut content analyses, but also through radiometric results. Transuranics bioaccumulation by sensitive species allowed some interesting comparisons on the different plutonium contamination of the southern hemisphere with respect to the northern one. Accepted: 25 April 2000     

Drug-metabolizing enzymes in liver, olfactory, and respiratory epithelium of cattle

The drug-metabolizing enzymes of olfactory and respiratory epithelium of cattle were determined. The data of nasal tissues were compared to those of bovine liver. Both oxidative and nonoxidative enzyme activities were investigated. Many compounds including testosterone were used as substrates for the P450-dependent monooxygenase activities. The results demonstrated that the P450 content and all the activities assayed including reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase were much higher in the olfactory than in the respiratory mucosa and for some activities (hexamethyl-phosphoramide and dimethylnitrosamine N-demethylase, aniline hydroxylase, and ethoxycoumarin O-deethylase) the values in the olfactory tissue were even markedly higher than those of liver. Also the activities of some nonoxidative enzymes such as glutathione S-transferase, uridine 5'-diphosphate (UDP)-glucuronyl-transferase, and epoxide hydrolase were higher in the olfactory than in the respiratory mucosa but lower than in liver. The results taken together suggest that the olfactory and respiratory epithelium of cattle, which contain in addition to a wide array of nonoxidative enzymes multiple forms of P450, can be useful and easily available tissues to study the biotransformation processes of odorants.     

Potentiation of temozolomide antitumor effect by purine receptor ligands able to restrain the in vitro growth of human glioblastoma stem cells

Glioblastoma multiforme (GBM), the most common and aggressive brain tumor in humans, comprises a population of stem-like cells (GSCs) that are currently investigated as potential target for GBM therapy. Here, we used GSCs isolated from three different GBM surgical specimens to examine the antitumor activity of purines. Cultured GSCs expressed either metabotropic adenosine P1 and ATP P2Y receptors or ionotropic P2X₇ receptors. GSC exposure for 48 h to 10–150 μM ATP, P2R ligand, or to ADPβS or MRS2365, P2Y₁R agonists, enhanced cell expansion. This effect was counteracted by the PY₁R antagonist MRS2500. In contrast, 48-h treatment with higher doses of ATP or UTP, which binds to P2Y_2/4R, or 2′(3′)-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), P2X₇R agonist, decreased GSC proliferation. Such a reduction was due to apoptotic or necrotic cell death but mostly to growth arrest. Accordingly, cell regrowth and secondary neurosphere formation were observed 2 weeks after the end of treatment. Suramin, nonselective P2R antagonist, MRS1220 or AZ11645373, selective A₃R or P2X₇R antagonists, respectively, counteracted ATP antiproliferative effects. AZ11645373 also abolished the inhibitory effect of Bz-ATP low doses on GSC growth. These findings provide important clues on the anticancer potential of ligands for A₃R, P2Y₁R, and P2X₇R, which are involved in the GSC growth control. Interestingly, ATP and BzATP potentiated the cytotoxicity of temozolomide (TMZ), currently used for GBM therapy, enabling it to cause a greater and long-lasting inhibitory effect on GSC duplication when readded to cells previously treated with purine nucleotides plus TMZ. These are the first findings identifying purine nucleotides as able to enhance TMZ antitumor efficacy and might have an immediate translational impact.     

Bright oligothiophene N-succinimidyl esters for efficient fluorescent labeling of proteins and oligonucleotides

The synthesis of multicolor fluorescent oligothiophene N-succinimidyl esters (TSEs) is reported, and their optical properties are discussed with the aid of ab initio calculations. The esters were coupled to proteins and to 3'-amino-modified oligonucleotides in mild conditions and with similar modalities. A comparative study of the bioconjugate of IgG1 anti-CD3 antibody labeled with a blue fluorescent TSE and with fluorescein isothiocyanate (FITC) is reported, showing that the former achieves higher photoluminescence intensity and optical stability than the latter. Fluorescence resonance energy transfer experiments with TSE-labeled oligonucleotides and examples of cellular imaging via TSE-labeled proteins are reported.     

Temperature effects on summer growth rates in the Antarctic scallop, Adamussium colbecki

Annual growth rates of Antarctic marine organisms are low compared to their relatives from warmer waters. Previous studies hypothesise that high food availability during austral spring–summer may enable Antarctic invertebrates to attain comparatively high short-term growth rates despite the low temperature. Neither a temperature-growth experiment with juvenile Adamussium colbecki (Smith 1902) nor the comparison of A. colbecki summer growth rates with an empirical scallop specific growth-to-temperature relationship could confirm this hypothesis. Hence, summer growth rates of young, immature A. colbecki are strongly affected by temperature, i.e. no uncoupling from temperature.     

A second outer-core region in Klebsiella pneumoniae lipopolysaccharide

Up to now only one major type of core oligosaccharide has been found in the lipopolysaccharide of all Klebsiella pneumoniae strains analyzed. Applying a different screening approach, we identified a novel Klebsiella pneumoniae core (type 2). Both Klebsiella core types share the same inner core and the outer-core-proximal disaccharide, GlcN-(1,4)-GalA, but they differ in the GlcN substituents. In core type 2, the GlcpN residue is substituted at the O-4 position by the disaccharide beta-Glcp(1-6)-alpha-Glcp(1, while in core type 1 the GlcpN residue is substituted at the O-6 position by either the disaccharide alpha-Hep(1-4)-alpha-Kdo(2 or a Kdo residue (Kdo is 3-deoxy-D-manno-octulosonic acid). This difference correlates with the presence of a three-gene region in the corresponding core biosynthetic clusters. Engineering of both core types by interchanging this specific region allowed studying the effect on virulence. The replacement of Klebsiella core type 1 in a highly type 2 virulent strain (52145) induces lower virulence than core type 2 in a murine infection model.     

Shallow- and deep-water mollusc distribution at Terra Nova Bay (Ross Sea, Antarctica)

The aim of this work is to improve the knowledge of the mollusc fauna of Terra Nova Bay (Ross Sea), on the basis of more than 100 stations sampled from 25 to 1100 m depth, during Italian Antarctic Expeditions (austral summers 1987/1988, 1989/1990, 1993/1994, 1994/1995, 1995/1996 and 1997/1998). In shallow waters, gastropod fauna is represented by 31 species, among which Neobuccinum eatoni, Onoba gelida, Powellisetia deserta, Philine cf. apertissima and Austrodoris kerguelenensis are the most abundant, while among bivalves (25 species) the commonest species are Adamussium colbecki, Yoldia eightsi, Laternula elliptica and “Montacuta”nimrodiana. At present, most of the Antarctic mollusc species are known as having circumantarctic distribution and are considered eurybathic. Moreover, in general, only scarce data are available on their substrate preferences, because of their occurrence in scattered sampling stations with different sediment features. In the present study carried out at Terra Nova Bay, based on a relatively high number of stations, a clear zonation of mollusc assemblages is shown, according to depth and sediment features. While gastropods characterise the superficial algal belts dominated by the red algae Iridaea cordata and Phyllophora antarctica, bivalve distribution is wider. Adamussium colbecki is the dominant species in the upper 100 m, both on soft and hard bottoms, if the slope is suitable (density up to 40–60 ind./m²). On coarse sands, this species is frequently accompanied by L. elliptica (density <20 ind./m²), while Y. eightsi characterises organic- enriched bottoms (density 70–80 ind./m²). Below 200 m depth Adacnarca nitens, Limatula hodgsoni, “Montacuta”nimrodiana and Mysella gibbosa are commonly found on relatively coarse sediments, while Thyasira dearborni and Yoldiella ecaudata prefer muddy bottoms. In order to identify bivalve assemblages, multivariate analyses were applied to stations grouped into depth ranges, sediment features and location. While shallow bathymetric ranges (25–200 m) are clearly defined, deeper clusters of stations are more influenced by the high degree of eurybathy of most of the species and by the scattered distribution of the sediment particle sizes. Accepted: 18 September 1999     

Assessment of Coupling between Trans-Abdominally Acquired Fetal ECG and Uterine Activity by Bivariate Phase-Rectified Signal Averaging Analysis

Couplings between uterine contractions (UC) and fetal heart rate (fHR) provide important information on fetal condition during labor. At present, couplings between UC and fHR are assessed by visual analysis and interpretation of cardiotocography. The application of computerized approaches is restricted due to the non-stationarity of the signal, missing data and noise, typical for fHR. Herein, we propose a novel approach to assess couplings between UC and fHR, based on a signal-processing algorithm termed bivariate phase-rectified signal averaging (BPRSA).

Methods

Electrohysterogram (EHG) and fetal electrocardiogram (fECG) were recorded non-invasively by a trans-abdominal device in 73 women at term with uneventful singleton pregnancy during the first stage of labor. Coupling between UC and fHR was analyzed by BPRSA and by conventional cross power spectral density analysis (CPSD). For both methods, degree of coupling was assessed by the maximum coefficient of coherence (C_PRSA and C_RAW, respectively) in the UC frequency domain. Coherence values greater than 0.50 were consider significant. C_PRSA and C_RAW were compared by Wilcoxon test.

Results

At visual inspection BPRSA analysis identified coupled periodicities in 86.3% (63/73) of the cases. 11/73 (15%) cases were excluded from further analysis because no 30 minutes of fECG recording without signal loss was available for spectral analysis. Significant coupling was found in 90.3% (56/62) of the cases analyzed by BPRSA, and in 24.2% (15/62) of the cases analyzed by CPSD, respectively. The difference between median value of C_PRSA and C_RAW was highly significant (0.79 [IQR 0.69–0.90] and 0.29 [IQR 0.17–0.47], respectively; p<0.0001).

Conclusion

BPRSA is a novel computer-based approach that can be reliably applied to trans-abdominally acquired EHG-fECG. It allows the assessment of correlations between UC and fHR patterns in the majority of labors, overcoming the limitations of non-stationarity and artifacts. Compared to standard techniques of cross-correlations, such as CPSD, BPRSA is significantly superior.