Microbial communities adhering to the obverse and reverse sides of an oil painting on canvas: identification and evaluation of their biodegradative potential.

In this study, we investigated and compared the microbial communities adhering to the obverse and the reverse sides of an oil painting on canvas exhibiting signs of biodeterioration. Samples showing no visible damage were investigated as controls. Air samples were also analysed, in order to investigate the presence of airborne microorganisms suspended in the indoor atmosphere. The diversity of the cultivable microorganisms adhering to the surface was analysed by molecular techniques, such as RAPD analysis and gene sequencing. DGGE fingerprints derived from DNA directly extracted from canvas material in combination with clone libraries and sequencing were used to evaluate the non-cultivable fraction of the microbial communities associated with the material. By using culture-dependent methods, most of the bacterial strains were found to be common airborne, spore-forming microorganisms and belonged to the phyla Actinobacteria and Firmicutes, whereas culture-independent techniques identified sequenced clones affiliated with members of the phyla Actinobacteria and Proteobacteria. The diversity of fungi was shown to be much lower than that observed for bacteria, and only species of Penicillium spp. could be detected by cultivation techniques. The selected strategy revealed a higher microbial diversity on the obverse than on the reverse side of the painting and the near absence of actively growing microorganisms on areas showing no visible damage. Furthermore, enzymatic activity tests revealed that the most widespread activities involved in biodeterioration were esterase and esterase lipase among the isolated bacterial strains, and esterase and N-acetyl-ß-glucosaminidase among fungi strains.

ETO-2, a new member of the ETO-family of nuclear proteins.

The t(8;21) is associated with 12-15% of acute myelogenous leukemias of the M2 subtype. The translocation results in the fusion of two genes, AML1 (CBFA2) on chromosome 21 and ETO (MTG8) on chromosome 8. AML1 encodes a DNA binding factor; the ETO protein product is less well characterized, but is thought to be a transcription factor. Here we describe the isolation and characterization of ETO-2, a murine cDNA that encodes a new member of the ETO family of proteins. ETO-2 is 75% identical to murine ETO and shares very high sequence identities over four regions of the protein with ETO (domain I-III and zinc-finger). Northern analysis identifies ETO-2 transcripts in many of the murine tissues analysed and in the developing mouse embryo. ETO-2 is also expressed in myeloid and erythroid cell lines. We confirmed the nuclear localization of ETO-2 and demonstrated that domain III and the zinc-finger region are not required for nuclear localization. We further showed that a region within ETO, containing domain II, mediates dimerization among family members. This region is conserved in the oncoprotein AML-1/ETO. The recent identification of another ETO-like protein, myeloid translocation gene-related protein 1, together with the data presented here, demonstrates that at least three ETO proteins exist with the potential to form dimers in the cell nucleus.

Reactions of protamine with the molecular chaperone DnaK.

Molecular chaperones of the 70 kDa family mediate protein-protein interactions by selectively binding to partially unfolded segments of other proteins in an ATP-dependent activity cycle. Previous investigations of chaperone substrate selectivity have shown that chaperones have a propensity to bind to partially unfolded segments of polypeptides that contain bulky hydrophobic residues. However, recent investigations have shown that 70 kDa chaperones such as DnaK, which is expressed by Escherichia coli, also bind short basic peptides and even polycations. We report here that DnaK specifically binds to the polycation protamine when [protamine]/[DnaK] is near unity, whereas protamine induces the aggregation of DnaK when [protamine]/[DnaK] > or = 20. Complexes between DnaK and protamine were detected using fluorescently labeled protamine (protamine*) in conjunction with high performance size exclusion chromatography. We found that: (i) an unlabeled peptide of known affinity for DnaK partially inhibited the formation of DnaK-protamine* complexes; (ii) Mg-ATP (and Mg-gamma-S-ATP) significantly reduced the affinity of protamine* for DnaK; and (iii) the rate of DnaK-protamine* complex dissociation is highly temperature-sensitive, with apparent activation enthalpies (delta H*) equal to 32 +/- 4 and 28 +/- 1 kcal mol-1 in the absence of added nucleotide and in the presence of ADP, respectively. The results are consistent with the specific binding of protamine* at the (poly)peptide binding site of DnaK. A model is proposed to account for the protamine-induced aggregation of DnaK.

Evidence against a hemodynamic role for serotonin in the dorsal motor nucleus of the vagus.

This study was performed to investigate the potential role of serotonin (5-HT) in the dorsal motor nucleus of the vagus (dmnX) in regulating peripheral hemodynamics. Microinjections (5 or 25 nmol in 50 nl) of the monoaminergic neurotransmitter were made into the dorsomedial medulla of the urethaneanesthetized rat during continuous recording of femoral arterial blood pressure. Heart rate was extracted electronically from the pressure waveform. Discrete injections of 5-HT placed directly in the dmnX were found to be entirely without effect on peripheral hemodynamics. In contrast, injections placed in the solitary tract nucleus, lying immediately above the dmnX, were found to have profound depressor and bradycardic effects, while the immediately subjacent hypoglossal nucleus appeared to contain both depressor and unresponsive sites. These findings cast doubt on the involvement of serotonin in the dmnX in the regulation of cardiovascular hemodynamics.

A rapid HPLC technique for determining levels of histamine in tears from normal and inflamed human eyes.

Because of the important role of histamine in the inflammatory process, the measurement of histamine levels in tears has been considered as a possible index of eye irritancy. Histamine levels have been measured using an HPLC procedure involving fluorimetric detection after fluorescamine derivatization. Analyses, at picomole levels, were carried out in less than 5 minutes using 10 microliter of tear samples collected from either normal or inflamed human eyes.

Bromide ion exchange with a Keggin polyoxometalate on functionalized polymeric membranes: a theoretical and experimental study.

Noncovalent interactions between the polyoxometalate [PMo12O40](3-) and acryloyloxyundecyltrimethyl ammonium bromide surfactant, used during membrane preparation, were evaluated in the frame of density functional theory. The electronic solvation energy of [PMo12O40](3) and bromide anions was also evaluated, at the same level of theory, in order to predict a probable exchange on the polymeric surface between these anions at the water/polymer interface. Energy balances were theoretically assessed, showing that the bromide cannot be exchanged with this nanosized polyanion in large extent. In order to validate this theoretical conclusion, ad hoc and accurate measurements were carried out by using homemade polymeric membranes and by dipping them in an ca. 0.4 mM solution of Na3[PMo12O40] for 4 days. The Br(-) concentration, released in a polyoxometalate solution, was followed at different times during the test period by gravimetrical analysis. The agreement between the theoretical prediction and experimental data was remarkable, as the quantum calculations correctly accounted for the short-range intermolecular interactions involved in this phenomenon. Bearing in mind that the achieved conclusion is based on an ab initio quantum approach, the findings of this study can be considered rather general and then exploitable for other similar systems.

Performance of PDMS membranes in pervaporation: effect of silicalite fillers and comparison with SBS membranes.

Laboratory-made silicalite filled PDMS membranes were tested by means of concentration and temperature influence on the membrane performance in removal of ethanol from ethanol/water mixtures. This allowed studying the applicability of solution-diffusion model in the transport mechanism description. Experiments were performed by varying the ethanol concentration in the feed and temperature. Two types of fillers were incorporated into the PDMS network: commercial zeolite silicalite (CBV 3002) and laboratory-made colloidal silicalite-1. Obtained results were then compared with data gathered for unfilled PDMS membranes to examine the effect of fillers incorporation. Moreover, the comparison with novel block co-polymer based porous and dense SBS membranes was done. It was found that the solution-diffusion model was a good representation of ethanol transport through both filled and unfilled PDMS membranes, whereas the water flux did not obey this model due to the swelling effects. Incorporation of the fillers increased membrane stability and improved the selectivity. Performance of the SBS membranes characterized by a dense structure was found to be similar to the performance of filled PDMS membranes.

Cardiovascular effects of serotonin in the nucleus of the solitary tract.

The cardiovascular regulatory role of serotonin [5-hydroxytryptamine (5-HT)] in the solitary tract nucleus (NTS) was investigated in urethan-anesthetized Wistar rats. Unilateral microinjection of 5-HT (5 nmol in 50 nl) into the NTS evoked depressions of both arterial pressure (-20 +/- 1 mmHg) and heart rate (-43 +/- 6 beats/min). Induction of bradycardia and hypotension was repeatable and consistently obtained with injections into the NTS but not into neighboring structures. Microinjection of the nonselective 5-HT receptor antagonist methiothepin or the 5-HT1A/5-HT1B antagonist pindolol prevented any cardiovascular change by subsequent microinjection of 5-HT into the NTS. In contrast, microinjection of the 5-HT2-selective antagonist ketanserin or the 5-HT1A antagonist spiroxatrine had no effect on the subsequent effects of 5-HT. Bilateral vagal denervation prevented the bradycardia induced by 5-HT, whereas the vasodepression remained intact. These data provide evidence that 5-HT in the NTS evokes vagal chronotropic cardioinhibition and sympathetic withdrawal and suggest that this action is mediated by 5-HT1 serotonergic receptors, possibly of the 5-HT1B subtype.

Large activation energy barriers to chaperone-peptide complex formation and dissociation.

To probe the mechanism of chaperone substrate selection, we have investigated the kinetics of complex formation and dissociation between the molecular chaperone DnaK and a short peptide (Cro, representing amino acids 1-12 of the cro repressor protein). The Cro protein was N-terminally labeled with the environmentally sensitive fluorophore dansyl chloride (Cro*), and steady-state and stopped-flow fluorescence spectroscopies and fluorescence-detected high-performance size exclusion chromatography (HPSEC) were used to monitor complex formation and dissociation over a range of temperatures in the absence of ATP. The results are summarized as follows: (i) Cro* binds to DnaK with a second-order rate constant, k(on), which varies from 8 to 200 M-1 s-1 between 15 and 37 degrees C. The slow on-rate is a consequence of a large activation energy barrier. The activation enthalpy (delta H*) and the prefactor [omega exp delta S*/R)] are 26 kcal mol-1 and 7 x 10(20) M-1 s-1, respectively. (ii) Once formed, DnaK-Cro* complexes are long-lived, especially at low temperatures (T < 15 degrees C). The off-rate is unusually temperature-sensitive, for example, there is a 478-fold increase in k(off) from 2.3 x 10(6) to 1.1 x 10(-3) s-1 over a range of only 30 degrees C (5-35 degrees C). The steep temperature-dependence of the off-rate is a consequence of a very large activation energy barrier to DnaK-Cro* complex dissociation [delta H* = 34.6 kcal mol-1 and omega exp (delta S*/R) = 2 x 10(21) s-1]. The relatively low affinity of the Cro* peptide for DnaK is due to a large kinetic barrier to binding. We discuss possible causes for these large kinetic barriers.

[Comparison of the Sensititre diagnostic system and the Bauer-Kirby test of sensitivity to chemotherapeutic agents in agar, on bacterial strains isolated from urinary infections]. / Comparazione tra il sistema diagnostico Sensititre e il test di sensibilità ai chemioterapici in agar Bauer-Kirby, su stipiti batterici isolati nel corso di infezioni urinarie.

The in vitro activity of nine antibiotics was tested against 50 bacterial strains isolated from urinary tract infections. The two methods showed a considerable percentage of diversity in an evaluation of their agreement. The Sensititre analytical method for the determination of minimum inhibitory concentrations is regarded as reliable and more useful to determine looked therapy. The authors suggest that the Sensititre system will be accepted in routine work.

Histamine determination in tears as an index of safety in use of cosmetic products.

Synopsis To assess ocular irritancy caused by chemical and cosmetic products a reliable method based on evaluation of histamine (Hm) in tears is presented. Hm is measured at picomole levels by HPLC and fluorimetric detection after fluorescamine-HM derivatization. In order to avoid any uncontrolled irritation and stimulation of the conjunctiva during sample collection, a procedure of conjunctiva lavage was developed. A balanced salt solution (50 mul) containing a known amount of Hm-fluorophore as reference standard is instilled in the conjunctiva fornix. After a few seconds 20 mul of tear fluid is collected: 10 mul are immediately analysed and 10 mul after derivatization reaction. In this way it is possible to evaluate tear dilution and to assess Hm content in less than 10 minutes. In a group of 20 normal subjects Hm has been determined in comparison with that of two volunteers after topical application of 50 mu of 0.2% and 0.4% sodium lauryl sulphate solution. A contact of 30 seconds of the cosmetic ingredient caused an immediate dose-dependent Hm release through a direct cytotoxic damage of cell membranes due to the surfactant action.

Substituted 4,7-dihydroxyindoles and related quinonoid derivatives as potential antimicrobial agents.

The antimicrobial activity of some 4,7-dihydroxyindole derivatives and indole-4,7-quinones was examined by determining the MIC against selected typical microorganisms (bacteria and fungi). A few of the compounds examined showed only a feeble inhibitory effect against some Gram-positive strains. Activity seems to be correlated with the lipophilic character of the molecules; in fact, all compounds containing a strong hydrophilic group, like a carboxyl, proved completely inactive.

Effects of carbohydrate feedings on plasma free tryptophan and branched-chain amino acids during prolonged cycling.

Brain serotonin (5-hydroxytryptamine, 5-HT) has been suggested to be involved in central fatigue during prolonged exercise. Changes in the ratio of plasma free tryptophan (free Trp) to branched-chain amino acids (BCAA) are associated with altered brain 5-HT synthesis. The purposes of this study were to describe systematically the effects of prolonged exercise on changes in plasma free Trp and BCAA and to examine the effects of carbohydrate (CHO) feedings on these same variables. Eight well-trained men [VO2max = 57.8 (SE 4.1) ml kg-1 min-1] cycled for up to 255 min at a power output corresponding to VO2 at lactate threshold (approximately 68% VO2max) on three occasions separated by at least 1 week. Subjects drank 5 ml kg-1 body wt-1 of either a water placebo, or a liquid beverage containing a moderate (6% CHO) or high (12% CHO) concentration of carbohydrate beginning at min 14 of exercise and every 30 min thereafter. Exercise time to fatigue was shorter in subjects receiving placebo [190 (SE 4) min] as compared to 6% CHO [235 (SE 10) min] and 12% CHO [234 (SE 9) min] (P < 0.05). Glucose and insulin decreased in the placebo group, and free Trp, free-Trp/BCAA, and free fatty acids increased approximately five- to sevenfold (P < 0.05). These changes were attenuated in a dose-related manner by the carbohydrate drinks.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipidemia en 150 cordones umbilicales para detectar un factor de riesgo coronario. / Lipidemia in 150 umbilical cords to detect a coronary risk factor

Se establecen las concentraciones de colesterol, trigliceridos, fosfolipidos, acidos grasos libres y lipoproteinas en un primer grupo de 100 recien nacidos (55 varones, 45 mujeres) considerados clinicamente normales, no habiendose podido demostrar la existencia de diferencias en los valores entre ambos sexos. Por medio del estudio lipidico determinado en un segundo grupo de 50 cordones ubilicales se pudo confirmar, mediante el metodo de las "rectas de alerta" y "rectas de accion" la validez estadistica de la cifra del colesterol asi como de los trigliceridos plasmaticos. En este segundo grupo se determinaron, ademas, el colesterol transportado por la alfa-lipoproteina (HDL) y beta-lipoproteina (LDL). Los valores hallados en la sangre del cordon umbilical de ambos grupos son muy inferiores a los que se encuentran en etapas mas avanzadas de la vida. Debido al hecho de que la frecuencia de las alteraciones del transporte lipidico caracteristica de la hiperlipoproteinemia de tipo IIa alcanzo una cifra del 4%, se concluye que seria conveniente estudiar la lipidemia del cordon umbilical en los recien nacidos, especialmente en aquellos de "alto riesgo" que presenten historia familiar positiva para aterosclerosis precoz o muerte subita y/o evidencias de alteraciones del transporte lipidico en uno o ambos progenitores