Production of an egg yolk antibody against Parietaria judaica 2 allergen.

Specific antibodies are essential tools for studying proteins as well as for diagnostic research in biomedicine. The egg yolk of immunized chicken is an inexpensive source of high-quality polyclonal antibodies. The 12-kDa Parietaria judaica 2 allergen was expressed as a fusion protein and was used to immunize Leghorn chickens. In this paper, we show, using 2-dimensional gel electrophoresis and immunoblotting, that chicken antibodies raised against a recombinant allergen can be used to recognize similar proteins from a pollen raw extract. Allergen identity was confirmed by nanoLC-nanospray-tandem mass spectrometry analysis. Our data demonstrate for the first time that a synergistic combination of molecular biology, 2-dimensional PAGE, and use of nonmammalian antibodies represents a powerful tool for reliable identification of allergens.

Occurrence and biological effects of cholesteryl sulfate on blood platelets.

Although the exact function of cholesteryl sulfate (CS) is unknown, it is present in low concentration in lipoproteins, in red blood cells and spermatozoa. In the present study, we investigated whether CS is present in blood platelets and its possible biological involvement in platelet function. Extensively washed platelets were prepared from rat and human blood. After lipid extraction and thin layer chromatography (TLC) on silica gel, a compound with the same mobility as authentic CS was isolated and identified by two different methods: (1) without hydrolysis, negative ion fast atom bombardment combined with tandem mass spectrometry (MS/MS); (2) after acidic hydrolysis, identification of cholesterol (Chol) by TLC and gas chromatography-MS. CS concentrations measured using beta-sitosteryl sulfate as internal standard in normal rat or human platelets were in the range of 164-512 pmol/10(9) platelets. This represented less than 1% of cell Chol. Biological effects of CS on platelet function were studied in vitro. CS incubated with rat platelets either as methanol solution or as albumin-bound complex potentiated the ADP- or thrombin-induced aggregation and serotonin secretion. The results of platelet sterol analysis indicated that CS was incorporated into platelet membrane and did not significantly change the platelet cholesterol composition. The potentiating effect of CS on platelet-induced aggregation and secretion was not obtained with cholesterol, cholesteryl acetate or estrone. In contrast, an inhibitory effect of estrone sulfate was observed. These results indicate that both the sulfate group and the cholesterol moiety are involved in the pro-aggregant property of CS. In addition, platelet mediators seem to be implicated in the mechanism since the thrombin-induced production of thromboxane B2, the stable end-product of arachidonic acid metabolism, was also enhanced in the presence of CS. These results suggest a new role for CS which may be involved in the modulation of platelet function.

Impedance spectroscopy of water solutions: the role of ions at the liquid-electrode interface.

We discuss the influence of the ions dissolved in a liquid on the impedance spectroscopy of a cell. Our analysis is performed in the small-voltage regime, where the actual bulk density of ions is only slightly perturbed by the external electric field. In this framework, we show that the presence of the ions can be taken into account by a surface density of charge. The agreement between the theoretical prediction, on the basis of the assumption that the ionic mobility is frequency independent, and the experimental data for the real and imaginary parts of the impedance is fairly good for frequencies larger than 100 Hz. In the low-frequency range, the agreement of the theory with the experiment is rather poor. In this region, the experimental data can be successfully fitted by introducing the impedance of the metal-electrolyte interface, which is accurately represented by Zi = w(i omega)(-nu), where w and nu are two constants, with 0 < nu < 1. From the analysis of the experimental data, we determine w and nu. The theoretical predictions of our model are in good agreement with the experimental data in the investigated frequency range.

Purification from human plasma of a tetrapeptide that potentiates insulin-like growth factor-I activity in chick embryo cartilage.

Human plasma has been shown to contain a low molecular weight factor that potentiates human IGF-I stimulation of glycosaminoglycan synthesis in chick embryo cartilage. The peptide was purified and characterized by Edman degradation and electrospray mass spectrometry. The primary structure determined was: Trp-Gly-His-Glu. A homologous synthetic peptide similarly promoted matrix biosynthesis in cartilage exposed to IGF-I.

Oxidative decarboxylation of benzoic acid by peroxyl radicals.

A chemical model based on the thermal decomposition of AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride is used for the production of peroxyl radicals. Peroxyl radicals induces the decarboxylation of [7-13C]benzoic acid and the production of 13CO2, which is measured by gas chromatography-isotope ratio mass spectrometry (GC-IRMS). The decarboxylation depends on temperature, AAPH, and benzoic acid concentrations. The decarboxylation also depends on the presence of oxygen. Electron spin resonance studies are performed to confirm the presence of peroxyl radicals under oxygen and of carbon-centered radicals in the absence of oxygen. Decarboxylation rates are measured in the presence of various antioxidants: ascorbate, dimethylsulfoxide, mannitol, and uric acid. It turns out that the decarboxylation is inhibited by each of these antioxidants. The ratio of decarboxylation rates, with and without the antioxidant, varies linearly with the antioxidant concentration. HPLC and GC-MS analyses of reaction products between benzoic acid and AAPH-derived radicals do not detect the presence of radical substitution products on the aromatic ring or the products derived from benzoic acid. There is no doubt that GC-IRMS is a powerful technique to investigate the effects of peroxyl radicals on benzoic acid. In addition, it is possible to follow the degradation of 13C-labeled chemical targets exposed to peroxyl radicals through the production of 13CO2.

Formation of cryptophanes from their precursors as viewed by liquid secondary ion mass spectrometry.

The formation of cryptophane-A (C1) and the deuterated cryptophanes C2-C6 from their respective precursors P1-P6 in a mass spectrometer ion-source was evidenced by liquid secondary ion mass spectrometry (LSIMS). Mass-analyzed ion kinetic energy experiments performed on the precursor molecular ions suggested that cryptophane formation occurred mainly in the liquid-matrix before desorption rather than in the gas phase. In addition, we observed that the presence of cations, such as lithium or sodium ions, inhibited the formation of the cryptophane molecular ions. In the light of these results we used the LSIMS technique to investigate the formation of the new cryptophanes C7-C13. All the data collected support the idea that a direct comparison can be made between these experimental findings and chemistry in solution.

Improved method of detection of testosterone abuse by gas chromatography/combustion/isotope ratio mass spectrometry analysis of urinary steroids.

The current approach to detection of doping with testosterone is based on measuring the testosterone to epitestosterone ratio (T/E) in urine by gas chromatography/mass spectrometry. The median T/E for healthy males who have not used T is about 1.0. In a single urine, a T/E lower than six leads to a negative report even though it does not exclude T administration. A value greater than six indicates possible T administration or a naturally elevated ratio. It has been shown previously that the carbon isotope ratio of urinary T changes after T administration. In this study a potential confirmation method for T abuse was optimized. Gas chromatography/combustion/carbon isotope ratio mass spectrometry (GC/C/IRMS) was used to analyze two T precursors (cholesterol and 5-androsten-3 beta, 17 beta-diol) and two T metabolites (5 alpha- and 5 beta-androstane-3 alpha, 17 beta-diol) in addition to T itself in each of 25 blind urines collected from eight healthy men before, during or after T administration. The carbon isotope ratios of T and the metabolites were lower after T administration. The relationships among the variables were studied using multivariate analysis and beginning with principal components analysis; cluster analysis revealed that the data are composed of two clusters, and classified the samples obtained after T administration in one cluster and the remainder in the other; discriminant analysis correctly identified T users. The measurement of carbon isotope ratios of urinary androgens is comparable to the T/E > 6 test and continues to show promise for resolving cases where doping with T is suspected.

Urinary testosterone (T) to epitestosterone (E) ratios by GC/MS. I. Initial comparison of uncorrected T/E in six international laboratories.

Six laboratories in six countries collaborated to investigate the analytical method for estimating the testosterone to epitestosterone ratio (T/E) in urine by gas chromatography/mass spectrometry in the context of detecting the application of T as a doping agent in sport. The protocol specified many but not all details of reagents and instrument conditions. The design included the distribution and analysis of four urines with different T/E values, three replicates per value, and one standard. The ranges of mean T/E values for the four urines estimated by peak area (PA) were 0.32-0.42, 0.72-0.94, 0.91-1.14 and 3.19-5.48. The analyses of variance for these data and for the peak height (PH) data were significant for the laboratory factor (p < 0.0001). In addition there was a significant interaction between the urine factor and the laboratory factor which indicates the complexity of the analysis. T/E calculated using PA was not significantly different from that using PH. For within-laboratory precision all values for PH and PA were < 8.3%, and for between-laboratory precision all values were < 11.7% except for one (20.1%). The data represent a baseline for future experiments designed to elucidate the sources of within-and between-laboratory variance, and to harmonize estimates of T/E.

Saponins from three species of Mimusops.

Six saponins were isolated from the seed kernel of Mimusops elengi, M. hexandra and M. manilkara. Their structures were determined using a combination of 1H NMR, 13C NMR and mass spectroscopy. Three of them are new compounds: 3-O-(beta-D-glucuronopyranosyl) 28-O-(alpha-L-rhamnopyranosyl (1-->3) beta-D-xylopyranosyl(1-->4) [alpha-L-rhamnopyranosyl(1-->3)] alpha-L-rhamnopyranosyl(1-->2) alpha-L-arabinopyranosyl) protobassic acid, 3-O-(beta-D-glucuronopyranosly) 28-O-(alpha-L-rhamnopyranosyl(1-->3) beta-D-xylopyranosyl(1-->4) alpha-L-rhamnopyranosyl(1-->2) alpha-L-arabinopyranosyl) 16-alpha-hydroxyprotobassic acid and 3-O-(beta-D-glucopyranosyl(1-->3) beta-D-glucopyranosyl) 28-O-(alpha-L-rhamnopyranosyl(1-->3) beta-D-xylopyranosyl(1-->4) alpha-L-rhamnopyranosyl(1-->2) alpha-L-arabinopyranosyl) protobassic acid.

Saponins from the stem bark of Filicium decipiens.

Four new saponins have been isolated from the stem bark of Filicium decipiens and identified as 3-O-{beta-D-glucopyranosyl(1-->2)-beta- D-glucopyranosyl}-28-O-{[alpha-L-arabinopyranosyl(1-->2)-beta- D-xylopyranosyl (1-->6)]. [4-O-angeloyloxy-alpha-L-arabinopyranosyl(1-->2)-alpha-L- rhamnopyranosyl(1-->2)]}-beta-D-glucopyranosyl gypsogenic acid, 3-O-{beta-D-glucopyranosyl(1-->2)-beta-D- glucopyranosyl}-28-O-{[alpha-L-arabinopyranosyl(1-->2)- beta-D-xylopyranosyl(1-->6)] [4-O-angeloyloxy-alpha-L- arabinopyranosyl(1-->2)-alpha-L-rhamnopyranosyl(1-->2)]}-beta-D- glucopyranosyl medicagenic acid, 3-O-{beta-D-glucopyranosyl(1-->2)-beta-D- glucopyranosyl}-28-O-{[alpha-L-arabinopyranosyl(1-->2)] [beta-D-xylopyranosyl(1-->4)]alpha-L-rhamnopyranosyl(1-->2)-4-O-[3'- hydroxy-2'-methyl-butyroyloxy)-3-hydroxy-2-methyl-butyroyloxy++ +]-beta-D- fucopyranosyl} medicagenic acid and 3-O-beta-D-glucopyranosyl-28-O- {[alpha-L-arabinopyranosyl(1-->2)] [beta-D-xylopyranosyl(1-->4)] alpha-L-rhamnopyranosyl(1-->2)-4- O-[(3'-hydroxy-2'-methyl-butyroyloxy)-3-hydroxy-2-methyl-butyro yloxy]-beta- D-fucopyranosyl} zanhic acid. These structures were elucidated by analysis of 2D-NMR spectra and of electrospray ionization mass spectra.

Structural determination of zinc dithiophosphates in lubricating oils by gas chromatography-mass spectrometry with electron impact and electron-capture negative ion chemical ionization.

Pentafluorobenzyl ester derivatives were used to identify zinc dialkyldithiophosphates and diaryldithiophosphates antiwear engine oil additives by GC-electron impact ionization (EI) MS and GC-electron-capture negative ion chemical ionization (ECNCI) MS analysis. GC-EI-MS of the dialkyldithiophosphate-pentafluorobenzyl derivatives afforded characteristic fragment ions corresponding to the cleavage of one and two alkyl radicals. In most cases, information was only obtained on one alkyl chain. Additional and complete information was obtained with retention time indices using synthetic derivatives and with GC-ECNCI-MS analysis. ECNCI afforded characteristic dithiophosphate anions which allowed the determination of the total number of carbon atoms in the alkyl radicals. The diastereoisomer mixtures of 2-hydroxy-sec.-alkyl radicals were completely separated on GC analysis.

Bloch wave approach to the optics of crystals.

The Bloch wave method is used to find the effective permittivity tensor epsilon of periodic liquid crystals and artificial structures whose period p is short with respect to the light wavelength lambda and whose optical properties are defined by a permittivity field epsilon(r). The main role of the multiple scattering within the periodic medium is evidenced, and very general expressions of epsilon, based on expansions in ascending powers of the ratio p/lambda and of the light wave vector k, are found. Such expansions allow to discuss the general properties of epsilon, to clarify the role of the spatial dispersions, i.e., to separate the part of epsilon explicitly depending on k from its k-independent part, and to find some interesting properties of crystals that are (i) periodic in only one direction, or (ii) locally isotropic. Finally, the limits of validity of the macroscopic model are discussed. Within these limits only a few terms of the power expansions are required, and their expressions are explicitly given. The obtained results are also useful to better understand the macroscopic optical properties of solid crystals.

Anomalous intensity dependence of optical reorientation in azo-dye-doped nematic liquid crystals.

The intensity dependence of optical nonlinearity in a nematic liquid crystal doped with an azo-dye is investigated. The reorientational part of the nonlinearity changes from self-defocusing to self-focusing character while the intensity passes through the saturation value of trans-cis photoisomerization. This observation, in accordance with previous models, indicates that the optical torque generated by the trans-isomers is of opposite sign than the torque generated by the cis-isomers. At very low intensities a further reorientational nonlinearity was found, which is attributed to light-induced orientational redistribution of adsorbed molecules at the surface.

Isolation and characterization of inositol sphingophospholipids from Phytophthora parasitica Dastur.

Several inositol sphingophospholipids (ISPL) were isolated from mycelia of Phytophthora parasitica Dastur, a phytopathogenic fungus of carnation. The ISPL structures were determined by fast atom bombardment. All ISPL consisted of ceramides linked to inositol phosphate. We investigated the effect of growth conditions on the ISPL produced in four different media that are commonly used for fungal cultures. We showed that P. parasitica Dastur synthesized four major classes of compounds with molecular weights of M(r) = 751, 807, 835, and 849 containing the 16:1 base and the 16:0 or 20:0 or 22:0 or 22h:1 N-acyl group. The relative abundance of the different ISPL is dependent on growth conditions.

Kinetic studies of cytoplasmic and mitochondrial aconitate hydratases from rat liver.

The kinetic properties of the cytoplasmic and mitochondrial aconitate hydratases of rat liver have been studied by measuring the formation of the two products from each of the three tricarboxylic acids used as substrate. The kinetic properties of the two enzymes are very similar; the similarity of the Km values for each of the three substrates is particularly remarkable. The results are discussed with particular reference to a possible role of the cytoplasmic aconitate hydratase in the process of gluconeogenesis. With both aconitate hydratases, substrate activation by citrate and D-isocitrate has been observed.

[Strategies for the development of the quality of integrated home care]. / Strategie di sviluppo della qualità dell'assistenza domiciliare integrata.

Home Health Care (HHC) is a strategy to provide holistic care for elderly, chronic and terminal patients and for patients early discharged from hospitals. The aim of this article is to give some advice to ensure social and health integration, coordination of procedures and family participation in HCC. Moreover a framework to ensure organization of a HHC service is required and a guideline to define functions and responsibilities, human and physical resources and procedures to deliver health care is provided. The first stage of a HHC project must be an educational programme for the multidisciplinary team involved in home care. The common education is the strategy to improve Quality of a HHC service, involving medical and social workers in the definition of their own "criteria and standards" for organization, resources and procedures.

[Multidimensional diagnosis in general medicine. Comparative study of the use of various instruments]. / La diagnosi multidimensionale in medicina generale. Studio comparativo sull'uso di alcuni strumenti.

This study was performed to test three instruments for functional status assessment in General Practice: the Dartmouth Coop Charts (COOP Charts), the Functional Status Questionnaire (FSQ) and the Duke University Health Profile (DUHP). All the instruments covered a score of functional aspects in physical, mental and social areas, providing a multidimensional measure of health status. We used these three instruments, validated by international studies, to acquire information concerning their feasibility and acceptability among patients from rural communities needing primary care and to test their validity in differentiating between patient subgroups. The COOP Charts, the FSQ and the DUHP were administered by physicians respectively to 98, 100 and 97 patients, waiting for a visit in the ambulatories of their General Practitioner. Answers relating to each instrument were analyzed according to sex, age and education of patients. All the instruments seemed to be feasible and acceptable, but only the COOP Charts and the FSQ were able to discriminate between different sex, age and scolarity groups. Taking into account the need to elaborate answers according to a formula when using the FSQ, we concluded that the best instrument for General Practice to provide a multidimensional measure of health status seems to be the COOP Charts.

[Study of plasma acylcarnitines using tandem mass spectrometry. Application to the diagnosis of metabolism hereditary diseases]. / Etude des acylcarnitines plasmatiques par spectrométrie de masse en tandem. Application au diagnostic des maladies héréditaires du métabolisme.

BACKGROUND: L-carnitine is known to transport long chain fatty acids through the mitochondrial membrane but also to export accumulated acyl-CoA's as acylcarnitine esters. Acylcarnitine identification in body fluids allows the diagnosis of mitochondrial inborn errors especially fatty oxidation defects. Tandem mass spectrometry represents a new method for isolation and identification of acylcarnitines in plasma or in blood spotted onto filter paper (Guthrie cards). MATERIAL AND METHODS: In order to validate our method, we studied 30 plasmas from children affected with 15 different inborn errors of metabolism and five amniotic fluids from fetuses affected with several organic acidurias. Fourty-six samples from children at risk for mitochondrial fatty oxidation disorders have been analyzed. We developed a method of tandem mass spectrometry with liquid secondary ion mass spectrometry using deuterated acylcarnitines as internal standards. RESULTS: This method is very sensitive (detection limit = 2 microM). In all affected patients specific acylcarnitine signals corresponding to the metabolic block were constantly found. This confirms the diagnosis and validates the method. Among the 46 at risk children, four defects of long chain fatty acid oxidation were identified. CONCLUSION: This new method is of great interest especially for the long chain fatty acid oxidation defects. These defects are very difficult to diagnose with classical methods as urinary organic acid profiling. A small amount of plasma (100 microL) or blood spotted onto paper is required. The acylcarnitine profile allows a rapid diagnosis if a dedicated apparatus is available.

[Health education through sports. Proposal for an intervention in the formative years]. / Educazione sanitaria allo sport. Proposta di intervento nell'eta' evolutiva.

The authors present the program: "Physical fitness and health education", direct to childhood of secondary school. The goal is to encourage physical activity and behaviors linked with exercise (caloric intakes and weight control, no smoking ...) to improve health and fitness. The accomplishments of the program need collaboration between physicians and gym teachers to determine methods of realizations and verifications of the program. The educational program has been based on the operative pattern introduced by Guilbert. It is developed through four stages: 1) Definition of pedagogical objectives. The authors point three intermediate objectives a) acquisition of theoretical and practical elements characterizing training exercises wealthy for growth and health; acquisition of the right attitudes not be lose the aforesaid educational contents. b) acquisition of theoretical and practical elements characterizing a diet wealthy for growth and efficiency in sports; acquisition of the right attitudes not be lose the aforesaid educational contents. c) acquisition of theoretical and practical elements for a correct way of life allowing growth and efficiency in sports; acquisition of the right attitudes not be lost the aforesaid educational contents. 2) Definition of methods of verification. The authors point out the following: a) written and oral test, questionnaires for the valuation of the "know" level b) practical tests for valuation of the "know how to do" level c) reports from the teachers of valuation of the "know how to be" level. 3) Organization and realization of the program. The authors point out educational contents to get the three intermediate objectives, together with the times for realization of the program. The fixed program is introduced to the young people at the beginning of secondary school in physical education periods all over the three years of the first grade course. 4) The authors point out the moments of verifications: beginning and end of each or the three years of course.

Impedance spectroscopy of conductive commercial hydrogels for electromyography and electroencephalography.

In biomedical applications such as the electroencephalogram, electrocardiogram and electromyogram, usually conductive hydrogels are applied to the skin providing a skin-electrode layer, with the aim of lowering contact impedance and improving the signal/noise ratio. It is important to know the electrical behavior of these materials, obtaining reliable values of their conductivity. It can be extracted by the whole cell impedance, which is affected not only by the bulk but also by the surface properties. The aim of the present work is to characterize the impedance of some currently available conductive hydrogels in the frequency range 5 Hz-1 MHz. For this purpose we have designed a novel cell, with silver electrodes, optical detection of the gap and guard ring. In this paper the results are compared with those obtained by means of a commercial cell with stainless steel electrodes, without a guard ring. In order to extract the different contributions of the bulk and the interface, we fitted the experimental data with a very simple model: the bulk properties are described by a single RC-parallel, whereas the interface is represented by a constant phase element.