Unique expression pattern of human nicotinamide mononucleotide adenylyltransferase isozymes in red blood cells.

Nicotinamide mononucleotide adenylyltransferase (NMNAT) catalyzes the formation of nicotinamide adenine dinucleotide (NAD). In humans, three isozymes have been identified: NMNAT1, which is widely expressed in all tissues, NMNAT2 and NMNAT3, which show a tissue-specific expression and whose mRNA levels are generally lower compared to NMNAT1. In the present study we determined the individual NMNAT isozymes activity in human red blood cells (RBCs) by using a biochemical discrimination assay based on the distinctive catalytic properties of the three proteins. We found that isozyme 3 predominates over isozyme 1, whereas isozyme 2 is absent. This high prevalence of NMNAT3 is cell-aging independent and was also confirmed by analyzing the mRNA and protein levels. RBC represent the first human cell type with a remarkable predominance of NMNAT3, and this unique expression pattern is discussed in light of the catalytic properties of the isozymes and in consideration of the biochemical microenvironment of RBC.

Nontuberculous mycobacteria in hospital water systems: application of HPLC for identification of environmental mycobacteria.

Nontuberculous mycobacteria (NTM), ubiquitous in water environments, are increasingly recognized as nosocomial pathogens. Our study reports a one-year survey of the water system of two hospitals, A and B, in a small town near Florence, Italy. NTM were found throughout the study period in both settings, but B showed a significantly higher mycobacterial load. Mycobacterium gordonae and Mycobacterium fortuitum were the most frequent species isolated. Identification was carried out by conventional techniques and by high performance liquid chromatography (HPLC) analysis of cell wall mycolic acids. HPLC profiling could be used as a first-choice method for identification of environmental mycobacteria.

Correction of emission spectra in microspectrofluorimetry using a reference lamp: computations.

Accurate correction of emission spectra in microspectrofluorimetry, using a reference lamp, may require that a factor for the emissivity of tungsten be introduced. This is only possible provided that the true temperature of the lamp filament is known. A method is given for obtaining the true temperature from the knowledge of the colour temperature. Also, the values of the spectral concentration of the radiance of the black body, either computed according to Planck's equation or taken from available published tables, have to be converted from energetic units to units of quanta since the photomultiplier is linear not to absorbed power but to units of quanta. When the fluorescence spectra to be corrected extend into the far red it is preferable to use a lower temperature (by lowering the supply voltage) than that for which the lamp is certified. It is possible to determine the new temperature (and then the corresponding spectral distribution) by taking a few pairs of measurements at different wavelengths both at the lower voltage and at the voltage for which the lamp is certified and then introducing these values in a non-linear regression soluble on a PC with a curve fitting program. The microscope tungsten halogen lamp can conveniently be used as a reference, thanks to its small size and its steady spectral characteristics. When high accuracy is required, however, the halogen lamp should be calibrated against a certified ribbon filament lamp.

Ethidium bromide fluorescence photo-enhancement in glycerol.

Frog erythrocyte nuclei were stained with ethidium bromide at concentrations of 10(-6) M to 10(-4) M and mounted in phosphate buffer or in glycerol. Ethidium fluorescence was excited with different irradiation powers at 514 nm from an argon laser, and the variation of fluorescence was measured with a microfluorimeter over the first twenty seconds of illumination. The following differences between glycerol and buffer mounting were observed: 1) fading was more delayed in glycerol at all dye concentrations and light powers; 2) fluorescence yield was significantly lower in glycerol compared to buffer at high dye concentration; 3) whereas from the start of illumination fluorescence exponentially declined in buffer, there was an initial fluorescence photo-enhancement in glycerol followed by exponential fading. Both retardation of fading and fluorescence photo-enhancement were more prominent with increasing dye concentration. It is suggested that the light induced fluorescence increase was caused by concentration dequenching and was observed in glycerol, not in buffer solution, through one or both of the following glycerol effects: 1) aggravation of self-quenching of the secondary type of binding of ethidium; 2) delayed fading. The concomitant attenuation of the inner filter effect through the photo-destruction of the non-fluorescent ethidium dimer, which absorbs within the excitation spectrum of the ethidium monomer, could also contribute to an apparent amplification of the fluorescence build-up.

Wavelength dependence of the time course of fluorescence enhancement and photobleaching during irradiation of ethidium bromide-stained nuclei.

The variation of fluorescence during irradiation of ethidium bromide-stained nuclei with the 458 nm argon laser line was measured at different wavelengths throughout the emission spectrum. When glycerol was used as a mountant, photoenhancement of fluorescence was observed at all wavelengths, but was greater at the shorter wavelengths. Fluorescence increased by almost one order of magnitude at 500 nm after 40 s of irradiation, compared with only about 10% at wavelengths longer than 600 nm after 2-3 s. In nuclei mounted in phosphate buffer, an initial photoenhancement of fluorescence was detected only at the shorter wavelengths, while continuous photobleaching was observed in the rest of the emission spectrum. When the spectra are normalized to maximum, so as to eliminate the effect of the concurrent photobleaching, it appears that the difference between the time course of fluorescence variation in buffer and glycerol depends largely on the lower photobleaching rate in glycerol. The photoenhancement of fluorescence at shorter wavelengths was found to consist of a band peaking at 485-491 nm in glycerol and at 495-496 nm in buffer. Attenuation of the inner-filter effect contributes minimally to the enhancement of fluorescence at shorter wavelengths. Since the dimer is known to be non fluorescent, the light-induced disaggregation of dimers to monomers cannot be an explanation for the large increase of fluorescence at the shorter wavelengths. The same laser beam that was used to excite the fluorescence of stained nuclei was also used for monitoring the concomitant variation of transmitted light, from which the variation of absorptance during irradiation was computed. While the expected decrease of absorptance was observed in glycerol, reflecting the photodestruction of the fluorophore, in buffer solution an unexpected initial increase was found, which may reflect the accumulation of an absorbing photoproduct.

Chemical composition of Schiff's reagent crystals.

Crystals were prepared by adding 0.04 M sulfuric acid to a Schiff's reagent made with 2.5 g pararosaniline (PR) chloride dissolved in a saturated SO2 solution. Elemental analysis of the crystals gave the composition C19H21N3S2O7.4H2O which corresponds to the sulfate of pararosanilinesulfonic acid (PRSA) tetrahydrate. The moisture content was ca. 5%. A reagent reconstituted by dissolving 0.2 grams of crystals in HCl 0.1 N contains ca. 3.5 x 10(-3) M or 0.11% PR. A solution prepared with 2.5 g PR in 100 ml O.1 N HCl plus 0.04 M M K2S2O5 gave only a few crystals after 0.04 M sulfuric acid was added. The PR content, determined colorimetrically, was 0.25% compared with 1.35% in saturated SO2. The per cent dye loss during charcoal purification was also higher. The low concentration of PR, caused both by the lower solubility and by the larger loss during charcoal purification explains the poor yield of crystals of a reagent prepared in HCl/K2S2O5, compared to a reagent prepared in saturated SO2. After crystallization is complete, the crystals are in equilibrium with a concentration of 0.2% of PR in the supernatant: when the initial concentration is close to this value crystallization is negligible or completely fails.

A simple procedure for crystallization of the Schiff reagent.

Formation of crystals in Schiff reagents prepared from SO2 gas previously has been reported either soon after preparation, using high dye concentrations and heating, or after long periods of storage at room temperature. With the first type of procedure only a low yield of crystals accompanied by dye precipitation was obtained. Crystallization without dye precipitation took place if the reagent, prepared with pararosaniline base or chloride in a saturated SO2 solution, was stored for a sufficient time at room temperature in partly filled flasks. These crystals remained colorless if washed with acid alcohol after being separated by filtration. Schiff reagents layered with paraffin oil or supplemented with 0.1 M hydroquinone took much longer to crystallize, suggesting that crystallization is promoted by the partial oxidation of sulfurous acid to sulfuric acid. A high yield of crystals can be obtained at room temperature after as little as 24 hr by adding 0.04 M of H2SO4 to a Schiff reagent prepared with 2% pararosaniline chloride in a saturated SO2 solution. A Schiff reagent prepared with only 0.2% of these crystals gives an intense staining in the Feulgen and in the Periodic acid-Schiff reactions.

[The role of infective intestinal complications on the course of Crohn's disease and ulcerative rectocolitis]. / Ruolo delle sovrapposizioni infettive intestinali in corso di morbo di Crohn e rettocolite ulcerosa.

The authors studied the incidence of protozoal and bacterial infection in 41 cases of Crohn disease und ulcerative colitis; the effect of anti infective therapy on the clinical course has also been tested. Patients with ulcerative colitis are more frequently involved in this type of complication than those with Crohn disease. Therapy has a positive influence on the course of the disease; therefore systematic study of the faeces for bacteria and parasites is necessary for a correct understanding of chronic inflammatory bowel disease and for specific treatment.

Autoradiographic thymidine-3H activity after successive steps of the Feulgen reaction.

Autoradiographs were prepared of sections of the ovary of Dytiscus marginalis labelled with thymidine-3H after each successive step of the Feulgen reaction and after treatment with each separate component of the Schiff's reagent. Results of grain counts over ovarian nurse cells showed that losses of thymidine-3H activity occur not only during hydrolysis but also during the successive steps of the Feulgen reaction. It is suggested that the latter decrease in radioactivity may depend on the extraction of fragments of apurinic acid from the sections. An emulsion desensitizing effect has also been observed in sections stained with basic fuchsin alone; this effect appears, however, to be strongly counteracted by the metabisulphite present in the Schiff's reagent.

Selection of optimal transmittances with the cytophotometric two-wavelength method.

With the help of a computer program using Newton's iterative method, it is possible to obtain a practically instantaneous solution for the two-wavelenght method equation even for values different from 2 for the ratio k of the extinction coefficients. The influence of the extinction ratio and of the mean transmittance on the correction of the distributional error and on the propagation of stochastic and systematic errors into the computed chromophore amount has been studied. After due precautions are taken to minimize chromatic and non-specific light loss errors, the selection of the optimal wavelenght couple will basically depend only on the compromise between correction of the distributional error and the least propagation of stochastic errors into the computed chromophore amount. In many instances, the use of interference filters will be convenient, since their use will eliminate the uncertainties of monochromator readjustment and will allow evenly illuminated field to be obtained more easily, particularly in the case of large measuring fields.

Substance use among the mentally ill: a comparison of Italian and American samples.

Thirty-six patients with long-standing psychotic illness randomly selected among attenders at two community centers of Bologna were interviewed as were their therapists, in order to determine prevalence and patterns of substance use, reasons for use and effects on illness. Protocols were the same as used in previous research carried out in Boulder, Colorado. For all substances, except stimulants and over-the-counter products, prevalence rates were lower in the Bologna sample than in Boulder. Overall lifetime rates of substance use were significantly lower in the Bologna sample. Nevertheless 47.2% of the Bologna sample had a lifetime moderate to severe drug use and nearly two-thirds of the sample had a current drug use of some severity. Reasons for use equally reflected attempts to alleviate unpleasant emotional states and recreational purposes. Patients with high degrees of psychopathology had significantly lower scores on severity of drug use.

[Quality of life assessment: validation of the Italian version of the WHOQOL-Brief]. / La valutazione della qualità della vita: validazione del WHOQOL-Breve.

OBJECTIVE: To test the psychometric properties of the Italian version of the WHOQOL-BRIEF (e.g., construct and internal validity, concurrent validity with the MOS SF-36 and test-retest reliability). The WHOQOL-BRIEF is a 26-items self-report instrument which assesses four domains assumed to represent the Quality Of Life (QOL) construct: physical domain, psychological domain, social relationships domain and environment domain, plus two facets for assessing overall QOL and general health. METHODS: Data have been collected in three sites (Bologna, Modena and Padua), located in the North of Italy, in the framework of the international WHOQOL project. According to the study design, the sample had to include about 50% males and 50% females, 50% of subjects below and 50% above the age of 45, all in contact with various health services. A subsample has been re-interviewed after 2-3 weeks in order to study test-retest reliability. After the WHOQOL-BRIEF, most subjects have also been administered the MOS-SF36 in order to test the concurrent validity between these two instruments. RESULTS: The instrument was administered to 379 subjects (1/6 healthy and 1/6 sick), chosen to be representative of a variety of different medical conditions. Seventy patients, who displayed stable health conditions, have been reassessed after 2-3 weeks to study test-retest reliability. The WHOQOL-BRIEF domains has shown good internal consistency, ranging from 0.65 for the social relationships domain to 0.80 for the physical domain; it has been able to discriminate between in- and out-patients and between the two age groups considered in the present study (< 45, > or = 45 years). Only physical and psychological domains were found to discriminate between healthy and ill subjects. No gender differences in the mean scores for the four domains were found. Concurrent validity between the WHOQOL-Brief and the MOS-SF-36 was satisfactory, and specific for the physical and psychological health domains. Test-retest reliability values were also good, ranging from 0.76 for the environment domain to 0.93 for the psychological domain. CONCLUSIONS: This study shows that the WHOQOL-BRIEF is psychometrically valid and reliable, and that it is also potentially useful in discriminating between subjects with different health conditions in clinical settings.