Hair testing is superior to urine to disclose cocaine consumption in driver's licence regranting.

In driver licence regranting, subjects with a history of cocaine use are requested to undergo laboratory testing to verify both current and past abstinence from the drug. Identification of cocaine use based only on urinalysis may miss some cases because of the short elimination half-life of the drug. Moreover, many abusers know how to time their cocaine consumption in such a way that they can "beat" the urinalysis, having a series of negative urine tests. We report on the use of hair testing to disclose sporadic cocaine consumption in seven subjects attending the Local Medical Commission to reobtain driver licence, with constant negative urinalysis. Even with one or two weekly negative urine screens along several months, all the subjects were positive using hair testing for cocaine and benzoylecgonine, above the internationally recommended limit of quantification: 0.5 ng/mg and 0.05 ng/mg for cocaine and benzoylecgonine, respectively (concentration range for cocaine: 0.51-2.23 ng/mg hair; concentration range for benzoylecgonine: 0.08-1.70 ng/mg hair). The obtained results support strongly the use of hair testing for cocaine in drug addicts and occasional abusers applying for regranting of driver licence in order to minimize social risk behaviours.

Diagnosis of diabetes mellitus at the hospital of Venice in 1863.

In 1674 Thomas Willis reported that the presence of urine 'as sweet as honey' was the pathognomonic sign of diabetes mellitus. In the 19th Century several reactions for the detection of glucose in urine were proposed and glucose measurement became common in the laboratories that were being set up in Europe. A case of diabetes mellitus, diagnosed by Namias, the head of the Women's Section of the Medicine Department of Venice Hospital, was reported in 1863 in the 'Giornale Veneto di Scienze Mediche' which contains clinical and laboratory information. A 34-year-old woman was admitted to the hospital for polydypsia, polyuria, bulimia and fatigue. Urine was weighed for 2 months (2-10 kgday(-1)) and the relative density ranged from 1.045 to 1.038. Glucose was measured in the urine using Moore, Trommer and Fehling reagents. A few days after admission a urine sample showed 7.69 parts/100 parts of urine and a blood sample showed 547 mg of glucose/100 g of serum. The assays were carried out in the Clinical Laboratory of Venice Hospital, founded in 1863, directed by Giovanni Bizio, one of the first chemists who graduated at Padua University. In 1863 chemical analyses were commonly carried out in Venice as in the other parts of Habsburg empire.

Determination of HbA1c in the presence of haemoglobin variants: comparison of three HPLC techniques.

The presence of Hb variants may cause analytical interference in HbA1c values measured with the HPLC techniques currently used in Italian laboratories. In this study performance of a new HPLC system, (HA-8140, Menarini) was compared with two other traditional HPLC systems (HA-8121, Menarini and Diamat, Bio-Rad). The HA-8140 system detects the Hb variant possibly present in an independent peak. The integration area of such a peak is not computed in the calculation of the percentage value of HbA1c. In this manner the underestimation of values obtained with traditional HPLC systems is avoided.

Morphogenetic effects induced by spermine and ruthenium red in yeasts.

Spermine (Sp) produces growth inhibition and wall malformation in Saccharomyces cerevisiae in response to oversynthesis of beta-glucans and chitin. The effect is related to the polycation nature of the molecule. In the present work, to verify this hypothesis, the yeast was treated with the abiogenic polycation ruthenium red (RR). The strict analogy observed between the RR- and Sp-induced alterations reinforced our earlier assumption that Sp interacted with the anionic sites of the plasmalemma determining a spurious activation of the two inserted enzymes beta-glucan and chitin synthases. This view was further confirmed by the aberrant accumulation of beta-glucans in Schizosaccharomyces pombe and of chitin in Rhodotorula glutinis treated with Sp and RR. In these micro-organisms spermidine, which bears three amino groups instead of the four encountered in Sp, was ineffective. It is inferred that at least four cation sites must be present in a compound in order to affect wall morphogenesis in yeasts.

Stimulation of the autophagic activity in blastospores of Candida albicans exposed in vitro to fluconazole.

Blastospores of Candida albicans were exposed in vitro to fluconazole, a bis-triazole which inhibits ergosterol biosynthesis in fungi by interfering with the cytochrome P-450 dependent 14 alpha-demethylase. Electron microscope examination revealed that a low dose (3 micrograms/ml), short treatment (1-3 h) with this compound greatly stimulated autophagic activity not accompanied by alterations of the cell organelles. Only rarely, wall thickenings and some damage to the membrane system, except for the plasma membrane, was noted. This unusual phenomenon suggests that in the presence of fluconazole, due to the depletion of ergosterol and the consequent accumulation of 14 alpha-methylsterols, changes are induced in the properties of the tonoplast. It seems that in the presence of the molecule the membrane is not able to distinguish what is to be degraded from what is still useful for intracellular metabolism with the consequent disintegration of cell compartmentation. Fluconazole may be useful for indicating the mechanism for controlling lytic activity and the homeostatic role of the vacuole in yeasts.

Interaction of monensin on dimorphic transition and hyphal growth in Candida albicans.

Monensin, an exocytosis perturber, was assayed on the dimorphic fungus Candida albicans in order to study its effects on wall synthesis and morphogenesis. In the presence of the drug, both germ-tube formation and hyphal elongation were markedly inhibited according to the dose and exposure time. The effect was accompanied by morphogenetic malformations which could be detected under UV light and by transmission electron microscopy. Enormous wall and septa thickenings revealed by cytochemical tests indicated an abnormal deposition of chitin. Since chitin synthase is inserted in the plasma membrane in a latent state and its activation depends on regulatory factors reaching the cell periphery through an orderly exocytosis, it is assumed that monensin affects the vesicular traffic leading to a prevalence of activators over inhibitors of the enzyme.

Effects of spermine and spermidine on cell division and wall morphogenesis in Saccharomyces cerevisiae.

The effect of polyamines (PA) on the synthesis and deposition of wall constituents in Saccharomyces cerevisiae was investigated. Spermidine (Spd) in various doses was ineffective whereas spermine (Sp) in the same concentrations caused a marked inhibition (25-60%) of cell proliferation accompanied by evident morphogenetic malformations. Sp-treated cells were elongated, grouped in small clusters, and showed malformed septa and aberrant wall thickenings. The PATAg technique revealed that the aberrations consisted of an abnormal accumulation of both reactive materials, like 1,3-beta-glucans, and unstained chitin components. Since 1,3-beta-glucan synthases and chitin synthases are inserted in the plasma membrane, whose anionic sites interacted with the cation groups of Sp, it is assumed that the molecule determines a condition resulting in an unregulated activation of the two enzymes. The fact that Spd, which contains three cationic groups instead of the four contained in Sp, is without effect suggests that a compound must have at least four cation sites in order to affect the cell division and wall morphogenesis of S. cerevisiae.

Metabolites from in vitro cultures of Cassia didymobotrya.

From suspension cultures of Cassia didymobotrya 7-acetylchrysophanol, chrysophanol-physcion-l0,l0'-bianthrone, (E)- and (Z)-3'-hydroxy-3,4,5'-trimethoxystilbene, (E)-4,3'-dihydroxy-3,5'-dimethoxystilbene and 7,4'-dihydroxy-3,5,3'-trimethoxyflavone have been isolated along with several known metabolites.

Aberrant development of Trichophyton mentagrophytes hyphae cultured in the presence of Congo red.

When Trichophyton mentagrophytes colonies were placed on a medium containing 150 micrograms/ml of Congo red, a dye which prevents chitin fibrillogenesis, their growth rate was reduced. The newly formed mycelium, examined under an ultraviolet microscope, consisted of thick, curled and branched hyphae endowed with swollen tips and subapical bulges. Short-time exposure revealed that the major sites of dye accumulation were the extension zones. Normally structured hyphae arose from aberrant mycelia when they were transferred onto a dye-free medium. The phenomena observed suggest that Congo red alters the wall properties of the extension zone, by inhibiting the gradual conversion of chitin chains, synthesized at the extreme tip, into microfibrils of increasing size and density.

Exocytosis in Saccharomyces cerevisiae treated with congo red.

When dividing cells of Saccharomyces cerevisiae were exposed to the polysaccharide-binding dye Congo red, the walls and septa became sites of chitin accumulation. In addition, the cytoplasm showed many vesicles that were different from those accumulating in the growing bud and from the lytic vacuoles of the untreated yeasts. To obtain information about these membranous structures, living cells were observed under phase contrast and UV light microscopes. Furthermore, ultrathin sections of Congo red-treated cells were processed by cytochemical techniques to reveal the chitin areas. Observations suggest that the aberrant vesicles were involved in a secretory process, and that pre-assembled chitin was not among the components transported to the cell periphery.

Separation and quantitation of protoanemonin in ranunculaceae by normal- and reversed-phase HPLC.

A method is described for the extraction and high-performance liquid chromatographic separation and quantitation of the lactone protoanemonin in leaves of Ranunculaceae. For samples with a protoanemonin concentration over 10 microg/g W. W., a reversed-phase technique using a Lichrosorb RP 18 column and a binary solvent system was developed. Alternatively, for samples with a lower lactone concentration, a normal-phase technique with a Lichrosorb Si 60 and quaternary elution system was elaborated. Protoanemonin was detected at 258 nm; its calibration curves were established, and its response factor was calculated using, as standard, the pure compound extracted from HELLEBORUS NIGER. A survey of ten different taxa of Ranunculaceae was performed and it showed the suitability of the method for routine work with high sensitivity limit.

Morphological anomalies induced by Congo red in Aspergillus niger.

Aspergillus niger germ tubes were exposed for 6 h to 0.15 mg/ml of Congo red, a stain which prevents chitin microfibril assembly. The most evident alterations, detected under ultraviolet light and by transmission and scanning electron microscopy, concerned the hyphal tips which burst or, most frequently, expanded into bulges. In the latter structures, new hyphal tips appeared which, after giving rise to more or less developed hyphae, were themselves converted into new bulges. Therefore, segments derived from isotropic and polarized growth alternated in the organisms exposed to the dye. An interpretation of these abnormalities is advanced based upon the assumption that the maintainance of a regular gradient of wall viscosity in the hyphal extension zone depends primarily on the capability of glycan chains to form crystalline aggregates of increasing complexity.

Autophagic vacuole development in Trichophyton mentagrophytes exposed in vitro to miconazole.

Trichophyton mentagrophytes was treated in vitro for 24 h with an inhibitory concentration of miconazole (10(-5) M). The youngest, apical hyphal cells showed alterations of the ultrastructure of their membranes and cell wall as well as several vacuoles of an autophagic nature. Cellular autophagy appeared as a multi-step process beginning with swelling of the endoplasmic reticulum, whose membranes later fused and gave rise to large vacuoles that entrapped previously demarcated portions of the cytoplasm. Sequestered materials, initially unaltered, were ultimately degraded. The initial event in vacuolation was interpreted as entry of water into the cells, especially into the luminal space of the endoplasmic reticulum, in response to a decrease of wall pressure on the cytoplasm.

Effects of erythromycin on Euglena gracilis as a model for testing the toxicity of antibacterial drugs.

Based on the main physiological and ultrastructural effects induced in Euglena gracilis by erythromycin, one of the least toxic of the commonly used antibiotics that specifically inhibit protein synthesis on 70S ribosomes of prokaryotic organisms, a symptomatologic picture is outlined which could be useful for the preliminary evaluation of the toxicity of antibacterial agents.

Two rapid fluorescence procedures for the detection of some thio pungent compounds in plant tissues.

Two rapid flourescence procedures are described for detecting sulphydryl, disulphide and isothiocyanate groups of scented and pungent principles present in the vacuolar sap of onion, garlic and cabbage. To localize compounds containing sulphydryl groups, fresh or fixed frozen sections of the plants were treated with mercurochrome. After the fluorochromization, strongly-positive sulphydryl sites emitted an intense orange-red fluorescence, while weakly-positive sites emitted a distinctive red-brown fluorescence. Disulphide groups were detected by first reducing with thioglycolic acid to thiol groups before treating with mercurochrome. To effect isothiocyanate localization, frozen sections were exposed to ammonia: isothiocyanates were converted to thioureas and the engendered amino groups were revealed with fluorescamine.

Coumarin-induced disturbances of morphological development and cell wall formation in Trichophyton mentagrophytes.

When Trichophyton mentagrophytes thalli are placed on a medium containing 300 microgram/ml of coumarin, their growth rate is drastically reduced and the newly formed mycelium consists of curled and branched hyphae showing subapical bulges and swollen tips. Under the electron microscope, the most relevant abnormalities concern the cell walls which are often thickened by aberrantly shaped zones that are usually smeared unevenly over the surface of the "primary" wall instead of being deposited in discrete ridges. An ultracytochemical analysis suggests that the irregular thickenings are formed of chitin. Arguments suitable to explain the phenomena observed are discussed.

Electron microscopic evidence of Phosfon D-induced alterations in the membranes of the dermatophytic fungus Microsporum cookei.

The dermatophytic fungus Microsporum cookei was cultivated for 24 h in the presence of subinhibitory and inhibitory concentrations (50 and 100 micrograms/ml) of Phosfon D, a growth retardant for higher plants also affecting fungal development, and its toxic effects were examined at the ultrastructural level. In both treatments, Phosfon D attacked the membranes, whose structural integrity was clearly compromised with damage of particular severity to mitochondria, nuclei and endoplasmic reticulum. In the instance of fungal growth suppression, the compound also caused plasmolytic and autolytic phenomena, sometimes accompanied by plasma membrane breakages. The submicroscopic effects observed confirm that Prosfon D is an antifungal compound which displays its toxic effects in the area of lipid metabolism, probably preventing the synthesis of fundamental components of the cellular membranes, such as unsaturated fatty acids and sterols.

Fungal toxicity of phosfon D and its reversion by lipids.

The growth retardant Phosfon D inhibits the growth of some yeasts and human pathogenic filamentous fungi. The toxic effects of the compound on the dermatophyte Microsporum cookei were completely reversed by adding ergosterol and oleic acid to the medium. The significance of these findings is discussed.