Regulation of gene expression during the vegetative incompatibility reaction in Podospora anserina. Characterization of three induced genes.

Vegetative incompatibility in fungi limits the formation of viable heterokaryons. It results from the coexpression of incompatible genes in the heterokaryotic cells and leads to a cell death reaction. In Podospora anserina, a modification of gene expression takes place during this reaction, including a strong decrease of total RNA synthesis and the appearance of a new set of proteins. Using in vitro translation of mRNA and separation of protein products by two-dimensional gel electrophoresis, we have shown that the mRNA content of cells is qualitatively modified during the progress of the incompatibility reaction. Thus, gene expression during vegetative incompatibility is regulated, at least in part, by variation of the mRNA content of specific genes. A subtractive cDNA library enriched in sequences preferentially expressed during incompatibility was constructed. This library was used to identify genomic loci corresponding to genes whose mRNA is induced during incompatibility. Three such genes were characterized and named idi genes for genes induced during incompatibility. Their expression profiles suggest that they may be involved in different steps of the incompatibility reaction. The putative IDI proteins encoded by these genes are small proteins with signal peptides. IDI-2 protein is a cysteine-rich protein. IDI-2 and IDI-3 proteins display some similarity in a tryptophan-rich region.

Statistical evaluation of diagnostic and prognostic features of CD30+ cutaneous lymphoproliferative disorders: a clinicopathologic study of 65 cases.

Several clinical and histopathologic features of 65 CD30+ cutaneous lymphoproliferations were evaluated for their diagnostic value between CD30+ primary versus secondary cutaneous lymphomas and for their prognostic significance. Primary cutaneous disease, spontaneous regression, and absence of extracutaneous spreading (but not age < or =60 years) were associated with a better prognosis. Epithelial membrane antigen, BNH9, CD15 or CBF.78 antigen were expressed in all types of cutaneous lymphoproliferations. However, epithelial membrane antigen immunoreactivity was more frequently expressed in CD30+ secondary cutaneous large-cell lymphoma. Among CD30+ primary cutaneous large-cell lymphoma, CD15 expression was only seen in localized skin lesions. P53 expression was not associated with spontaneous regression, extracutaneous spreading, or survival. Nested reverse transcriptase-polymerase chain reaction allowed the detection of NPM-ALK transcripts in 10 of 26 CD30+ primary and in 3 of 11 secondary cutaneous large-cell lymphomas. The ALK protein was detected in only 1 of 50 primary and in 4 of 15 secondary cutaneous CD30+ lymphoproliferations. In CD30+ primary cutaneous lymphoproliferation, NPM-ALK transcripts might be expressed by very rare normal or tumoral cells that are undetectable by immunohistochemistry. However, the expression of either NPM-ALK transcripts or ALK-protein was not correlated with prognosis or age in CD30+ cutaneous lymphoproliferations.

Identification of novel trkA variants with deletions in leucine-rich motifs of the extracellular domain.

The peripheral expression of trkA encoding for NGF receptor was investigated by RNase protection assay. A thymus-specific protected fragment was identified. Using 5' rapid amplification of cDNA ends, three different trkA fragments were characterized. The longer fragment corresponded to the classical trkA L3 transcripts while the two shorter fragments lacked sequences encoding for leucine-rich motifs of the extracellular domain of TrkA, similarly to the trkB L1 and L0 variants. RT-PCR analysis of adult rat tissues showed the expression of trkA L1 transcripts in the thymus, testis, lung and kidney but not in the central nervous system. Their combined expression with trkA L3 transcripts suggests that specific peripheral TrkA oligomers may modulate NGF binding and function in non-neuronal cells.

Expression of NGF receptors in normal and pathological human thymus.

The expression of NGF receptors was investigated in normal human thymus and in thymic hyperplasias, thymomas and thymic carcinomas. By RT-PCR, we detected TrkAI transcripts encoding for the high-affinity NGF receptor. Western blot analysis showed the presence of both TrkA and p75NGFR proteins. In normal thymuses, epithelial subcapsular and medullar cells were TrkA immunoreactive. Interdigitated medullar cells were stained for both TrkA and p75NGFR. While epithelial cells of normal thymuses or benign thymomas exhibited a TrkA positive-p75NGFR negative phenotype, a switch to a TrkA negative-p75NGFR positive phenotype was observed in malignant epithelial cell tumours and was associated with cell proliferation-associated MIB1 expression. Our results argue for a local role of NGF and its receptors on thymic stromal cells both in normal and neoplastic conditions.

Obesity and beta-blockers: influence of body fat on their kinetics and cardiovascular effects.

Beta-blockers are among the most widely used antihypertensive drugs. They differ from each other in regard to several factors such as: beta-agonist activity, beta 1-selectivity and solubility. Aim of this work was to evaluate the influence of obesity on the kinetics and the antihypertensive effect of two Beta-blockers with different solubility such as: the water-soluble, atenolol and the liposoluble, metoprolol. The study was carried out according to an open randomized cross-over design. Eight obese hypertensive patients, after a two week washout period, were randomly allocated to a four week treatment. After a two week intermediate washout period, each patient switched to the other treatment for an additional four week period. On the first and the last day of each treatment the subjects were hospitalized to collect blood samples for the assay of the two drugs and to measure cardiovascular parameters. Obesity does not exert any effect on the kinetics of the water-soluble beta-blocker, atenolol, while markedly interferes with that of the liposoluble, without any apparent influence on its anti-hypertensive effect. These findings extend to obese hypertensives the concept that the plasma concentrations of beta-blocking agents are not reliable predictors of their therapeutic effect.

Differential expression of NGF receptors in human thymic epithelial tumors.

NGF receptor (TrkA and p75NGFR) expression was investigated in human thymuses, including normal thymuses, thymic hyperplasias, thymomas and thymic carcinomas. TrkAI but not TrkAII transcripts were demonstrated by RT-PCR. In normal thymuses, immunohistochemistry revealed a restricted TrkA-immunoreactivity to epithelial and interdigitated reticular cells, while only interdigitaded reticular cells were immunoreactive for p75NGFR. Thymocytes were negative for both receptors. A switch from the normal TrkA positive-p75NGFR negative phenotype to a TrkA negative-p75NGFR positive phenotype was found in histologically aggressive epithelial cell tumors, suggesting that NGF and its receptors are potentially involved in thymus stroma organogenesis and proliferation.

Rapid and highly selective GC/MS/MS detection of heroin and its metabolites in hair.

A direct treatment of methanol-washed hair with a silylating solution is proposed to extract heroin, O-6-monoacetylmorphine, morphine, acetylcodeine, and codeine, obtaining the simultaneous derivatization of the hydroxylated metabolites and reducing potential sample contamination. Analysis is performed by capillary gas chromatography-tandem mass spectrometry (GC/MS/MS) using multiple selected reaction monitoring. Owing to the selectivity and sensitivity of the GC/MS/MS analysis, and to the extremely simple treatment of the sample, the method fulfils the requirements of both clinical and forensic diagnosis of heroin use.

Hair analysis for opiates, cocaine and metabolites. Evaluation of a method by interlaboratory comparison.

The aim of this study was to evaluate the performance of a technique for the simultaneous testing of opiates, cocaine and metabolites in hair by interlaboratory comparison. Sixteen forensic and clinical laboratories with different degrees of experience in hair analysis participated voluntarily in the study (no selection criteria were applied). The suggested analytical procedure, the one routinely used in our laboratory, consisted of incubation in HCl 0.1N (45 degrees C, overnight), solid phase extraction (with Bond Elut Certify) cartridges), derivatisation (trimethylsilyl (TMS) derivatives) and GC-MS analysis. Three different mixtures of finely cut (1 mm or less) hair were prepared using drug-users' and drug-free hair: one 'negative' sample (<0.1 ng/mg for morphine, 6-acetylmorphine (6AM), cocaine and benzoylecgonine (BE)), one 'low concentration' sample (between 0.5 and 2 ng/mg) and one 'high concentration' sample (>3 ng/mg). Accuracy and precision (CV% lower than 5.1, 9.9, 5.2, 3.8, 7.3 and 8.3% for morphine, 6AM, codeine, cocaine, BE, and methylecgonine (ME), respectively; range 0.5-5 ng/mg) of the method and homogeneity of the mixtures were evaluated in our laboratory by intraday (CV% lower than 12% for all analytes) and interday analyses (CV% lower than 17% for all analytes except 6AM, 25%). Participants in the study were grouped into: (1) laboratories (n = 6) obtaining the best qualitative and quantitative values, corresponding to those with long experience in hair analysis; (2) laboratories (n = 5) with no reported false positive and/or false negatives; (3) laboratories (n = 5) with one or more reported false positives/false negatives. The results obtained by the labs of the first group were used as reference values. The scatter of data was similar to those obtained in other published studies.

Simultaneous hair testing for opiates, cocaine, and metabolites by GC-MS: a survey of applicants for driving licenses with a history of drug use.

A sensitive GC-MS method for the simultaneous determination of opiates, cocaine, and metabolites in hair at a cut-off level of 0.1 ng/mg was adopted to assess past exposure to these drugs in applicants for driving licenses with a history of drug use. The sampling protocol consisted of collection of one hair (sample A, 5-cm length) and one urine sample. When hair and urine (EMIT Syva, cut-off levels: 0.3 mg/l for opiates, 0.15 mg/l for cocaine, GC-MS confirmation of positives) were both positive or negative the protocol was concluded. In the other cases, the assessment of 'current exposure' to drugs was carried out, in order to avoid seriated random urinalysis, by collecting a second hair sample (sample B) 6 weeks later and analysing the proximal 1-cm segment. Out of the 214 'A' hair samples analyzed, 14 (6.5%) tested positive for morphine and/or 6-acetylmorphine (6AM), and 26 (12%) for cocaine and/or benzoylecgonine (BE), whereas none of the samples tested positive for both drugs. Levels between 0.1 and 1 ng/mg of the single analytes were found in eight out of the 14 morphine-6AM positives (57%) and in 18 out of the 26 cocaine-BE positives (69%). The time course of positive cases showed a progressive decrease of morphine-6AM positives and a corresponding increase of cocaine-BE positives within the study period September 1995-February 1999. No cases with positive urine and negative hair were observed. Among the 40 positive cases, seven (four and three for opiates and cocaine, respectively) were found to be 'currently exposed to drug', four by urinalysis (three and one) and three by analysis of the hair sample B (1 and 2).

The role of alcohol abuse in the etiology of heroin-related deaths. Evidence for pharmacokinetic interactions between heroin and alcohol.

In order to evaluate pharmacokinetic interactions between heroin and alcohol and their role in the etiology of heroin-related deaths (HRD), the alcohol concentration in blood (BAC), the free (FM) and total morphine (TM) concentrations in blood (determined by DPC Coat-A-Count radioimmunoassay before and after enzymatic hydrolysis), and the TM concentration in urine and bile (DPC Coat-A-Count after enzymatic hydrolysis) in a population of 39 lethal cases included in the records of the Department of Legal Medicine and Public Health at the University of Pavia from the period January 1997-April 1998 were examined. The cause of death in each case was attributed to either heroin or associated heroin-ethanol intoxication. Cases were arbitrarily divided into two groups according to BAC (low-ethanol group, LE, BAC < or = 1000 mg/L and high-ethanol group, HE, BAC > 1000 mg/L). The differences in the FM and TM concentrations in blood, bile, and urine and in the FM/TM ratios between the two . groups were statistically evaluated (Mann-Whitney U test). A similar statistical evaluation was carried out on data from a previously published study concerning the disposition of heroin and its metabolites (6-acetylmorphine and morphine) in blood and urine in 23 lethal cases attributed to either heroin or heroin and alcohol intoxication. The values of the following variables in the LE and HE groups were compared: FM, TM, and 6-acetylmorphine concentrations in blood (6-AM); the FM/ (FM + 6-AM) ratio; the FM/TM ratio; and the urinary concentrations of heroin, 6-acetylmorphine, and free morphine. Statistical analyses of data indicated that high BACs are associated with reduced hydrolysis of 6-AM to morphine (FM/[FM + 6-AM], p = 0.0022) and that a good inverse correlation exists between BAC and hydrolysis of 6-AM to morphine (r2 = 0.67). High BACs were also found to be associated with an increased FM/TM ratio and with reduced excretion of free and total morphine. These results suggest the hypothesis that pharmacokinetic interactions between heroin and alcohol do occur in individuals exposed to high doses of these substances.

A fatal case of trichlorofluoromethane (Freon 11) poisoning. Tissue distribution study by gas chromatography-mass spectrometry.

A case of lethal poisoning due to trichlorofluoromethane (FC11) inhalation is described. The fluorocarbon was determined in biological tissues by headspace gas chromatography-mass spectrometry. FC11 was detected in all the examined tissues, with decreasing levels in heart, lung, brain, liver, blood, kidney, and spleen. The highest concentration measured in heart could be related to the mode of toxic action of fluorocarbons postulated by many authors, characterized by the sensitization of the myocardium to the catecholamines producing arrhythmia and cardiac arrest. Nevertheless the aspecific picture of the anatomo-pathological and histological findings does not exclude that the described accidental fatality may have been caused by the combination of direct from toxicity with hypoxemic asphyxiation, due to the saturation of the atmosphere by FC11 in the closed environment in which the intoxication occurred.

Distribution of embutramide and mebezonium iodide in a suicide after tanax injection.

Tanax is a veterinary formulation for euthanasia comprising embutramide, mebezonium iodide and tetracaine. A 37-year-old female was found dead on her bed, with three empty used syringes and a bottle of Tanax beside her body. Three needle puncture marks were observed on the body. The aim of this study was to evaluate the distribution of embutramide and mebezonium iodide in different biological matrices (femoral and cardiac blood, liver, muscle and vitreous humor) using a chromatographic method for the simultaneous determination of the two drugs. A direct and sensitive liquid chromatography-tandem mass spectrometry method was developed in multiple reaction monitoring mode with positive ionization. Lidocaine was used as an internal standard. Limits of detection and quantitation of 0.01 and 0.05 mg/L, respectively, were reached for both compounds. Embutramide levels ranged from 2.74 mg/L in vitreous humor to 5.06 mg/L in femoral blood, while mebezonium iodide was found at widely differing concentrations (ranging from 2.80 mg/kg in muscle to 24.80 mg/kg in liver). The chromatographic method developed for this study provides a very simple and sensitive means for the simultaneous determination of embutramide and mebezonium iodide, the emetic concentrations of which were consistent with suicides reported in the literature.

The standardization of results on hair testing for drugs of abuse: An interlaboratory exercise in Lombardy Region, Italy.

Hair testing for drugs of abuse is performed in Lombardy by eleven analytical laboratories accredited for forensic purposes, the most frequent purposes being driving license regranting and workplace drug testing. Individuals undergoing hair testing for these purposes can choose the laboratory in which the analyses have to be carried out. The aim of our study was to perform an interlaboratory exercise in order to verify the level of standardization of hair testing for drugs of abuse in these accredited laboratories; nine out of the eleven laboratories participated in this exercise. Sixteen hair strands coming from different subjects were longitudinally divided in 3-4 aliquots and distributed to participating laboratories, which were requested to apply their routine methods. All the participants analyzed opiates (morphine and 6-acetylmorphine) and cocainics (cocaine and benzoylecgonine) while only six analyzed methadone and amphetamines (amphetamine, methamphetamine, MDMA, MDA and MDEA) and five Δ(9)-tetrahydrocannabinol (THC). The majority of the participants (seven labs) performed acidic hydrolysis to extract the drugs from the hair and analysis by GC-MS, while two labs used LC-MS/MS. Eight laboratories performed initial screening tests by Enzyme Multiplied Immunoassay Technique (EMIT), Enzyme-linked Immunosorbent Assay (ELISA) or Cloned Enzyme Donor Immunoassay (CEDIA). Results demonstrated a good qualitative performance for all the participants, since no false positive results were reported by any of them. Quantitative data were quite scattered, but less in samples with low concentrations of analytes than in those with higher concentrations. Results from this first regional interlaboratory exercise show that, on the one hand, individuals undergoing hair testing would have obtained the same qualitative results in any of the nine laboratories. On the other hand, the scatter in quantitative results could cause some inequalities if any interpretation of the data is required.

Ethyl glucuronide and ethyl sulfate in meconium and hair-potential biomarkers of intrauterine exposure to ethanol.

This study investigated ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentration in meconium and in maternal and neonatal hair (HEtG and HFAEEs, respectively) as potential markers of intrauterine exposure to ethanol together with meconium fatty acid ethyl esters (FAEEs) in a cohort of 99 mother-infant dyads, 49 coming from the Arcispedale of Reggio Emilia (Italy) and 50 from the Hospital del Mar of Barcelona (Spain). FAEEs, EtG and EtS were measured in meconium samples using liquid chromatography-tandem mass spectrometry. A head space-solid phase microextraction-gas chromatography-mass spectrometry was used to test HEtG and HFAEEs in hair samples from mothers and their newborns. Eighty-two meconium samples (82.8%) tested positive for EtG, 19 (19.2%) for EtS while 22 (22.2%) showed FAEEs levels higher than 2 nmol/g, the cut-off used to differentiate daily maternal ethanol consumption during pregnancy from occasional or no use. Although EtG and EtS in meconium did not correlate with total FAEEs concentration, a good correlation between EtG, EtS and ethyl stearate was observed. Moreover, EtG correlated well with ethyl palmitoleate, while EtS with ethyl laurate, myristate and linolenate. Neither maternal nor neonatal hair appears as good predictors of gestational ethanol consumption and subsequent fetal exposure in these mother-infant dyads. In conclusion, these data show that meconium is so far the best matrix in evaluating intrauterine exposure to ethanol, with EtG and EtS being potentially good alternative biomarkers to FAEEs.

Segmental hair analysis in order to evaluate driving performance.

On the 31st of July 2002 the Lombardy local government issued a memorandum, C.R. 35/SAN, providing "guidelines to investigate drugs of abuse addiction in order to judge driving performance". About hair samples, this memorandum advises that the proximal lock of 6 cm-length would be analysed for opiates, cocaine, cannabinoids, amphetamine and derivatives, divided into two segments of 3 cm each. The Local Medical Driving Licence Commissions (CML) can decide whether or not to enforce these instructions; from our survey it resulted that most CMLs do not abide by the memorandum, not requiring segmental analysis. The purpose of our study was to verify whether this procedural discordance could affect analytical results and, consequently, the evaluation of the subject's driving performance. We analysed hair samples taken from subjects who were requesting the renewal of their driving licence in our Laboratory during the period from 1 August 2002 to 31 December 2006. We divided samples into two groups: (1) samples previously analysed in one single segment which resulted positive for at least one analyte, but under the cut-off (0.5 ng/mg), were re-analysed in accordance with the guidelines; (2) samples previously processed following guidelines which resulted positive in one of the segments were newly analysed in a single segment. Comparing the new results with the original ones, an increase of positive results emerged in the first group. The second set of results fully supported the first ones. These results underscore the importance of the 35/SAN memorandum, so if the guidelines had been followed there would have been a larger amount of driving licence renewal denied.

Fatal sotalol poisoning.

Sotalol [4-(2-isopropylamino-1-hydroxyethyl)-methane-sulfonanilide hydrochloride] is a beta-adrenergic blocking agent. Despite the widespread use of these drugs, poisonings are not frequent. In this report the authors describe the first recorded case of fatal sotalol overdosage to their knowledge. The results of toxicological analysis, performed by a specially developed method, are presented, and compared with findings in fatal intoxications with other beta-adrenergic blocking agents.

Improved methods for genotype determination of human alcohol dehydrogenase (ADH) at ADH 2 and ADH 3 loci by using polymerase chain reaction-directed mutagenesis.

The human gene for producing alcohol dehydrogenase (ADH; EC 1.1.1.1) is polymorphic at ADH 2 and ADH 3 loci. Until now, the study of this polymorphism required liver biopsy or allele-specific radioactive probes. We have used directed mutagenesis by the polymerase chain reaction (PCR) to amplify and analyze the genotype of ADH 2 and ADH 3 loci. Thus, we could determine easily and unambiguously the complete genotype at these two loci by using a microsample of blood and restriction fragment length polymorphism after DNA amplification by PCR.

Determination of clenbuterol in urine as its cyclic boronate derivative by gas chromatography-mass spectrometry.

A rapid and reliable gas chromatographic-mass spectrometric method for the determination of clenbuterol in urine is described. Penbutolol was used as internal standard. Four derivatization procedures have been tested, of which 1-butaneboronic acid gave the best results. The method includes extraction of the alkalinized urine (3 ml) with tert.-butyl methyl ether-n-butanol (9:1), derivatization with 1-butaneboronic acid (15 min at room temperature), and analysis in the selected-ion monitoring mode of the derivatives of clenbuterol at m/z 243, 327 and 342 and of penbutolol at m/z 342 and 357. The detection limit is 0.5 ng/ml and the recovery better than 90%.