Relevance of perceived childhood neglect, 5-HTT gene variants and hypothalamus-pituitary-adrenal axis dysregulation to substance abuse susceptibility.

The hypotheses of (1) gene x environment interaction in the susceptibility to experiment with drugs and (2) hypothalamus-pituitary-adrenal (HPA) axis involvement in mediating the effects of early adverse experiences and gene variants affecting serotonin function on substance abuse vulnerability were tested by investigating in 187 healthy adolescents the possible relevance of 5-HTT "S" polymorphism, childhood parental neglect reported retrospectively and HPA axis function to the susceptibility to experiment with illicit drugs. Higher frequency of the 5-HTT SS genotype seems to be associated with an increased susceptibility to use illegal psychotropic drugs among the adolescents. At the same time, reduced maternal care perception was found to represent a key intermediate factor of the association between SS polymorphism and drug use, suggesting that genetic factors and parental behavior concur to drug use susceptibility. Our results also confirm the relationship between basal plasma levels of cortisol and adrenocorticotropic hormone (ACTH) on the one hand, and retrospective measures of neglect during childhood: the higher the mother and father neglect CECA-Q scores, the higher the plasma levels of the two HPA hormones. Such positive relationship has been proved to be particularly effective and important when associated to the S-allele, both in homozygote and heterozygote individuals. However, when tested together with genotype and parental neglect, the effect of HPA hormones such as cortisol and ACTH was not found to improve significantly the explanatory power of the risk model.

A forensic image processing environment for investigation of surveillance video.

We present an image processing software suite, based on the Matlab environment, specifically designed to be used as a forensic tool by law enforcement laboratories in the analysis of crime scene videos and images. Our aim is to overcome some drawbacks which normally appear when using standard image processing tools for this application, i.e. mainly the lack of full control and documentation on the operations which have been performed on the images, and the absence of new, more sophisticated algorithms which can provide improved performances and "make the difference" in critical cases.

Perceived parenting behavior in the childhood of cocaine users: relationship with genotype and personality traits.

Low parental care during childhood, a pattern characteristic of an "affectionless control" rearing style was frequently reported in the history of addicted individuals. Parents' childrearing regimes and children's genetic predispositions, with their own behavioral characteristics, have been seen to be closely interwoven, probably affecting children's development and addictive behavior susceptibility. In the present study, parents care perception, aggressive personality traits, and genotype (serotonin transporter promoter gene--5-HTTLPR) have been investigated in cocaine users and healthy control subjects. PBI scores (maternal and paternal care) were lower and BDHI scores (aggressiveness) higher in cocaine users in comparison with controls and significant differences in the perception of either paternal or maternal care were observed between cocaine users and non-users. The short-short (SS) genotype frequency was significantly higher among cocaine users compared with control subjects (P = 0.04). Logistic regression proves that persons bearing the SS genotype have a risk of becoming cocaine user almost three times higher than those having the LL genotype. Estimations of the effects of other factors potentially affecting the risk of being cocaine addicted clearly prove the significant impact of aggressiveness: the highest the score, the highest the risk of becoming cocaine user. Moreover, paternal and maternal care perception significantly improve the fit of the model (the log likelihood decreases passing from -105.9 to -89.8, LR test = 32.17, P-value = 0.0000). Each unit increase in the PBI score yields a significant 12% and 10% decrease of the risk of becoming cocaine user, respectively for paternal and maternal care. Interestingly, once controlled for the PBI score, the relative risk associated to the SS genotype drops strikingly and becomes no longer statistically significant. On the whole, our preliminary data suggest that the association between 5-HT transporter polymorphism and psycho-stimulant use may be mediated by mother-child relationship and parental attachment perception, both being environmental and genetic factors involved in the proneness to substance use disorders, particularly in aggressive-antisocial individuals.

Association of the serotonin transporter promoter polymorphism with smoking behavior among adolescents.

Serotonin transporter promoter polymorphism (5-HTTLPR) genotype was previously found associated with smoking behavior, difficulty in quitting smoking, and nicotine addiction; with non-replicated findings and contrasting results. Aim of the present study was to evaluate the possible association between 5-HTTLPR genotype and smoking behavior among adolescents, in relationship with psychological characteristics. Two hundred and ten Caucasian high school students (aged 14-19 years); 103 non-smokers, who have never smoked nicotine; and 107 tobacco smokers have been genotyped. Aggressiveness levels and temperamental traits were measured in both smokers and non-smokers, respectively, utilizing Buss-Durkee Hostility Inventory (BDHI) and Cloninger Three-Dimensional Personality Questionnaire (TPQ). Data about school performance have been also collected. The short-short (SS) genotype frequency was significantly higher among smokers compared with non-smokers (P = 0.023). The odds ratio for the SS genotype versus the long-long (LL) genotype frequency was 1.17 [95% CL (0.30-2.05)], when smokers were compared with non-smokers. The SS genotype frequency was significantly higher among heavy smokers with early onset, compared with moderate smokers with late onset (P = 0.042). BDHI irritability scores, NS scores at TPQ, and school failure frequency were significantly higher in smokers than in non-smokers. Multivariate model-fitting analysis evidenced a significantly greater relationship of genotype with irritability levels (BDHI scores) (0.34, P < 0.001) and temperament traits (NS scores) (0.36, P < 0.001), than with school performance (rate of school under-achievements) (0.18, P < 0.05) and nicotine smoking (number of cigarettes) (0.24, P < 0.01). Accordingly, factor-analysis showed that gene polymorphism contributes more directly to BDHI scores and NS scores (0.73; 0.71) than to smoking behavior and school under-achievement (0.54; 0.51). Our data suggest that a decreased expression of the gene encoding the 5-HTT transporter, due to "S" promoter polymorphism, may be associated with smoking behavior among adolescents and increased risk to develop nicotine dependence, possibly in relationship to personality traits, temperamental characteristics, and school under-achievements.

Serotonin transporter promoter polymorphism genotype is associated with temperament, personality traits and illegal drugs use among adolescents.

Serotonin transporter promoter polymorphism (5-HTTLPR) genotype was previously found associated with substance use disorders, particularly in the subjects with comorbid antisocial behavior, and with temperament and personality traits at risk for substance abuse. Aim of the present study was to evaluate the possible association between 5-HTTLPR genotype and the availability to experiment illegal drugs among adolescents, in relationship with psychological characteristics. 216 caucasian high school students (aged 14-19 ys), 125 abstinent subjects, who have never experimented psychotropic drugs, and 91 experimenters of illegal drugs have been genotyped. Aggressiveness levels and temperamental traits were measured in both abstinent subjects and experimenters utilizing respectively Buss-Durkee-Hostility-Inventory (BDHI) and Cloninger Three-dimensional Personality Questionnaire (TPQ). Data about school performance have been also collected. The short-short (SS) genotype frequency was significantly higher among experimenters compared with abstinent subjects (p = 0.001). The odds ratio for the SS genotype vs the long-long (LL) genotype frequency was 4.67, 95% Cl (1.97-11.04), when experimenters were compared with abstinent students. The SS genotype frequency was significantly higher among aggressive/novelty seeker (NS) experimenters with poor school achievements, compared with drugs experimenters without aggressiveness and school failure (p = 0.02). When evaluated on the entire sample, BDHI mean total scores, NS scores at TPQ and school failure frequency were significantly higher in SS individuals, in comparison with LL subjects. Our data suggest that a decreased expression of the gene encoding the 5-HTT transporter, due to "S" promoter polymorphism, may be associated with an increased availability to experiment illegal drugs among adolescents, particularly in the subjects with more consistent aggressiveness, NS temperament and learning disabilities.

Analysis of monoamine oxidase A (MAO-A) promoter polymorphism in male heroin-dependent subjects: behavioural and personality correlates.

The promoter of the monoamine oxidase A (MAO-A) gene was analysed to test whether length variation of the repeat polymorphism contributes to variation in individual vulnerability to aggressive-criminal behaviour, and liability to heroin dependence. The repeat number of the MAO-A polymorphism was assessed in 199 male subjects of Italian descent, a sample comprising 95 healthy subjects and 104 heroin-dependent subjects including 52 addicted individuals with violent behaviour and antisocial personality disorder. The frequency of the low-activity 3-repeat allele was significantly higher in violent offenders among heroin addicts, compared to addicted individuals without antisocial behaviour (34.6 vs. 15.4%; p<0.03) and controls (18.9%; p<0.05). No significant difference was evidenced in the frequencies of the MAO-A alleles between heroin-dependent subjects in general and control subjects. High activity 4-repeat allele frequency was significantly higher in addicted individuals without antisocial behavior compared to antisocial-aggressive heroin-dependent subjects (76.9 vs. 55.8%; p<0.02). Buss Durkee Hostility Inventory (BDHI) mean total scores were significantly higher in heroin addicts than in controls (p<0.001), and in antisocial-violent heroin addicts in comparison with addicted individuals without antisocial behaviour (p<0.005). Among heroin addicts BDHI irritability, suspiciousness and resentment subscales scores were found significantly higher in low activity 3-repeat allele subjects than in high activity alleles subjects (p<0.001; p<0.05; p<0.05, respectively). No association was found between MAO-A polymorphism and suicide history. Our findings suggest that the low-activity 3-repeat allele of the MAO-A promoter polymorphism confers increased susceptibility to antisocial-violent behavior and aggressiveness, rather than drug dependence per se, in heroin-dependent males.

Association between low-activity serotonin transporter genotype and heroin dependence: behavioral and personality correlates.

In previous studies, serotonin (5-HT) system disturbance was found involved in a variety of behavioral disorders, psychopathologies, and substance use disorders. A functional polymorphism in the promoter region of the human serotonin transporter gene (5-HTTLPR) was recently identified and the presence of the short (S) allele found to be associated with a lower level of expression of the gene, lower levels of 5-HT uptake, type 2 alcoholism, violence and suicidal behavior. In the present study, 101 heroin addicts (males, West European, Caucasians) and 101 healthy control subjects matched for race and gender, with no history of substance use disorder, have been genotyped. Aggressiveness levels were measured in both heroin addicts and controls utilizing Buss-Durkee-Hostility-Inventory (BDHI). Data about suicide attempt and violent criminal behavior in subject history have been collected. The short-short (SS) genotype frequency was significantly higher among heroin dependent individuals compared with control subjects (P = 0.025). The odds ratio for the SS genotype versus the long-long (LL) genotype frequency was 0.69, 95% Cl (0.49-0.97), when heroin addicts were compared with healthy controls. The SS genotype frequency was significantly higher among violent heroin dependent individuals compared with addicted individuals without aggressive behavior (P = 0.02). BDHI mean total scores and suspiciousness and negativism subscales scores were significantly higher in SS individuals, in comparison with LL subjects, among heroin addicts. No association was found between SS genotype and suicide history. Our data suggest that a decreased expression of the gene encoding the 5-HTT transporter, due to "S" promoter polymorphism, may be associated with an increased risk for substance use disorders, particularly in the subjects with more consistent aggressiveness and impulsiveness.

Genetic analysis of a Sicilian population using 15 short tandem repeats.

The genetic structure of the population of Alia (Sicily, Italy) was analyzed using 15 short tandem repeats: TPOX, D2S1338, D3S1358, FIBRA, D5S818, CSF1PO, D7S820, D8S1179, TH01, VWA, D13S317, D16S539, D18S51, D19S433, and D21S11. Two of these markers, D2S1338 and D19S433, have never before been used in research on population genetics and only recently have they been put to use in forensic medicine. Results of the analysis underline the genetic isolation of the Alia population and show it to be a recent bottleneck as a consequence of a cholera epidemic in 1837. While comparing the Alia population with other populations from Sicily, a genetic heterogeneity within Sicily was uncovered, thus confirming previous results obtained from the analysis of classical markers. This heterogeneity underlines the existence of genetic boundaries within the island. Comparisons with other Italian, Mediterranean, and European populations highlight the differentiation of the Sicilian population, reflecting the presence of a genetic boundary that separates Sicily from northern and central Italy and from the western Mediterranean basin.

Development of latent fingerprints on metallic surfaces using electropolymerization processes.

We propose a new process for developing latent fingerprints on metal items, applicable to unfired weapons made of Ergal in particular. The method is based on the presence of fatty acids that are contained in fingerprints and act as an insulator on the surface where fingerprints are to be developed. The process of polymerization occurs on the metal portions left untouched by finger contact. Hence, the developing process results as a negative pattern of the original fingerprint. The reaction consists in the electropolymerization of pyrrole and substituted porphyrins, i.e., tetra (o-aminophenyl) porphyrine: radical-cations are generated on superficial nucleation sites by oxidation of monomer, close to the electrode surface; subsequently, the radical species react with the neutral monomer, which begins to diffuse to the electrode. We have also studied the polymer's morphology by means of SEM and AFM, in order to find a correlation between the reagent to be used and the quality of the enhancement process. These are only preliminary results; however, they show that the suggested method is a new way to increase the rate of success in developing latent fingerprints on metal surfaces. In this regard, it may be considered complementary to other conventional procedures, due to the low costs of the instruments and reagents, and the rapidity and simplicity of the treatment.

Italian population data on two new short tandem repeat loci: D2S1338 and Penta E.

A population study on two new short tandem repeat (STR) loci D2S1338 (a tetranucleotide repeat) and Penta E (a pentanucleotide repeat) was performed on 208 unrelated Italian Caucasians. The DNA was amplified by polymerase chain reaction (PCR) and separation and detection of the amplified STR fragments were carried out by use of a PE/ABD PRISM 377 DNA Sequencer 377 automated system (Applied Biosystems Division/Perkin-Elmer). Both loci meet Hardy-Weinberg expectations. There is no evidence for departures from expectations between the two loci. The combined Probability of Discrimination and Probability of Exclusion for the two STR loci are 0.999155 and 0.944925, respectively. The results demonstrate that these two regions can be useful for differentiating among individuals, particularly in concert with other STR loci.

Italian population data on two new short tandem repeat loci: D6S477 and D19S433.

A population study on two new short tandem repeat (STR) loci D6S477 and D19S433 was performed on 214 unrelated Italian Caucasians. The DNA was amplified by PCR and separation and detection of the amplified STR fragments were carried out by use of a PE/ABD PRISM 377 DNA sequencer 377 automated system (Applied Biosystems Division/Perkin Elmer). Both loci meet Hardy-Weinberg expectations. There is no evidence for departures from expectations between the two loci. The combined probability of discrimination and probability of exclusion for the two STR loci are 0.997161 and 0.883183, respectively. The results demonstrate that these loci can be useful for human identification in forensic cases in Italy.

Italian population data on thirteen short tandem repeat loci: HUMTH01, D21S11, D18S51, HUMVWFA31, HUMFIBRA, D8S1179, HUMTPOX, HUMCSF1PO, D16S539, D7S820, D13S317, D5S818, D3S1358.

A population study on thirteen short tandem repeat (STR) loci was performed on 223 unrelated Italian Caucasians. The DNA was amplified by PCR. Separation and detection of the amplified STR fragments was carried out by use of 377 automated system (Applied Biosystems Division/Perkin Elmer). All loci meet Hardy-Weinberg expectations, and the data show only five departures out of seventy-eight pairwise locus tests which is close to expectations of 5% (5/78 = 6.4%). When correcting for multiple tests, there is little evidence for departures from expectations between loci. The combined Power of Exclusion for the thirteen STR loci is 0.99999270. The results demonstrate that these loci will be very useful for human identification in forensic cases in Italy.

Ion trap mass spectrometry for the characterization of N-methyl-1- (3,4-methylene-dioxyphenyl)-2-butanamine and N-ethyl-3,4- methylenedioxyamphetamine, two widely distributed street drugs.

The potential of ion trap mass spectrometry has been evaluated for the characterization and distinction of two isomeric amphetamines drugs, namely N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine and N-ethyl-3,4-methylenedioxyamphetamine. Whereas the electron impact spectra of the two molecules lack specificity, collisional experiments on the ionic species at m/z 72 allows unequivocal distinction between the two isomers. Analogous results are achieved by positive ion chemical ionization and collisional experiments on the protonated molecules. All the different approaches have been successfully applied to the gas chromatography/mass spectrometry analysis of a tablet of illicit drug.

Italian population data on the polymarker system and on the five short tandem repeat loci CSF1PO, TPOX, TH01, F13B, and vWA.

A population study on five short tandem repeat (STR) loci and five sequence specific polymorphism loci was performed on unrelated Italian Caucasians. Separation and detection of the amplified STR fragments were carried out by high resolution vertical denaturing polyacrylamide gel electrophoresis (PAGE) and silver staining, respectively. The sequence specific loci were analyzed using the AmpliType PM Typing Kit (Perkin Elmer, Foster City, CA). All loci, except Gc (p = 0.031), meet Hardy-Wienberg expectations. In addition, there is no evidence for association of alleles between pairs of loci. The combined power of discrimination for the five STR loci is 0.9999862 and for the PM loci is 0.99503. The results suggest that these loci may be useful for human identification cases in Italy.

Streptomyces peucetius daunorubicin biosynthesis gene, dnrF: sequence and heterologous expression.

The dnrF gene, responsible for conversion of aklavinone to epsilon-rhodomycinone via C-11 hydroxylation, was mapped in the daunorubicin (Dnr) gene cluster of Streptomyces peucetius ATCC 29050, close to drrAB, one of the anthracycline-resistance genes. The dnrF gene was sequenced and should encode a protein of 489 amino acids with a molecular mass of 52 kDa. The deduced DnrF protein shows significant similarities with bacterial FAD- and NADPH-dependent hydroxylases either required to introduce hydroxyl groups into polycyclic aromatic polyketide antibiotics or involved in catabolism of aromatic compounds. Heterologous expression of dnrF in Streptomyces lividans TK23 and in Escherichia coli demonstrated that the gene encodes a NADPH-dependent hydroxylase catalysing the hydroxylation of aklavinone to yield epsilon-rhodomycinone. The enzyme is inactive on anthracyclines glycosylated at position C-7 and its activity decreases to a different extent with other substrate modifications, indicating that DnrF has a significant substrate specificity.

Characterization of a saporin mitotoxin specifically cytotoxic to cells bearing the granulocyte-macrophage colony-stimulating factor receptor.

When granulocyte-macrophage colony-stimulating factor (GM-CSF) is chemically conjugated to the ribosome-inactivating protein saporin, the resulting protein conjugate is highly toxic for cells expressing the GM-CSF receptor. Structural and Western blot analyses of the purified conjugate establish that it contains equimolar amounts of the starting materials and is free of any contamination by the non-conjugated components. The resulting bifunctional reagent is specifically cytotoxic to cells expressing the GM-CSF receptor, but is ineffective to cells that do not express the receptor. The cytotoxic activity is inhibited in a dose-dependent manner by GM-CSF, but not by any one of five other peptide growth factors. This is the first report of a mitotoxin for cells that express the GM-CSF receptor and which promises to be a valuable tool to study the expression of the GM-CSF receptor in normal and pathological states.

Experience with the preclinical assessment of basic fibroblast growth factor (bFGF).

Repeated intravenous administrations were carried out in cynomolgus monkeys and rats (S.D.) for a maximum of 4 weeks at doses of 1, 10 and 100 micrograms/kg/day in stable formulation. Three main target organs were identified: red blood cells (RBC), kidney glomeruli (KG) and bone at the top dose level. RBC: Normochromic normocytic anaemia started in rats and monkeys during the second week of treatment (decrease in red blood cell production). The kinetics of this anaemia, as well as its recovery, will be discussed. Bone: Dramatic hyperostosis in rats was present by day 10 in long or spongious bone. This became marked on day 29 and regressed after treatment was stopped. KG: In the rat glomerular lesions were present starting from day 16. They consisted of enlargement and vacuolation of podocytes with loss of foot processes and adhesions between glomerular tuft and Bowman's capsule. Proteinuria was a striking feature. In the monkey the lesions were hyperplasia of the parietal epithelium of Bowman's capsule which involved replacement of normally flattened epithelium by cuboidal cells, with some pseudostratification. Proteinuria also occurred in monkeys, accompanied by a lowering of serum protein (albumin). In two animals, death (by day 15) was preceded by high levels of urea and blood creatinine. The above lesions (KG) disappeared almost completely over a recovery period. It is suggested that these phenomena are not the expression of direct toxicity in the form of lethal insults, but rather a manifestation of a change in cell activity.

Expression of doxorubicin-daunorubicin resistance genes in different anthracycline-producing mutants of Streptomyces peucetius.

Two DNA fragments, ric1 and ric2, were isolated from the Streptomyces peucetius 7600 mutant, which produces daunorubicin and doxorubicin, on the basis of their abilities to confer doxorubicin and daunorubicin resistance to Streptomyces lividans. These two fragments are unrelated by restriction mapping and do not show any homology by Southern analysis, yet both of them increase the level of resistance 10-fold in transformed S. lividans. Functional analysis revealed that ric1 also contains two genes of daunorubicin biosynthesis: one coding for the aklavinone C-11 hydroxylase and the other corresponding to the putative dnrR2 regulatory gene of wild-type S. peucetius ATCC 29050 (K. J. Stutzman-Engwall, S. L. Otten, and C. R. Hutchinson, J. Bacteriol. 174:144-154, 1992). Northern (RNA) blot experiments, performed with a ric1 fragment containing daunorubicin-doxorubicin resistance gene(s), revealed a transcript of about 2,100 nucleotides that is present only during the phase of anthracycline metabolite production. The amount of this transcript is higher in strain 7600 than in strain 7900, a mutant which produces 5-fold more daunorubicin and 10-fold less doxorubicin than 7600. Furthermore, two 7900-derived blocked mutants, 8600 and 9700, do not express the 2,100-nucleotide transcript in spite of the absence of gross rearrangements in the ric1 region such as occur with the 7900 parental strain.

Ribosome-inactivating proteins from plant cells in culture.

1. Ribosome-inactivating proteins were found in high amounts in one line of cells of Phytolacca americana (pokeweed) cultured in vitro and, in less quantity, in lines of Saponaria officinalis (soapwort) and of Zea mays (corn) cells. 2. The main ribosome-inactivating protein from pokeweed cells was purified to homogeneity. It is a protein with Mr 29,000 and basic pI, similar to the 'pokeweed antiviral protein' (PAP), a ribosome-inactivating protein from pokeweed leaves. We propose to call the pokeweed antiviral protein isolated from pokeweed cells PAP-C. 3. PAP-C inactivates ribosomes in a less-than-equimolar ratio, thus inhibiting protein synthesis by a rabbit reticulocyte lysate with an IC50 (concentration causing 50% inhibition) of 0.067 nM (2 ng/ml), and modifies rRNA in a manner apparently identical to that of ricin and other ribosome-inactivating proteins. It inhibits protein synthesis by intact cells with an IC50 of 0.7-3.4 microM, and is toxic to mice with an LD50 of 0.95 mg/kg.