Nerve growth factor ß polypeptide (NGFB) genetic variability: association with the methadone dose required for effective maintenance treatment.

Opioid addiction is a chronic disease with high genetic contribution and a large inter-individual variability in therapeutic response. The goal of this study was to identify pharmacodynamic factors that modulate methadone dose requirement. The neurotrophin family is involved in neural plasticity, learning, memory and behavior and deregulated neural plasticity may underlie the pathophysiology of drug addiction. Brain-derived neurotrophic factor (BDNF) was shown to affect the response to methadone maintenance treatment. This study explores the effects of polymorphisms in the nerve growth factor (ß polypeptide) gene, NGFB, on the methadone doses required for successful maintenance treatment for heroin addiction. Genotypes of 14 NGFB polymorphisms were analyzed for association with the stabilizing methadone dose in 72 former severe heroin addicts with no major co-medications. There was significant difference in methadone doses required by subjects with different genotypes of the NGFB intronic single-nucleotide polymorphism rs2239622 (P=0.0002). These results may have clinical importance.

Association of polymorphisms of the cannabinoid receptor (CNR1) and fatty acid amide hydrolase (FAAH) genes with heroin addiction: impact of long repeats of CNR1.

Alterations in expression of a cannabinoid receptor (CNR1, CB1), and of fatty acid amide hydrolase (FAAH) that degrades endogenous ligands of CB1, may contribute to the development of addiction. The 385C>A in the FAAH gene and six polymorphisms of CNR1 were genotyped in former heroin addicts and control subjects (247 Caucasians, 161 Hispanics, 179 African Americans and 19 Asians). In Caucasians, long repeats (>or=14) of 18087-18131(TAA)(8-17) were associated with heroin addiction (P=0.0102). Across three ethnicities combined, a highly significant association of long repeats with heroin addiction was found (z=3.322, P=0.0009). Point-wise significant associations of allele 1359A (P=0.006) and genotype 1359AA (P=0.034) with protection from heroin addiction were found in Caucasians. Also in Caucasians, the genotype pattern, 1359G>A and -6274A>T, was significantly associated with heroin addiction experiment wise (P=0.0244). No association of FAAH 385C>A with heroin addiction was found in any group studied.

Physical mapping of rDNA and heterochromatin in chromosomes of 16 Coffea species: a revised view of species differentiation.

The chromosome organization among 15 wild diploid Coffea species and cultivated tetraploid C. arabica was determined by fluorochrome banding (CMA, DAPI) and double fluorescence in-situ hybridization (FISH) of 5S and 18S rDNA achieved on the same chromosome plates. Two to five chromosome pairs (plus one putative chromosome B) are marked. Overall, there are two SAT-chromosome pairs for East African species and one for the Malagasy and the West and Central African species. 18S rDNA loci are telomeric and strongly marked the SAT-chromosome pairs. Generally, only one pericentromeric 5S rDNA locus characterized East African species, while an additional minor locus co-localized with the 18S rDNA-SAT locus for the Malagasy species and West and Central African species. A combination of rDNA FISH plus CMA and DAPI banding patterns enables identification of almost all the species, even those for which the genetic or botanical status is still being discussed. C. arabica clearly appears to be an allotetraploid species, including one genome from East Africa and one from West and Central Africa. However, since the minor 5S rDNA-SAT locus present in West/Central African genomes is not detected, two evolutionary hypotheses could be put forward for C. arabica. Considering only the diploid species, global trends are obvious in rDNA signal patterns, genome size variations, and geographic distribution of the species, but there are no clear evolutionary trends. However, complex interactions between these factors and environmental growing conditions exist, which have resulted in loss and gain of rDNA loci and probably also in copy repeat number variations in each rDNA family.

Polymorphisms in C2, CFB and C3 are associated with progression to advanced age related macular degeneration associated with visual loss.

BACKGROUND: Age related macular degeneration (AMD) is a leading cause of blindness. AMD is a complex disorder caused by genetic and environmental factors in which single nucleotide polymorphisms (SNPs) in the genes CFH and LOC387715/HTRA1/ARMS2 have prognostic importance for progression to advanced AMD (with visual loss). CFH may also have a pharmacogenetic role by affecting treatment response to widely used nutritional supplements. This paper examines other AMD susceptibility genes to determine if these genotypes influenced disease progression and treatment response. METHODS: Three cohorts, totalling 3137 individuals, were genotyped for SNPs in 13 genes previously published to be associated with advanced AMD (other than CFH and LOC387715/ARMS2/HTRA1). Those genes found associated were then evaluated for their involvement in disease progression. Interactions between the genes and with AREDS (Age-Related Eye Disease Study) nutritional supplements were investigated. RESULTS: Positive independent associations were noted in SNPs in the genes C2 (p = 0.0001, odds ratio (OR) 0.35, 95% confidence interval (CI) 0.2 to 0.6), CFB (p = 0.0001, OR 0.35, 95% CI 0.2 to 0.6), C3 (p = 0.0001, OR 3.91, 95% CI 1.94 to 7.88), APOE (epsilon4, p = 0.01, OR 0.50, 95% CI 0.29 to 0.86) and VEGFA (p = 0.01, OR 2.23, 95% CI 1.06 to 4.68). C2/CFB and C3 were independently related to progression from early/intermediate to advanced AMD with OR 0.32 (95% CI 0.14 to 0.73) and 3.32 (95% CI 1.46 to 7.59), respectively. Gene-gene and pharmacogenetic interactions were not observed. No preferential associations were observed with geographic atrophy or choroidal neovascularisation. CONCLUSION: This study provides insights into the genetic pathogenesis of AMD. Five genes have now been shown to be independently involved in progression from intermediate disease (before vision loss has occurred) to advanced disease in which blindness is frequent.

MHC fine mapping of human type 1 diabetes using the T1DGC data.

AIM: The human Major Histocompatibility Complex (MHC) is a highly polymorphic genomic region occupying approximately 4 Mb on chromosome 6p21.3. The relationship between human MHC and type 1 diabetes (T1D) has been previously investigated. To fine map the disease locus in this region, we carried out both linkage and association analyses using the Type 1 Diabetes Genetics Consortium data. METHODS: Two-point linkage analysis was performed with a set of microsatellite markers assuming a fully recessive inheritance model, where we found clustering of high LOD (logarithm of the odds) scores across the MHC region. To narrow down the linkage region, we performed association analyses using both microsatellite and two sets of single nucleotide polymorphism (SNP) markers. We focused on the nuclear families containing a discordant sib-pair (an affected and unaffected sib). For the microsatellite markers, we computed the average repeat length for each individual and carried out a paired t-test. RESULTS: Microsatellite marker D6S2884 showed the highest association in a sharp peak with a p value of 3.15E-24. We confirmed this finding when using also SNP markers performing a McNemar's test for association. The SNPs that showed the most significant evidence of association mapped to almost the same location as the microsatellite markers. CONCLUSIONS: Besides the main goal of fine mapping of T1D genes, our results also illustrated the differences and the advantage of using both linkage and association analyses. After the identification of a wide peak with linkage analysis, we were able to dramatically narrow down the region by performing association analysis.

The contribution of individual and pairwise combinations of SNPs in the APOA1 and APOC3 genes to interindividual HDL-C variability.

Apolipoproteins (apo) A-I and C-III are components of high-density lipoprotein-cholesterol (HDL-C), a quantitative trait negatively correlated with risk of cardiovascular disease (CVD). We analyzed the contribution of individual and pairwise combinations of single nucleotide polymorphisms (SNPs) in the APOA1/APOC3 genes to HDL-C variability to evaluate (1) consistency of published single-SNP studies with our single-SNP analyses; (2) consistency of single-SNP and two-SNP phenotype-genotype relationships across race-, gender-, and geographical location-dependent contexts; and (3) the contribution of single SNPs and pairs of SNPs to variability beyond that explained by plasma apo A-I concentration. We analyzed 45 SNPs in 3,831 young African-American (N=1,858) and European-American (N=1,973) females and males ascertained by the Coronary Artery Risk Development in Young Adults (CARDIA) study. We found three SNPs that significantly impact HDL-C variability in both the literature and the CARDIA sample. Single-SNP analyses identified only one of five significant HDL-C SNP genotype relationships in the CARDIA study that was consistent across all race-, gender-, and geographical location-dependent contexts. The other four were consistent across geographical locations for a particular race-gender context. The portion of total phenotypic variance explained by single-SNP genotypes and genotypes defined by pairs of SNPs was less than 3%, an amount that is miniscule compared to the contribution explained by variability in plasma apo A-I concentration. Our findings illustrate the impact of context-dependence on SNP selection for prediction of CVD risk factor variability.

Two skeletal alpha-tropomyosin transcripts with distinct 3'UTR have different temporal and spatial patterns of expression in the striated muscle lineages of Xenopus laevis.

The Xenopus laevis alpha-tropomyosin (TM) gene, like its vertebrates counterparts, encodes muscle and non-muscle isoforms through two promoters and alternatively spliced exons. In the present study we describe a cDNA clone (XTMalpha7) encoding a skeletal muscle isoform of the gene that differs from the previously described skeletal TM transcript (XTMalpha2) by its 3'UTR sequence. The two skeletal alpha-TM encoding mRNAs are generated through distinct 3'end processing using different polyA signals and distinct patterns of exon splicing. Using RNAse protection and RNA in situ hybridization, we have analysed the developmental and spatial expression of the two transcripts. Both are expressed in the embryo, but XTMalpha7 is by far the most prevalent of the two. In contrast, only XTMalpha2 is expressed in adult striated muscle tissues. In the embryo, the spatial expression of XTMalpha7 is restricted to the somites whereas XTMalpha2 is expressed in both somites and embryonic heart.

Serum lipid abnormalities in heart transplant recipients: predominance of HDL2-like particles in the HDL pattern.

Accelerated coronary atherosclerosis is a major risk limiting long-term survival after heart transplantation and is commonly associated with dyslipoproteinemia even in subjects who were not dyslipoproteinemic before intervention. The purpose of this study was to analyse the abnormalities in the lipid profiles of 2 different groups of heart-transplanted males: 18 subjects with underlying ischemic heart disease (IHD) and 19 subjects with non-obstructive cardiomyopathy of unknown aetiology (CM). Both groups were compared to 33 healthy males. All patients were under immunosuppressive therapy including prednisone, cyclosporin A and azathioprine. A moderate hyperlipidemia was found in all transplant recipients, associated with high HDL-cholesterol concentrations in the CM group (1.80 +/- 0.37 vs. 1.29 +/- 0.23 mmol/l) and normal HDL-cholesterol levels in the IHD group (1.40 +/- 0.23 mmol/l). HDL subfractionation showed a marked increase in HDL2-cholesterol (CM: 1.12 +/- 0.32; IHD: 0.69 +/- 0.28; control: 0.40 +/- 0.17 mmol/l) while HDL3-cholesterol was significantly lower than in the control group. Analysis of HDL particle sizes showed in all transplant subjects an increase of an intermediate size particle HDL2a (diameter 9.0 +/- 0.10 nm) which is a minor form in control subjects. In the CM group, both the common HDL2b (10.2 +/- 0.13 nm) and HDL2a were abundant in 13 of 17 patients. The pattern was more heterogeneous in the IHD group but witnessed to a high frequency of HDL2a particles either alone (5/14) or associated with larger HDL2b (4/14) or with small HDL3 (4/14).(ABSTRACT TRUNCATED AT 250 WORDS)

Inclusion bodies and crystals of Bacillus sphaericus mosquitocidal proteins expressed in various bacterial hosts.

Mosquitocidal proteins of 42 and 51 kDa from Bacillus sphaericus were produced in acrystallogenic B. sphaericus and B. subtilis strains. In both species, transformants containing each protein expressed individually were non-toxic for mosquito larvae and produced small inclusions which did not have a crystalline ultrastructure. When both components of the binary toxin were expressed together, toxicity was restored: oval and round amorphous inclusions were produced in B. subtilis, and typical native-type crystals were synthesized in B. sphaericus. In B. subtilis, native-type crystals were produced only when the two components of the binary toxin were synthesized as a 93-kDa fusion protein.

Introgression of self-compatibility from Coffea heterocalyx to the cultivated species Coffea canephora.

Self-compatibility segregation was assessed in two successive backcross progenies originating from an interspecific cross between Coffea canephora (self-incompatible) and Coffea heterocalyx (self-compatible). After self- and cross-pollination, pollen tube behaviour in styles was observed under ultraviolet fluorescence microscopy and fruit-set was determined at harvesting time. Segregation ratios in the two progenies were consistent with monofactorial control of self-compatibility. Self-compatible plants exhibited higher fruit-set than self-incompatible ones in open-pollination conditions. Segregation of AFLP markers was scored in the first backcross progeny. By molecular linkage analysis, the S locus could be mapped to a short linkage group.

Comparative flow cytometric estimation of nuclear DNA content in oil palm (Elaeis guineensis Jacq) tissue cultures and seed-derived plants.

Flow cytometric analysis performed on two different crosses of dura×pisifera oil palm gave an accurate estimation of nuclear DNA content. The genome size of Elaeis guineensis was found to be 2C=3.76±0.09 pg and therefore ca. 3.4×109 bp. Embryogenic calli and plants showed the same ploidy level, but the measured 2C DNA values differed significantly. No variation in the ploidy level between three different types of calli originating from foliar explants, namely nodular compact callus, fast-growing callus and friable callus was observed. Since fast-growing callus (FGC), already identified as a source of `mantled' phenotype variants, did not show any difference in their ploidy level, these results are consistent with the hypothesis of an epigenetic origin for this type of somaclonal variant.

Vertebrate safety of Clostridium bifermentans serovar malaysia, a new larvicidal agent for vector control.

The safety of bacterial cells of Clostridium bifermentans serovar malaysia, which is highly toxic to mosquito larvae, was tested on mice, guinea pigs, rabbits, and goldfish. Inoculations of at least 1 x 10(8) cells per animal by routes recommended by World Health Organization (subcutaneous, percutaneous, inhalation, force-feeding, intraperitoneal, intravenous) and tests of subacute toxicity, anaphylactic shock, persistence in heart blood, and virulence by successive passages, were performed on mice, guinea pigs, or both. Growth was monitored for 1 mo before necropsy. Ocular irritation and skin scarification were tested with rabbits. C. bifermentans serovar malaysia did not induce any mortality or abnormal reactions in mammals at a dose 1,000 times higher than the level established by W.H.O. for the demonstration of safety. Bacterial cells are not toxic to goldfish at a dose 1,000 times higher than the LD50 for the target-mosquito larvae. We conclude that C. bifermentans serovar malaysia bacterial cells are safe for laboratory mammals and goldfish.

Bacterial control of mosquito larvae: investigation of stability of Bacillus thuringiensis var. israelensis and Bacillus sphaericus standard powders.

Bacillus thuringiensis var. israelensis and Bacillus sphaericus products were assayed against their respective reference powders IPS82 and SPH88. Since their production in 1982 and 1988, the potency and larvicidal activity of these standard powders have been regularly checked on their test insects Aedes aegypti (for IPS82) or Culex pipiens (for SPH88). Over the 16-year evaluation period of IPS82 and 10-year evaluation period of SPH88, their potencies were considered stable. The global mean of each year's mean showed a coefficient of variation of less than 20%. Larval rearing was the most important factor in the reproducibility of the bioassay, although some variation also originated from the person performing the bioassay. This study demonstrated that the SPH88 standard could be kept in a stock suspension at 4 degrees C for 3 years without loss of potency. Moreover, after 9 years of storage in suspension, only a 2-fold decrease in the potency of SPH88 was detected.

Host range of Clostridium bifermentans serovar. malaysia, a mosquitocidal anaerobic bacterium.

Clostridium bifermentans serovar. malaysia (C.b.m.) is toxic to mosquito larvae. In this study, we quantified its toxicity to the mosquitoes, Aedes aegypti, Ae. albopictus, Ae. caspius, Ae. detritus, Anopheles stephensi, An. gambiae, Culex pipiens and Cx. quinquefasciatus. Anopheles larvae are the most susceptible, followed by Ae. detritus and Ae. caspius, then Culex and other Aedes larvae. According to mosquito species, the LC50 varies from 7 x 10(3) to 1.3 x 10(6) cells/ml. Three concentrations (10(7), 10(6) and 10(5) cells/ml) of C.b.m., Bacillus thuringiensis var. israelensis (B.t.i.) and Bacillus sphaericus were tested on Ae. aegypti, An. stephensi and Cx. pipiens larvae in order to determine the time necessary for each concentration to kill 50 and 90% of the population. Ninety percent of the 3 mosquito populations are killed within 4-15 h by the C.b.m. concentrations. Whatever the concentrations, C.b.m. kills at least 10 times less rapidly than B.t.i. but always quicker than B. sphaericus. Bioassays of C.b.m. bacterial cells or final whole culture were not toxic to Musca domestica and Drosophila melanogaster (Diptera) as well as to Phaedon cochleariae (Coleoptera) and Spodoptera littoralis (Lepidoptera).

Beneficial effect of post -thawing osmoconditioning on the recovery of cryopreserved coffee (Coffea arabica L.) seeds.

Osmoconditioning-controlled rehydration of seeds in a solution with low osmotic potential -has been shown to reinvigorate aged seeds. The present work aimed at investigating the effect of osmoconditioning on the germination of cryopreserved seeds of Coffea arabica, whose viability and vigour are drastically affected by cryopreservation. For cryopreservation, seeds were desiccated to 0.21 g H2O/g dw, cooled at 1 degree C/min to -50 degree C, then immersed rapidly in liquid nitrogen. After rapid rewarming, seeds were osmoconditioned for 1 to 6 weeks using solutions with osmotic potentials between -1 and -4 MPa. The time to produce half of the final percentage of normal seedlings, T50, was about three fold lower with osmoconditioned seeds than with non-osmoconditioned seeds (12-14 d vs 36 d). Moreover, after a 6-week osmoconditioning treatment with solutions with osmotic potential of -1 and -1.25 MPa, the percentage of seedlings recovered from cryopreserved seeds was 64-74%, against 13-16% only for cryopreserved seeds which were not osmoconditioned.

A variant in ANKK1 modulates acute subjective effects of cocaine: a preliminary study.

This study aimed to evaluate whether functional variants in the ankyrin repeat and kinase domain-containing 1 (ANKK1) gene and/or the dopamine receptor D2 (DRD2) gene modulate the subjective effects (reward or non-reward response to a stimulus) produced by cocaine administration. Cocaine-dependent participants (N = 47) were administered 40 mg of cocaine or placebo at time 0, and a subjective effects questionnaire (visual analog scale) was administered 15 min prior to cocaine administration, and at 5, 10, 15 and 20 min following administration. The influence of polymorphisms in the ANKK1 and DRD2 genes on subjective experience of cocaine in the laboratory was tested. Participants with a T allele of ANKK1 rs1800497 experienced greater subjective 'high' (P = 0.00006), 'any drug effect' (P = 0.0003) and 'like' (P = 0.0004) relative to the CC genotype group. Although the variant in the DRD2 gene was shown to be associated with subjective effects, linkage disequilibrium analysis revealed that this association was driven by the ANKK1 rs1800497 variant. A participant's ANKK1 genotype may identify individuals who are likely to experience greater positive subjective effects following cocaine exposure, including greater 'high' and 'like', and these individuals may have increased vulnerability to continue using cocaine or they may be at greater risk to relapse during periods of abstinence. However, these results are preliminary and replication is necessary to confirm these findings.

Variation within the serotonin (5-HT) 5-HT2C receptor system aligns with vulnerability to cocaine cue reactivity.

Cocaine dependence remains a challenging public health problem with relapse cited as a major determinant in its chronicity and severity. Environmental contexts and stimuli become reliably associated with its use leading to durable conditioned responses ('cue reactivity') that can predict relapse as well as treatment success. Individual variation in the magnitude and influence of cue reactivity over behavior in humans and animals suggest that cue-reactive individuals may be at greater risk for the progression to addiction and/or relapse. In the present translational study, we investigated the contribution of variation in the serotonin (5-HT) 5-HT2C receptor (5-HT2CR) system in individual differences in cocaine cue reactivity in humans and rodents. We found that cocaine-dependent subjects carrying a single nucleotide polymorphism (SNP) in the HTR2C gene that encodes for the conversion of cysteine to serine at codon 23 (Ser23 variant) exhibited significantly higher attentional bias to cocaine cues in the cocaine-word Stroop task than those carrying the Cys23 variant. In a model of individual differences in cocaine cue reactivity in rats, we identified that high cocaine cue reactivity measured as appetitive approach behavior (lever presses reinforced by the discrete cue complex) correlated with lower 5-HT2CR protein expression in the medial prefrontal cortex and blunted sensitivity to the suppressive effects of the selective 5-HT2CR agonist WAY163909. Our translational findings suggest that the functional status of the 5-HT2CR system is a mechanistic factor in the generation of vulnerability to cocaine-associated cues, an observation that opens new avenues for future development of biomarker and therapeutic approaches to suppress relapse in cocaine dependence.

Pharmacogenetic randomized trial for cocaine abuse: disulfiram and α1A-adrenoceptor gene variation.

Disulfiram is a cocaine addiction pharmacotherapy that inhibits dopamine ß-hydroxylase (DßH) and reduces norepinephrine production. We examined whether a functional variant of the ADRA1A gene (Cys to Arg at codon 347 in exon 2, Cys347Arg) may enhance treatment response through decreased stimulation of this α1A-adrenoceptor, since antagonists of this receptor show promise in reducing cocaine use. Sixty-nine cocaine and opioid co-dependent (DSM-IV) subjects were stabilized on methadone for two weeks and subsequently randomized into disulfiram (250 mg/day, N=32) and placebo groups (N=37) for 10 weeks. We genotyped the ADRA1A gene polymorphism (rs1048101) and evaluated its role for increasing cocaine free urines in those subjects treated with disulfiram using repeated measures analysis of variance, corrected for population structure. The 47 patients who carried at least one T allele of rs1048101 (TT or TC genotype) reduced their cocaine positive urines from 84% to 56% on disulfiram (p=0.0001), while the 22 patients with the major allele CC genotype showed no disulfiram effect. This study indicates that a patient's ADRA1A genotype could be used to identify a subset of individuals for which disulfiram and, perhaps, other α1-adrenoceptor blockers may be an effective pharmacotherapy for cocaine dependence.

Modifying the role of serotonergic 5-HTTLPR and TPH2 variants on disulfiram treatment of cocaine addiction: a preliminary study.

Disulfiram is a cocaine pharmacotherapy that may act through increasing serotonin, benefiting patients with genetically low serotonin transporter levels (5-HTTLPR, S' allele carriers) and low serotonin synthesis (TPH2, A allele carriers). We stabilized 71 cocaine and opioid co-dependent patients on methadone for 2 weeks and randomized them into disulfiram and placebo groups for 10 weeks. We genotyped the SLC6A4 5-HTTLPR (rs4795541, rs25531) and TPH2 1125A>T (rs4290270) variants and evaluated their role in moderating disulfiram treatment for cocaine dependence. Cocaine-positive urines dropped from 78% to 54% for the disulfiram group and from 77% to 76% for the placebo group among the 5-HTTLPR S' allele carriers (F = 16.2; df = 1,301; P < 0.0001). TPH2 A allele carriers responded better to disulfiram than placebo (F = 16.0; df = 1,223; P < 0.0001). Patients with both an S' allele and a TPH2 A allele reduced cocaine urines from 71% to 53% on disulfiram and had no change on placebo (F = 21.6; df = 1,185; P < 0.00001).