Chemical modification of cyclomaltodextrin glucanotransferase from Bacillus circulans var. alkalophilus.

Counting of integral numbers of cysteine residues of the reduced and denaturated form of cyclomaltodextrin glucanotransferase (CGTase) from Bacillus circulans var. alkalophilus (ATCC 21783) showed two cysteine residues per enzyme molecule. Titrations of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) led to the same result. No free SH-group was detected in denatured form of CGTase, indicating that the two cysteine residues are linked by one disulfide bridge. Cyclizing activity of the GdmCl-denaturated and reduced enzyme was 13% of that of the native one. Incubation of CGTase with diethylpyrocarbonate (DEP) showed a pseudo-first-order inhibition with second-order rate constant of 3.2 M-1 s-1. Reaction with hydroxylamine and spectroscopic studies implied that inactivation of CGTase by DEP is due to modification of one histidine residue concomitantly with a 50% decrease in the cyclizing activity (t1/2 = 10.8 min). The inhibition was partially reversible. CGTase was protected against inactivation by alpha- and beta-cyclodextrins suggesting that the modified histidine residue is at or near the active site. Conversion of starch with DEP-modified enzyme resulted in a decreased formation of cyclodextrins while the relative amount of reducing sugars increased. Preliminary results on modification of CGTase with other reagents, e.g., Woodward's reagent K, 2,3-butanedione and carbodiimide are included.

The dopamine D2 receptor 5'-flanking variant, -141C Ins/Del, is not associated with reduced dopamine D2 receptor density in vivo.

The Del allele of the -141C (Ins/Del) polymorphism located in the immediate 5'-flanking region of the human dopamine D2 receptor gene has been reported to be associated with reduced promoter activity in vitro. However, genetic association studies of the -141C (Ins/Del) polymorphism with schizophrenia and alcoholism have yielded conflicting results. In this report, we explored the effect of the Del allele on the D2 receptor binding characteristics in vivo in healthy volunteers using positron emission tomography and D2 receptor antagonist, [11C]raclopride. No difference in D2 receptor density was observed between the Del allele carriers compared to the individuals with the Ins/Ins genotype, indicating that the genetic variation at the -141C (Ins/Del) site does not affect D2 receptor expression level in vivo.

Sex differences in the striatal dopamine D2 receptor binding characteristics in vivo.

OBJECTIVE: The authors investigated whether striatal dopamine D2 receptor binding characteristics in vivo are similar in men and women and whether there are sex-related differences in the decline in D2 receptor density due to aging. METHOD: Striatal D2 receptor density (Bmax), affinity (Kd), and binding potential (Bmax/Kd) were measured with positron emission tomography and [11C]raclopride in 54 healthy subjects (33 men and 21 women). RESULTS: Women had generally lower D2 receptor affinity than men, and this difference was statistically significant in the left striatum. Bmax and Bmax/Kd tended to decline with age twice as fast in men as in women, but the difference did not reach statistical significance. CONCLUSIONS: These results confirm the age-related reduction of D2 receptor density and binding potential in both sexes in vivo. The lower D2 receptor affinity suggests an increased endogenous striatal dopamine concentration in women. This may have implications for the differential vulnerability of men and women to psychiatric disorders like schizophrenia and alcohol and substance dependence.

Lack of association between the functional variant of the catechol-o-methyltransferase (COMT) gene and early-onset alcoholism associated with severe antisocial behavior.

Addictive drugs, including ethanol, increase the brain's dopaminergic transmission, and catechol-o-methyltransferase (COMT) enzyme has a crucial role in dopamine inactivation. A common functional polymorphism in the COMT gene results in a three- to four-fold variation in enzyme activity. In a previous study, we found an association between type 1 (with late-onset but without prominent antisocial behavior) alcoholism and the low activity allele of the COMT gene. In this work we analyzed whether the COMT polymorphism has any effect on the development of type 2 (with early-onset and habitual impulsive violent behavior) alcoholism. The COMT genotype was determined in 62 impulsive violent recidivist offenders with early-onset (type 2) alcoholism, 123 late-onset nonviolent (type 1) alcoholics, and 267 race and gender-matched controls. The allele and genotype frequencies of these groups were compared with each other and also with previously published data from 3,140 Finnish blood donors. The type 2 alcoholics did not differ from either the blood donors or the controls. The low activity (L) allele frequency was higher among type 1 alcoholics (chi(2) = 4.98, P = 0.026) when compared with type 2 cases. The odds ratio for type 1 alcoholism as compared with type 2 alcoholism for those subjects with the LL genotype versus the HH genotype was 3.0 (95% confidence interval 1.1-8.4, P = 0.017). The results suggest that COMT genotype has no major role in the development of early-onset alcoholism with severe antisocial behavior.

Allelic association between D2 but not D1 dopamine receptor gene and alcoholism in Finland.

We studied the relationship of D2 and D1 receptor gene polymorphisms and alcoholism in male Finnish alcoholics and assessed male controls. Seventy alcoholics entering a detoxification programme and 50 control individuals were recruited. Forty-three per cent of the alcoholic patients, but only 22% of controls, had the D2 receptor gene TaqI A restriction fragment length polymorphism A1 allele. The frequency of the A1 allele was significantly higher in alcoholics (p = 0.039). In comparison, no association between alcoholism and the D1 receptor gene EcoRI restriction fragment length polymorphism alleles was found. A logistic regression analysis of the alcoholic population failed to support the idea that the presence of the A1 allele would be linked to estimates of alcohol dependence severity rated with the Severity of Alcohol Dependence Questionnaire or the Michigan Alcoholism Screening Test. In conclusion, allelic association between the D2 but not D1 receptor gene and alcoholism in a genetically relatively homogenous population of male Finns was found. The results are in agreement with the view that the D2 receptor locus is involved in genetic susceptibility to alcoholism but does not give support to a special association of severe alcohol dependence and the A1 allele of the D2 receptor gene.

Regulation of dopamine D1 receptors by chronic administration of structurally different D1 receptor antagonists: a quantitative autoradiographic study.

The effects of chronic treatment (18 days) with the novel D1 antagonists, the benzonaphthazepine SCH 39166 (2 mg/kg per day) and the tetrahydroisoquinoline A-69024 (10 mg/kg per day), on D1 and D2 receptor binding in the rat brain were studied by quantitative receptor autoradiography. The benzazepine derivatives, SCH 23390 (0.5 mg/kg per day) and SKF 38393 (20 mg/kg per day), the prototype D1 antagonist and agonist, respectively, were also included in the experiment. Chronic treatment with SCH 23390 increased D1 receptor binding, studied with [3H]SCH 23390, in the nucleus accumbens and in all subregions of the anterior caudatus-putamen. However, chronic treatment with SKF 38393 did not alter D1 receptor binding in the brain areas studied. Interestingly, chronic treatment with SCH 39166 increased D1 receptor binding in the anterior caudatus-putamen but not in the nucleus accumbens. In contrast, chronic treatment with A-69024 did not alter D1 receptor binding in the brain areas studied. Treatment with SCH 23390, SCH 39166, A-69024 or SKF 38393 failed to alter D1 receptor binding in the posterior caudatus-putamen and the tuberculum olfactorium. Neither the D1 antagonists nor the D1 agonist investigated altered D2 receptor binding, studied with [125I]sulpiride, in the caudatus-putamen and nucleus accumbens. In summary, the benzonaphthazepine D1 antagonist, SCH 39166, as well as the benzazepine D1 antagonist, SCH 23390, can increase D1 receptor binding without influencing D2 receptor binding. However, a tetrahydroisoquinoline, A-69024, failed to increase D1 receptor binding, suggesting a differential regulation of D1 receptors after treatment with this putative D1 antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Intraocular pressure after phacoemulsification and intraocular lens implantation in nonglaucomatous eyes with and without exfoliation.

PURPOSE: To evaluate intraocular pressure (IOP) after phacoemulsification and intraocular lens (IOL) implantation in nonglaucomatous eyes with and without exfoliation. SETTING: Department of Ophthalmology, Helsinki University Central Hospital, Helsinki, Finland. METHODS: One hundred ninety-six eyes that had phacoemulsification with IOL implantation were examined. Eyes with a history of intraocular disease or surgery that could affect IOP were excluded. The study comprised the remaining 160 eyes: 23 with exfoliation (EXF group) and 137 without exfoliation (non-EXF group). Follow-up data were available for 136 eyes. The same surgeon performed all surgeries. Patients were examined on the first postoperative day and after 1 week, 4 months, and 1.0 to 2.7 years. RESULTS: One day postoperatively, IOP rose in the EXF group from a mean preoperative level of 16.3 mm Hg +/- 2.7 (SD) to 21.0 +/- 8.5 mm Hg, a 28.4% increase (P =.0061). In the non-EXF group, mean IOP rose from 16.2 +/- 3.4 mm Hg to 20.5 +/- 5.7 mm Hg, a 29.9% increase (P =.001). In 4 eyes (17.4%) in the EXF group and 8 eyes (5.8%) in the non-EXF group, IOP increased to 30 mm Hg or higher at 1 day. After this, significant IOP decreases occurred in both the EXF and non-EXF group, respectively, as follows: 14.2 +/- 3.0 mm Hg (12.0% decrease from preoperative value; P =.001) and 15.0 +/- 2.9 mm Hg (5.9%; P =.001) 1 week postoperatively; 12.8 +/- 2.7 mm Hg (20.2%; P =.0002) and 13.8 +/- 2.7 mm Hg (13.2%; P =.001) after 4 months; and 12.3 +/- 2.6 mm Hg (23.2%; P =.0001) and 12.7 +/- 2.7 mm Hg (21.2%; P =.001) after 1.0 to 2.7 years. There was no significant difference between the 2 groups. CONCLUSION: After phacoemulsification with IOL implantation, IOP decreased significantly and remained lower than preoperatively in eyes with and without exfoliation. One day postoperatively, transient pressure peaks were more common in eyes with exfoliation. One eye without exfoliation developed glaucoma.

Posterior capsular opacification in pseudophakic eyes with a silicone or acrylic intraocular lens.

PURPOSE: To compare posterior capsular opacification in eyes with IOL of two different materials--silicone or acrylic. METHODS: Eighty consecutive eyes undergoing cataract surgery were prospectively randomized in two groups, 40 eyes receiving a silicone (Sl--30NB) and 40 eyes an acrylic (Acrysof MA60BM) intraocular lens (IOL). The same surgeon performed phacoemulsification and the intraocular lens (PHACO IOL) operation in all cases. Patients were re-examined on the first postoperative day, after one week, four months, and 1-2.4 years. Seven eyes were lost to late control. RESULTS: Clinically significant posterior capsular opacification (PCO) (including eyes with capsulotomy already performed) was equally common in both groups; 25% in the silicone group and 19% in the acrylic group (p=0.53). The posterior capsule remained clear in 61% of the silicone and 76% of the acrylic IOL eyes (p=0.18). In the whole study group, 29% of eyes with and 14% without concurrent ocular diseases had significant PCO (p=0.13). In the silicone IOL group, PCO was more common in eyes with concurrent ocular diseases (44%) than eyes without other diseases (10%) (p=0.049). Eyes with acrylic IOL showed no difference in significant PCO, with or without other diseases (18% and 20%, respectively). CONCLUSIONS: In a consecutive series of 80 cataract eyes central PCO was equally common in eyes receiving a silicone or an acrylic IOL. In the silicone IOL group, however, significant PCO was more common if there was concurrent ocular disease, while with the acrylic IOL concurrent ocular disease did not seem to increase the risk of PCO.

Phacoemulsification and intraocular lens implantation in eyes with open-angle glaucoma.

PURPOSE: To study the effect of phacoemulsification and intraocular lens implantation (PHACO IOL) on intraocular pressure (IOP) and glaucoma medication in open-angle glaucoma (OAG) eyes. METHODS: 38 open-angle glaucoma (OAG) eyes with cataract underwent phacoemulsification and intraocular lens implantation (PHACO IOL) performed by one surgeon (RJU). None of the patients had prior intraocular surgery. Surgery was performed by scleral incision on 37% and by clear corneal incision on 63%. Patients were re-examined on the first postoperative day, after one week, 4 months, and in 29 cases 1-3.7 (mean 2.8) years after the operation. RESULTS: The mean preoperative IOP was 18.4+/-3.3 mmHg with a mean of 1.7 glaucoma medications. On the first postoperative day, the mean IOP rose to 28.2 +/- 12.5 mmHg. IOP > or = 30 mmHg occurred in 39.5% of the eyes. After one week, IOP had returned to the preoperative level. After 4 months, IOP had further decreased to 16.1 +/- 3.8 mmHg (p = 0.0027). After a mean follow-up of 1-3.7 (mean 2.8) years, the average postoperative IOP was 15.1 +/- 2.9 mmHg, being significantly (p = 0.001) lower than the preoperative IOP with 86% of the patients having a mean of 1.6 drugs on average. The type of incision (scleral vs. corneal) did not affect the postoperative IOP level. Using the criteria of Bigger and Becker (1971) the long-term IOP control after PHACO-IOL surgery was improved or unchanged in 86% and worse in 14% of the preoperatively well-controlled OAG eyes. CONCLUSIONS: In OAG eyes PHACO IOL is associated with a significant decrease in IOP with less medication up to 1-3.7 (mean 2.8) years.

The A1 allele of the D2 dopamine receptor gene is associated with high dopamine transporter density in detoxified alcoholics.

The A1 allele of TaqI A restriction fragment length polymorphism (RFLP) in the D2 receptor (DRD2) gene locus has been suggested to be associated with low D2 receptor density in man. Striatal dopamine transporter (DAT) densities were studied with [(123)I]2-beta-carbometoxy-3beta(4-iodophenyl)tropane and single-photon emission tomography in 29 detoxified alcoholics, who were also genotyped for the two alleles of TaqI A RFLP at the DRD2 receptor gene locus. Alcoholics with the A1/A2 genotypes (n = 10) had statistically significantly higher DAT densities than subjects with the A2/A2 genotypes [n = 19; 8.0 +/- 1.2 (mean +/- SD) vs 6.9 +/- 1.1, P = 0.035]. We suggest that the TaqI A RFLP is in linkage disequilibrium with a gene variant modifying DAT density in alcoholics.

Dopamine D2 receptor gene expression in rat lines selected for differences in voluntary alcohol consumption.

A selective breeding program has led to the establishment of the alcohol-preferring AA (Alko, Alcohol) and alcohol-avoiding ANA (Alko, Nonalcohol) rat lines. To reveal putative baseline differences in dopamine receptor gene expression and dopamine receptor binding profile in the AA and ANA rat lines, we assessed striatal D2 mRNA levels in these two rat lines. Autoradiographical studies on dopamine D1 and D2 receptors in the striatum and nucleus accumbens were also performed with [3H]SCH 23390 and [125I]iodosulpiride/[3H]spiperone, respectively. The baseline differences in D1 or D2 receptor binding and D2 receptor gene expression between AA and ANA rat lines are marginal, and are not likely to play a role in the genetic background of the differential alcohol drinking behavior of these rat lines.

Ethanol consumption and DRD2 gene TaqI a polymorphism among socially drinking males.

The dopaminergic system in the human brain is thought to play a major role in the development of alcohol consumption habits and alcoholism. It has been reported that homozygous D2-/- knock-out mice lacking D2 receptors consume about 50% to 60% less ethanol than wild-type D2+/+ mice, and heterozygous mice have an intermediate level of alcohol consumption. The DRD2 gene TaqI A polymorphism has been suggested to associate with a low D2 receptor density in post mortem and in vivo measurements. Numerous association studies on this polymorphism and alcoholism have shown most controversial results. We studied whether DRD2 TaqI A genotype affects alcohol consumption in an ethnically homogeneous, representative sample of 1,019 Finnish Caucasian males. After excluding the abstainers from the study, the self-reported alcohol consumption among the remaining 884 non-abstainers was compared in the TaqI A genotype groups (A1/A1, A1/A2, A2/A2). The alcohol consumption of the homozygous A1/A1 group was about 30% lower than in A1/A2 group, and 40% lower than in A2/A2 group (P = 0.042 and 0.041 in a sociodemographic variable-adjusted multivariate model). The results indicate an association between DRD2 genotype and alcohol consumption habits in humans. These results in the large sample of non-alcoholic males are also opposite to some previous findings on the higher A1 allele frequency among alcoholic populations.

The A1 allele of the human D2 dopamine receptor gene predicts low D2 receptor availability in healthy volunteers.

Positron emission tomography (PET) studies have revealed significant interindividual variation in dopamine D2 receptor density in vivo in human striatum. Low D2 receptor binding in vivo has been found to associate with alcohol/substance dependence. It has been suggested that the A1 allele of human D2 receptor gene might be associated to a specific type of alcoholism and possibly to a reduced D2 receptor density in vitro. We have determined D2 dopamine receptor-binding density (Bmax), affinity (Kd) and availability (Bmax/Kd) in 54 healthy Finnish volunteers using PET and [11C]raclopride in order to determine whether the A1 allele is associated with a 'baseline' difference in D2 receptor characteristics in vivo. A statistically significant reduction in D2 receptor availability reflecting an alteration in receptor density was observed in the A1/A2 genotype group compared to the A2/A2 group. There was no difference in apparent Kd between the two groups. In conclusion, the association between the A1 allele and low D2 receptor availability in healthy subjects indicates that the A1 allele of the TaqIA polymorphism might be in linkage disequilibrium with a mutation in the promoter/regulatory gene element that affects dopamine D2 receptor expression. This study provides an in vivo neurobiological correlate to the A1 allele in healthy volunteers.

Antagonist binding characteristics of the Ser311-->Cys variant of human dopamine D2 receptor in vivo and in vitro.

We report in vivo and in vitro antagonist binding characteristics of the naturally occurring Ser311-->Cys variant of the human D2 dopamine receptor. Striatal receptor binding characteristics in vivo were measured with positron emission tomography and the D2 antagonist [11C]raclopride. The in vitro affinity of raclopride for the Ser311-->Cys variant and the wild type receptor was studied in membrane binding assays from stably transfected cell lines. One healthy male carrying the heterozygous Ser311-->Cys (TCC-->TGC) substitution was identified with denaturing gradient gel electrophoresis and DNA sequencing. The striatal D2 receptor binding characteristics in vivo in this subject were normal. This was supported by the in vitro data as the Ki values of raclopride for the Ser311-->Cys variant and the wild type receptor were identical. Our data suggest that the Ser311-->Cys variant of the human D2 receptor does not influence antagonist-receptor recognition in vivo or in vitro.

Association between low activity serotonin transporter promoter genotype and early onset alcoholism with habitual impulsive violent behavior.

A common 44-base pair insertion/deletion polymorphism in the promoter region of the human serotonin transporter (5-HTT) gene has been observed to be associated with affective illness and anxiety-related traits. This biallelic functional polymorphism, designated long (L) and short (S), affects 5-HTT gene expression since the S promoter is less active than the L promoter. Since there is strong evidence of a disturbance in brain serotonergic transmission among antisocial, impulsive, and violent type 2 alcoholic subjects, we decided to test the hypothesis that the frequency of the S allele, which is associated with reduced 5-HTT gene expression, is higher among habitually violent type 2 alcoholics when compared with race and gender-matched healthy controls and non-violent late-onset (type 1) alcoholics. The 5-HTT promoter genotype was determined by a PCR-based method in 114 late onset (type 1) non-violent alcoholics, 51 impulsive violent recidivistic offenders with early onset alcoholism (type 2), and 54 healthy controls. All index subjects and controls were white Caucasian males of Finnish origin. The S allele frequency was higher among type 2 alcoholics compared with type 1 alcoholics (chi2 = 4.86, P = 0.028) and healthy controls (chi2 = 8.24, P = 0.004). The odds ratio for SS genotype vs LL genotype was 3.90, 95% Cl 1.37-11.11, P = 0.011 when type 2 alcoholics were compared with healthy controls. The results suggest that the 5-HTT 'S' promoter polymorphism is associated with an increased risk for early onset alcoholism associated with antisocial personality disorder and impulsive, habitually violent behavior.

Association between the functional variant of the catechol-O-methyltransferase (COMT) gene and type 1 alcoholism.

Catechol-O-methyltransferase (COMT) is an enzyme which has a crucial role in the metabolism of dopamine. It has been suggested that a common functional genetic polymorphism in the COMT gene, which results in 3 to 4-fold difference in COMT enzyme activity, may contribute to the etiology of mental disorders such as bipolar disorder and alcoholism. Since ethanol-induced euphoria is associated with the rapid release of dopamine in limbic areas, it is conceivable that subjects who inherit the allele encoding the low activity COMT variant would have a relatively low dopamine inactivation rate, and therefore would be more vulnerable to the development of ethanol dependence. The aim of this study was to test this hypothesis among type 1 (late-onset) alcoholics. The COMT polymorphism was determined in two independent male late onset (type 1) alcoholic populations in Turku (n = 67) and Kuopio (n = 56). The high (H) and low (L) activity COMT genotype and allele frequencies were compared with previously published data from 3140 Finnish blood donors (general population) and 267 race- and gender-matched controls. The frequency of low activity allele (L) was markedly higher among the patients both in Turku (P = 0.023) and in Kuopio (P = 0.005) when compared with the general population. When all patients were compared with the general population (blood donors), the difference was even more significant (P = 0.0004). When genotypes of all alcoholics (n = 123) were compared with genotypes of matched controls, the odds ratio (OR) for alcoholism for those subjects having the LL genotype vs those with HH genotype was 2.51, 95% CI 1.22-5.19, P = 0.006. Also, L allele frequency was significantly higher among alcoholics when compared with controls (P = 0.009). The estimate for population etiological (attributable) fraction for the LL genotype in alcoholism was 13.3% (95% CI 2.3-25.7%). The results indicate that the COMT polymorphism contributes significantly to the development of late-onset alcoholism.