Biomedical subjects
D Goldman
Publications and source records attributed to D Goldman.
Clozapine response and the 5HT2C Cys23Ser polymorphism.
The presence of the 5HT2C receptor allele, Ser23, has recently been reported to predict favorable response to the antipsychotic drug, clozapine. This finding is of interest as Ser23, compared with the more abundant Cys23, alters pharmacological characteristics of the receptor and therefore may provide insights into the mechanism of action of antipsychotic drugs. We determined 5HT2C receptor genotype at the Cys23Ser locus in 66 subjects participating in double-blind studies of clozapine. There was no relationship between Ser23 and clozapine response (p = 0.30, Fisher's exact) nor was there any effect of Ser23 upon absolute levels of psychiatric symptoms after 10 weeks of clozapine treatment (t = -0.57, p = 0.57). These data suggest that this 5HT2C receptor polymorphism is not associated with clozapine response.
Arylsulfatase A pseudodeficiency-associated mutations: population studies and identification of a novel haplotype.
Pseudodeficiency of arylsulfatase A is characterized by reduction of arylsulfatase A activity without neurodegeneration, making it an important complication when diagnosing metachromatic leukodystrophy. Two DNA substitutions are associated with arylsulfatase A pseudodeficiency. One, 1788A-->G, results in the loss of an N-glycosylated asparagine in the protein, and the second, 2723A-->G, removes the polyadenylation signal site of the mRNA. Previously, the polyadenylation signal site variant was observed only in the presence of the N-glycosylation site variant, although the latter has been reported to occur in the absence of the polyadenylation signal site variant. We investigated the frequencies of these alleles and their linkage disequilibrium in a number of populations and in psychiatric patients. While the N-glycosylation site variant had a high frequency in the Bantu-speaking people from Southern Africa (0.44), the San of Southern Africa (0.22), African Americans (0.37), and Cheyenne Indians (0.375), the polyadenylation signal site variant was absent in these groups. The mutated polyadenylation signal site was found only in the Caucasian groups surveyed. Two Caucasian sibs were identified with the pseudodeficiency polyadenylation signal site variant in the absence of the N-glycosylation site variant, indicating that linkage disequilibrium between the two polymorphisms is not perfect.
Isolation and characterization of murine vav2, a member of the vav family of proto-oncogenes.
We describe the isolation and characterization of a cDNA encoding murine vav2. vav2 shares 63% and 55% identity at the nucleic acid and amino acid levels, respectively, with vav, a proto-oncogene that plays an essential role in embryonic development and hematopoietic signal transduction. The 100 kDa Vav2 protein contains the characteristic array of structural motifs found in Vav. However, unlike vav, vav2 transcripts are widely distributed in both hematopoietic and non-hematopoietic tissues. In the adult, vav2 mRNA is found at high levels in the spleen, liver, testes and placenta. Northern blot analysis reveals two vav2 mRNA species (designated alpha and beta). The alpha species is expressed throughout development while the alpha and beta species are expressed tissue-specifically in adults. Transfection of NIH3T3 cells with expression vectors containing vav2 deletions demonstrate that elimination of 183 amino terminal residues of Vav2 is sufficient to activate its oncogenic potential. Vav2-induced transformation is characterized by the appearance of foci composed of cells in which cytokinesis and karyokinesis are uncoupled. This phenotype is comparable, but not identical, to morphological changes induced by Vav and other members of the DbI family of oncoproteins. Our results suggest that Vav family members mediate functions important in the regulation of cell architecture and proliferation in most, if not all, tissues.
5HT 2a receptor T102C polymorphism and schizophrenia.
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Identification and characterization of a 47 base pair activity-dependent enhancer of the rat nicotinic acetylcholine receptor delta-subunit promoter.
Nicotinic acetylcholine receptor (nAChR) genes are regulated by muscle electrical activity. E-box sequences found in their promoters are necessary for this regulation. However, many muscle genes contain E-boxes, yet are not regulated by muscle depolarization. This suggests that other elements are necessary, perhaps working in conjunction with E-boxes, to confer depolarization-dependent control onto promoter activity. We have used direct DNA injection into muscle as an in vivo assay to identify and characterize these additional elements. Mutagenesis and expression assays identified multiple elements within the first 81 base pairs (bp) of the nAChR delta-subunit promoter that contribute to its regulation by muscle electrical activity. Within this 81 bp sequence, two regions of DNA were identified that were capable of conferring activity-dependent regulation onto a heterologous promoter. The stronger of these two putative enhancers was characterized further. It is a 47 bp sequence that contains an E-box along with sequences similar to the SV40 core enhancer and an SP1 site. Site-directed mutagenesis identified residues within each of these sequences that were necessary for enhancer activity. Furthermore, methylation interference DNA footprinting assays showed increased nuclear protein binding to sequences within both these enhancers after muscle denervation, and this pattern of binding was very similar to that observed with nuclear protein isolated from myotube extracts. These latter results suggest that similar mechanisms may mediate increased nAChR expression during muscle development and after muscle denervation.
Opsin gene expression and regulation in retinal transplants.
In the present study we investigated the expression and regulation of the opsin gene in retinal transplants. Embryonic retinae were transplanted to intracranial locations in neonatal rodents in which they either reliably projected to the superior colliculus, or in locations (such as the cerebral cortex) in which they did not project to subcortical visual nuclei. Our results show that, regardless of the graft location, the developmental schedule of opsin gene expression in the outer nuclear layer was similar to normal, and that it was maintained in transplants for at least 6 months. To test if ambient light affected opsin gene expression, we dark-reared rats containing a retinal transplant for up to 26 days before assaying for opsin transcripts. In situ hybridization experiments showed that opsin gene expression in the transplants of these dark-reared recipients was not different either from transplants in animals reared in cyclic light conditions, or from the retina in situ. These observations support the hypothesis that the opsin gene is activated and maintained by molecular mechanisms intrinsic to the photoreceptor.
Cloning and characterization of a novel transcriptional repressor of the nicotinic acetylcholine receptor delta-subunit gene.
We have identified a negative cis-acting regulatory element in the nicotinic acetylcholine receptor delta-subunit gene's promoter. This element resides within a previously identified 47-base pair activity-dependent enhancer. Proteins that bind this region of DNA were cloned from a lambdagt11 innervated muscle expression library. Two cDNAs (MY1 and MY1a) were isolated that encode members of the Y-box family of transcription factors. MY1/1a RNAs are expressed at relatively high levels in heart, skeletal muscle, testis, glia, and specific regions of the central nervous system. MY1/1a are nuclear proteins that bind specifically to the coding strand of the 47-base pair enhancer and suppress delta-promoter activity in a sequence-specific manner. These results suggest a novel mechanism of repression by MY1/1a, which may contribute to the low level expression of the delta-subunit gene in innervated muscle. Finally, the gene encoding MY1/1a, Yb2, maps to the mid-distal region of mouse chromosome 6.
DNA mismatch repair and DNA methylation in adult brain neurons.
DNA repair is essential for maintaining the integrity of the nucleotide sequence of cellular DNA over time. Although much information has accumulated recently on the mechanisms of DNA repair in cultured cells, little is known about the DNA repair capabilities of cells in the adult brain. In the present study, we have investigated the capacity of nuclear extracts from adult rodent brain neurons to carry out DNA mismatch repair. We focused on the repair of G.T and G.U mismatches, which are caused by deamination of 5-methyl cytosine to thymine, or cytosine to uracil, respectively, because these are the only types of mismatches that can arise in nondividing cells. We found that nuclear extracts from adult brain neurons can correct G.T and G.U mismatches, restoring them to G:C base pairs. Several other types of DNA mismatches could not be processed. These data provide the first direct demonstration that neurons in the adult mammalian brain have the capability to carry out DNA mismatch repair. We also we report that adult brain contains high levels of DNA methyltransferase (MTase) activity. We propose that one function of DNA MTase in the adult brain is to remethylate newly incorporated cytosine residues from G.T mismatch repair after deamination of 5-methyl cytosine, thereby maintaining the original pattern of DNA methylation. The high levels of brain DNA MTase suggest further that this enzyme has additional functions in the brain.
Imipramine treatment of alcoholics with primary depression: A placebo-controlled clinical trial.
BACKGROUND: Depressive disorders are commonly comorbid with alcoholism, particularly in treatment-seeking samples. If antidepressant treatment were safe and improved the treatment outcome in the subset of actively drinking alcoholics with depression, this would be of clinical importance. METHODS: We conducted a randomized, 12-week placebo-controlled trial of imipramine hydrochloride combined with weekly relapse prevention psychotherapy. The subjects were 69 actively drinking alcoholic outpatients with current depressive disorders. The first onset of depression was either antecedent to the abuse of alcohol or occurred during prolonged periods of sobriety. Depression and drinking outcomes at 12 weeks, as well as their relationship, were measured. RESULTS: Imipramine treatment was safe and associated with improvement in depression in both adequately treated and intention-to-treat samples. While there was no overall effect on drinking outcome, patients whose mood improved showed decreased alcohol consumption that was more marked in those treated with imipramine. CONCLUSIONS: Imipramine treatment is effective for primary depression among actively drinking alcoholic outpatients, and may improve alcoholic outcome for those whose depression responds to treatment.
TPH replication study: not!
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A dual function activity-dependent, muscle-specific enhancer from rat nicotinic acetylcholine receptor delta-subunit gene.
Nicotinic acetylcholine receptors (nAChR) mediate communication between nerve and muscle. The expression of these receptors increases dramatically during muscle development when myoblasts are fusing into multinucleated myotubes. The molecular mechanisms mediating this muscle developmental stage specific expression are not well understood. We report here the identification of nAChR delta-subunit promoter DNA sequences that differentially interact with nuclear proteins isolated from myoblasts, myotubes, and nonmuscle cells. The functional role these sequences play in mediating muscle-specific expression was explored using mutagenesis and enhancer assays. These studies resulted in the identification of a 47-bp muscle-specific enhancer that mediates increased expression of the nAChR delta-subunit gene during myotube formation. This enhancer contains an E-box and an element with similarity to the SV40 core enhancer (SVCE). Point mutations throughout this 47-bp enhancer showed that the E-box and the SVCE sequence are both necessary for conferring muscle-specific expression onto a heterologous promoter. Interestingly, this same DNA sequence also functions as an activity-dependent enhancer.
Mass allele detection (MAD) of rare 5-HT1A structural variants with allele-specific amplification and electrochemiluminescent detection.
A strategy is described that exploits allele-specific amplification (ASA-PCR) and electrochemiluminescence (ECL) detection technology to rapidly and cheaply screen large numbers of DNAs arranged in pooled matrices in order to identify individual nucleotide sequence variants. To demonstrate this strategy, a large genomic DNA collection was screened for two nucleotide variants in the 5-HT1A serotonin receptor gene and individual heterozygotes were identified. Conversion of two SSCP variants to allele-specific PCR polymorphisms was accomplished, and PCR product capture and ECL detection were enabled by the covalent addition of biotin to allele-specific PCR primers and ruthenium to the nonspecific PCR primer. A two-level DNA pooling strategy was used to reduce the number of individual PCR reactions required. Pooling experiments established that ASA-PCR with ECL detection is sufficiently sensitive to reproducibly detect a single specific allele in the presence of a 40-fold excess of genomic DNA from individuals negative for the specific allele. The detection sensitivity of the ECL device and the design of the pooled DNA arrays reduced the number of PCRs required to detect the rare individuals with the variant sequences by approximately 90%. This strategy is called mass allele detection (MAD).
Serotonin in alcoholic violent offenders.
Finnish alcoholic, impulsive, habitually violent offenders have been found to have low brain serotonin (5-hydroxytryptamine; 5-HT) turnover which is associated with impaired impulse control, a history of suicide attempts, hypoglycaemic tendency after an oral glucose load and diurnal activity rhythm dysregulation or hyperactivity. Relatively high cerebrospinal fluid (CSF) free testosterone concentration is a further characteristic of the offenders with antisocial personality disorder. The impulsive offenders may represent a behaviourally extreme group of type 2 alcoholics as defined by Cloninger. A large cohort of 800 subjects, including alcoholic violent offenders, their relatives and male controls, has now been gathered from Finland with support from the National Institute on Alcohol Abuse and Alcoholism. About 200 subjects have provided CSF samples. Leukocytes from the whole cohort have been harvested and immortalized. Genes regulating 5-HT functions are now being systematically analysed from these samples. Thus far, polymorphisms of the tryptophan hydroxylase (TPH) and 5-HT2C receptor genes have been the most informative findings.
Direct analysis of candidate genes in impulsive behaviours.
Antisocial behaviour is both heterogeneous and the product of interacting genetic and environmental factors acting at different levels of causation. Heritability studies show that individual differences in predisposition to antisocial behaviour are transmitted vertically in families by genetic mechanisms. Owing to aetiological heterogeneity and complexity, study of a variety of other behavioural phenotypes may shed more light on the antecedents of antisocial behaviour than direct studies on antisocial behaviour. Identification of genetic vulnerability factors would clarify mechanisms of vulnerability and the role of the environment. Direct gene analysis and genetic linkage analysis have identified structural variants in genes involved in neurotransmitter function, and some progress has been made towards relating these genetic variants to antisocial personality and other behaviours. Thyroid hormone receptor variants can cause attention deficit/hyperactivity disorder, and a monoamine oxidase A variant leads to aggressive behaviour in one family. Direct gene analyses have revealed non-conservative amino acid substitutions and structural variants (generally rare) at DRD2, DRD3 and DRD4 dopamine receptors and 5-HT1A, 5-HT2A, 5-HT2C and 5-HT7 serotonin receptors. The stage is set to identify the phenotypic significance of these as well as genetic variants at other loci which may be relevant as candidate genes for antisocial behaviour and related behavioural differences.
Mannose-binding protein genetic polymorphisms in black patients with systemic lupus erythematosus.
OBJECTIVE: To determine whether dysfunctional or deficient mannose-binding protein (MBP) variants are found with increased frequency in black patients with systemic lupus erythematosus (SLE) compared with controls. METHODS: Allele-specific polymerase chain reaction amplification of 4 different polymorphic sites was performed on samples from 92 black SLE patients and 86 geographically matched black controls. RESULTS: Two structural polymorphisms of MBP, associated with low serum levels of MBP, were found with significantly increased frequency in the SLE patient population compared with controls. In contrast, a promoter haplotype associated with particularly high serum levels of MBP was negatively associated with SLE. CONCLUSION: Deficiencies of MBP predispose individuals to SLE.
Identification of a Ser857-Asn857 substitution in DRK1 (KCNB1), population frequencies and lack of association to the low voltage alpha EEG trait.
A nonconservative amino acid substitution (Ser857Asn) in the human delayed-rectifier potassium channel DRK1 (KCNB1 locus), a candidate gene for the low voltage alpha electroencephalogram (EEG) trait locus (LVEEG1) at 20q13.2, and its frequency in ethnic population samples are described. The frequency of Asn857 in seven different ethnic population samples, totalling more than 1600 individuals, ranged from zero to greater than 3%. However, no association was found between Asn857 and the low voltage alpha EEG trait (LVA) in a population of 105 subjects assessed for the EEG, 24 of whom actually had LVA.
An anxiolytic action of oxytocin is enhanced by estrogen in the mouse.
The established role of oxytocin (OT) in facilitation of steroid-modulated reproductive and affiliative behaviors led to the speculation that it may have anxiolytic actions under certain hormonal conditions. NIH-Swiss mice were tested for responsiveness to OT in two behavioral tests of anxiety, the holeboard apparatus and elevated plus-maze. Dose-response assessment indicated that 3 mg/kg was the optimal dose for peripherally administered (IP) OT on the elevated plus-maze. There were no consistent effects at any dose on the holeboard apparatus. In ovariectomized mice pretreated with estradiol (E2), peripherally administered OT increased the number of entrances onto the open arms, as well as the amount of time on the open arms compared to other groups (ANOVA; p < 0.05). There was little to no effect of OT in ovariectomized animals not pretreated with E2. When OT was administered intracerebroventricularly (ICV), there was an increase in entrances and time on the open arms compared to that of females infused with arginine vasopressin (AVP). This increase was apparent in ovariectomized females, but was further enhanced in those pretreated with E2 (ANOVA; p < 0.05). In contrast, the combination of E2 pretreatment and ICV AVP decreased the number of entrances and time spent on the open arms of the elevated plus-maze compared to those receiving OT, suggesting an estrogen-modulated anxiogenic action of AVP. Analyses of [125]I-OVTA binding density indicated a significant increase in binding density in the lateral septum of E2-treated females compared to OIL-treated controls (ANOVA; p < 0.05). There was no effect of E2 treatment on [125]I-OVTA binding density in the amygdala or ventromedial nucleus of the hypothalamus. Taken together, these data indicate that OT exerts an anxiolytic action that is enhanced in the presence of circulating estrogen. This behavioral effect may be mediated by estrogen-induced increases in OT binding density in the lateral septum and may be important to the facilitation of social interactions.