Association between SNAP-25 gene polymorphisms and cognition in autism: functional consequences and potential therapeutic strategies.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is involved in different neuropsychiatric disorders, including schizophrenia and attention-deficit/hyperactivity disorder. Consistently, SNAP-25 polymorphisms in humans are associated with hyperactivity and/or with low cognitive scores. We analysed five SNAP-25 gene polymorphisms (rs363050, rs363039, rs363043, rs3746544 and rs1051312) in 46 autistic children trying to correlate them with Childhood Autism Rating Scale and electroencephalogram (EEG) abnormalities. The functional effects of rs363050 single-nucleotide polymorphism (SNP) on the gene transcriptional activity, by means of the luciferase reporter gene, were evaluated. To investigate the functional consequences that SNAP-25 reduction may have in children, the behaviour and EEG of SNAP-25(+/-) adolescent mice (SNAP-25(+/+)) were studied. Significant association of SNAP-25 polymorphism with decreasing cognitive scores was observed. Analysis of transcriptional activity revealed that SNP rs363050 encompasses a regulatory element, leading to protein expression decrease. Reduction of SNAP-25 levels in adolescent mice was associated with hyperactivity, cognitive and social impairment and an abnormal EEG, characterized by the occurrence of frequent spikes. Both EEG abnormalities and behavioural deficits were rescued by repeated exposure for 21 days to sodium salt valproate (VLP). A partial recovery of SNAP-25 expression content in SNAP-25(+/-) hippocampi was also observed by means of western blotting. A reduced expression of SNAP-25 is responsible for the cognitive deficits in children affected by autism spectrum disorders, as presumably occurring in the presence of rs363050(G) allele, and for behavioural and EEG alterations in adolescent mice. VLP treatment could result in novel therapeutic strategies.

Genomic organization and transcription of the human retinol dehydrogenase 10 (RDH10) gene.

A cDNA clone up-regulated in hydraulic lung edema in rabbit showed high similarity with human RDH10 mRNA, which encodes a protein involved in retinoic acid metabolism. We defined the organization of the human gene, which includes a unique transcriptional start site, a coding region with six translated exons and a 3' untranslated region containing at least two used polyadenylation sites. The two poly(A) signals are responsible for the production of the 3 and 4 kb RDH10 mRNA isoforms detected in several human tissues and cell lines.

Sp proteins and Phox2b regulate the expression of the human Phox2a gene.

Phox2a is a vertebrate homeodomain transcription factor that is involved in the specification of the autonomic nervous system. We have isolated the 5' regulatory region of the human Phox2a gene and studied the transcriptional mechanisms underlying its expression. We first identified the minimal gene promoter by means of molecular and functional criteria and demonstrated that its activity relies on a degenerate TATA box and a canonical Sp1 site. We then concentrated on the region immediately upstream of the promoter and found that it stimulates transcription in a neurospecific manner because its deletion caused a substantial decline in reporter gene expression only in neuronal cells. This DNA region contains a putative binding site for homeodomain transcription factors, and its mutation severely affects the transcriptional activity of the entire 5' regulatory region, thus indicating that this site is necessary for the expression of Phox2a in this cellular context. The use of the electrophoretic mobility shift assay showed that Phox2b/PMX2b is capable of specifically interacting with this site, and cotransfection experiments demonstrated that it is capable of transactivating the human Phox2a promoter. Many data obtained from knock-out mice support the hypothesis that Phox2a acts downstream of Phox2b during the development of most of the autonomic nervous system. We have provided the first molecular evidence that Phox2b can regulate the expression of Phox2a by directly binding to its 5' regulatory region.

Neuronal and extraneuronal expression and regulation of the human alpha5 nicotinic receptor subunit gene.

The mRNA encoding the human alpha5 nicotinic subunit was detected in several structures of the nervous system but appeared to be mainly expressed in cerebellum, thalamus, and the autonomic ganglia. For the first time, the alpha5 transcript was also detected in several non-neuronal tissues, with maximal expressions being found throughout the gastrointestinal tract, thymus, and testis. Many other extraneuronal sites expressed alpha5, but there were also nonexpressing organs, such as the liver, spleen, and kidney. To understand the transcriptional mechanisms controlling such a diversified expression of alpha5 in neuronal and nonneuronal cells, we isolated the 5'-regulatory region of the human gene and characterized its properties. Here we identify the alpha5 core promoter and demonstrate that the DNA regions surrounding it contain elements (with positive or negative activities) that work in a tissue-specific fashion. In particular, the segment specifying the 5'-untranslated region in neuronal cells has most of the properties of an enhancer because it activates a heterologous promoter in a position- and orientation-independent fashion. We therefore conclude that the expression of alpha5 relies on a highly complex promoter that uses distinct regulatory elements to comply with the different functional and developmental requirements of the various tissues and organs.

Transcriptional regulation of the human alpha5 nicotinic receptor subunit gene in neuronal and non-neuronal tissues.

The human alpha5 nicotinic receptor subunit gene appears to be expressed in several structures of the nervous system, but also in a number of non-neuronal tissues, with maximal expressions occurring in the entire gastrointestinal tract, thymus and testis. To understand whether specific transcriptional mechanisms are involved in the tissue-specific expression of the alpha5 subunit in neuronal and non-neuronal cells, we isolated the 5'-regulatory region of the human gene and characterized its functional properties. We demonstrate that specific DNA elements, with positive or negative activities depending on the cell type, are responsible for the diversified expression of the alpha5 subunit in different tissues. We therefore conclude that the expression of the alpha5 subunit relies on a highly complex promoter that uses distinct regulatory elements to comply with the different functional and developmental requirements of the various tissues and organs.

Neurosecretory cells without neurosecretion: evidence of an independently regulated trait of the cell phenotype.

Neurosecretion competence is a fundamental property that enables differentiated neurones and professional neurosecretory cells to store neurotransmitters and hormones in specialized organelles, the synaptic-like vesicles and dense granules, and to release them by regulated exocytosis. In our laboratory, the study of rat phaeochromocytoma (PC12) clones that fail to express the above organelles or any other components involved in neurosecretion, whilst maintaining most of the general markers of the parental population, has served to demonstrate that this trait is controlled independently from the rest of the phenotype. The present review focuses on recent advances in elucidating the molecular mechanisms governing neurosecretion competence. Moreover, the opportunities that such neurosecretion-defective PC12 clones offer for the investigation of new aspects of regulated exocytosis and the localization of its components are summarized.

Neurosecretion competence, an independently regulated trait of the neurosecretory cell phenotype.

Neurosecretion competence is intended as the ability of neurosecretory cells to express dense and clear vesicles discharged by regulated exocytosis (neurotransmitter release). Such a property, which so far has never been studied independently, is investigated here by a heterotypic cell fusion approach, using a clone of rat pheochromocytoma PC12 cells totally incompetent for neurosecretion that still largely maintains its typical molecular and cellular phenotype. When fused with wild-type partners of various species (rat, human) and specialization (PC12, neuroblastoma SH-SY5Y, HeLa), the defective cells reacquire their competence as revealed by the expression of their secretion-specific proteins. Fused wild-type cells therefore appear able to complement defective cells by providing them with factor(s) inducing the reactivation of their secretory program. The mechanism of action of these factors may consist not in a coordinate unblocking of transcription but in the prevention of a rapid post-transcriptional degradation of the mRNAs for secretion-specific genes.

Combined effects of HDL cholesterol, triglyceride, and total cholesterol concentrations on 18-year risk of atherosclerotic disease.

BACKGROUND: Whether the combination of a low level of HDL cholesterol (HDL-C) and high level of triglyceride (TG) confers increased risk of cardiovascular disease and whether risk varies across levels of total cholesterol (TC) are not well established. Combined effects of HDL-C, TG, and TC on the incidence of atherosclerotic disease were examined prospectively in Japanese-American men from the Honolulu Heart Program. METHODS AND RESULTS: Among 1,646 men aged 51 to 72 years who were free of coronary heart disease (CHD), stroke, and cancer and were not taking lipid-lowering medication, 318 developed atherosclerotic events (angina, coronary insufficiency, aortic aneurysm, definite CHD, or thromboembolic stroke) and 170 developed definite CHD between 1970 and 1988. Subjects were stratified by TC level (desirable, < 200 mg/dL; borderline high, 200 to 239 mg/dL; high, > or = 240 mg/dL), HDL-C level (< 35 and > or = 35 mg/dL), and TG level (< 200 and > or = 200 mg/dL). With Cox regression with high HDL-C and low TG as reference, age-adjusted relative risks (RR) of atherosclerotic events were significantly elevated in men with low HDL-C and high TG at borderline-high (RR, 2.46; 95% CI, 1.48 to 4.09) and high (RR, 2.21; 95% CI, 1.34 to 3.66) TC levels but not in men with desirable TC levels (RR, 0.89; 95% CI, 0.38 to 2.09). Elevated risks were independent of blood pressure, obesity, fat distribution, diabetes, smoking, and alcohol. Results were not materially altered by exclusion of subjects with angina alone and were similar but somewhat weaker for CHD. CONCLUSIONS: Risk of atherosclerotic disease appears elevated in subjects with low HDL-C and high TG levels when TC is borderline high or high, independent of other cardiovascular risk factors. These findings support recent cholesterol screening recommendations and suggest that joint effects of HDL-C and TG may be important to consider.

Lifestyle and biologic factors associated with atherosclerotic disease in middle-aged men. 20-year findings from the Honolulu Heart Program.

OBJECTIVES: To examine the association between a variety of baseline lifestyle and biologic factors in a middle-aged cohort of Japanese-American men and the 20-year incidence rates of total atherosclerotic end points and each of the initial clinical manifestations of this disease, including fatal and nonfatal coronary heart disease, angina pectoris, thromboembolic strokes, and aortic aneurysms. DESIGN: Prospective epidemiologic study. POPULATION: Japanese-American men (N = 2710) between the ages of 55 and 64 years at the time of the initial clinical examination of the Honolulu Heart Program (1965 through 1968) free from evidence of coronary heart disease, cerebrovascular disease, cancer, or aortic aneurysms. RESULTS: Among the men studied, 602 atherosclerotic events developed during the 23-year period of follow-up (1965 through 1988). After adjustment for each of the baseline characteristics examined, significant positive associations between quartile cutoffs of body mass index, systolic blood pressure, serum levels of cholesterol, triglycerides, glucose, and uric acid, as well as cigarette smoking, and the occurrence of any atherosclerotic end point were seen, while an inverse association with alcohol consumption was observed. Characteristics associated with the development of other fatal and nonfatal clinical events in this cohort, including coronary heart disease, thromboembolic stroke, and aortic aneurysms are presented with accompanying relative and attributable risks. CONCLUSIONS: The results of this prospective epidemiologic study provide insights to the long-term predictive utility of the commonly accepted risk factors for coronary heart disease in relation to the different clinical manifestations of atherosclerosis in a middle-aged male cohort followed up for approximately 20 years. These results provide additional support for risk factor modification in middle-aged men and for the encouragement of positive long-term lifestyle changes.

To what extent do cardiovascular risk factor values measured in elderly men represent their midlife values measured 25 years earlier? A preliminary report and commentary from the Honolulu Heart Program.

In a Honolulu Heart Program cohort of 1,604 elderly men aged 70-90 years who were sampled from the most recent follow-up examination (1991-1992), current risk factor values were compared with those obtained 25 years earlier when the same men were between ages 45-64 and free of clinically diagnosed cardiovascular disease. Cardiovascular disease risk factors studied included systolic blood pressure, diastolic blood pressure, serum cholesterol, cigarette smoking, body mass index, and alcohol intake. For systolic pressure, 65% of the men had moved into a different quartile by old age, with 25% changing by more than one quartile. For diastolic pressure, 68% had moved into another quartile, with 28% moving more than one quartile, and for body mass index, 53% had moved into another quartile, with 14% moving more than a quartile. Less than 1% started to smoke, while 27% were reclassified from smokers to nonsmokers. Only 4% started to drink alcohol, while 30% were reclassified from drinkers to nondrinkers. When the men were stratified into cardiovascular disease, noncardiovascular disease, and healthy follow-up groups, modest deviations from the overall pattern were observed, with morbidity groups showing a greater tendency to reduction in risk factor levels. The results show that there is a substantial redistribution of major cardiovascular disease risk factor values between midlife and old age. Since midlife values are more likely to represent lifelong exposure values that, in turn, make the main contribution to the development of atherosclerosis, investigators and clinicians may need to be cautious in using risk factor values measured late in life as the only means of assessing risk for subsequent disease.

Topoisomerase I action on the heterochromatic DNA from the brine shrimp Artemia franciscana: studies in vivo and in vitro.

The genomes of higher eukaryotes contain various amounts of tandem repeated DNA sequences (satellite DNA) typically located in the constitutive heterochromatin, the most highly condensed region of interphase chromosomes. We have previously demonstrated that an AluI DNA family of repeats is the major component of constitutive heterochromatin in the brine shrimp Artemia franciscana. The analysis of cloned heterochromatic fragments revealed that this repetitive DNA shows a stable curvature conferring a solenoidal geometry to the double helix. In this paper we provide evidence, using the antitumour drug camptothecin, that, in vivo, topoisomerase I cleaves heterochromatin with a frequency comparable with that observed in the whole genome. The analysis of the break sites shows that the enzyme cleaves heterochromatic DNA at specific sites characterized by a degenerate consensus sequence. Moreover the enzyme-mediated breaks have, in vitro, a degenerate consensus sequence similar to, but not identical with, the in vivo one. Some of these sites are influenced by the DNA flanking the heterochromatic insert, suggesting that structural variations could modify the enzyme specificity.

Elevated serum cholesterol is a risk factor for both coronary heart disease and thromboembolic stroke in Hawaiian Japanese men. Implications of shared risk.

BACKGROUND AND PURPOSE: The relation between total serum cholesterol level and thromboembolic or nonhemorrhagic stroke is controversial. The Honolulu Heart Program cohort of Japanese-American men provides data which show that elevated serum cholesterol is an independent predictor of thromboembolic stroke as well as coronary heart disease (CHD). The data are presented to suggest that the association of elevated cholesterol with stroke is sometimes underestimated or underreported partly because of competing or shared risk with CHD, the other major atherosclerotic end point. METHODS: The data are based on 6352 men (aged 51 to 74 years) at baseline examination (1971 to 1974) who were free of clinical CHD and stroke and were followed an average of 15 years for new cases of both end points. Relative risks of serum cholesterol for CHD and thromboembolic stroke were calculated, controlling for other major cardiovascular covariates. RESULTS: There was a continuous and progressive increase in both CHD and thromboembolic stroke rates with increasing levels of serum cholesterol. The relative risk between the highest and lowest quartiles of serum cholesterol was 1.7 (95% confidence interval, 1.4 to 2.0) for CHD and 1.4 (95% confidence interval, 1.1 to 1.9) for thromboembolic stroke. There was a decline in the difference in relative risks between CHD and thromboembolic stroke in older men (aged 60 years and older) compared with younger men (aged younger than 60 years). CONCLUSIONS: These data provide additional evidence that elevated serum cholesterol should be considered a primary risk factor for thromboembolic stroke, presumably through its effect on both coronary and cerebrovascular atherosclerosis. It is suggested that this association is sometimes underestimated or underreported partly because of shared or competing risk with CHD, the clinical manifestation of atherosclerosis that generally occurs earlier in life and with greater frequency than thromboembolic stroke.

Studies of cardiovascular disease and cause-specific mortality trends in Japanese-American men living in Hawaii and risk factor comparisons with other Japanese populations in the Pacific region: a review.

The Honolulu Heart Program (HHP) is a long-term prospective epidemiologic study of cardiovascular disease (CVD) in male descendants of Japanese migrants to Hawaii. The article is a review of data from recent and past HHP studies relevant to the Seventeenth Pacific Science Congress symposium "Changes in Disease Patterns in the Western Pacific and Southeast Asia." The Ni-Hon-San Study, which compared CVD rates and risk factors in Japanese men living in Japan, Hawaii (HHP), and California, showed that coronary heart disease (CHD) and stroke mortality rates in Hawaii were intermediate between rates in Japan and California. Gradients in CVD risk factors were similar to the gradients in disease rates. From 1966 to 1984 trends in incidence rates for CHD, stroke, and cause-specific mortality were compared for the 8006 participants and 3130 non-participants in the HHP. CHD and stroke rates declined by about 40% for the total HHP cohort. There was a larger decline for CHD mortality (over 60%) in the nonparticipants. There was also a much greater decline in total mortality and cancer mortality rates in the nonparticipants. The results of the reviewed studies show that the subjects, although sharing a common ethnic background, experience different rates of disease when living in diverse geographic and cultural locales. This finding supports evidence that environmental and behavioral factors influence chronic disease rates and provides a basis for intervention and prevention. The finding that nonparticipants in epidemiologic studies can show different incidence trends suggests that caution should be used in interpreting trends limited only to participants.

Do coronary heart disease risk factors measured in the elderly have the same predictive roles as in the middle aged. Comparisons of relative and attributable risks.

Risk factors for the 12-year incidence of definite coronary heart disease (CHD) among 3440 men who were middle-aged (51 to 59 years old) and 1419 men who were elderly (65 to 74 years old) at baseline examination were examined for differences in predictive values in terms of both relative risk and attributable (excess) risk of the highest versus the lowest quartile or appropriate categories. In multivariate models using Cox life-table regression procedures, serum cholesterol level, cigarette smoking, systolic blood pressure, and history of treatment for diabetes were significant predictors of incident CHD for both age groups. Alcohol consumption when modeled as drinker versus nondrinker showed a protective effect in both younger and older men. There was no dose relationship, however, among elderly drinkers. While the relative risks for the variables studied were similar between the two age groups, the excess risk was typically between 1.5 to 2.0 times higher for the older than the middle-aged men. In contrast, the detrimental effect of adiposity as measured by body mass index appeared to decline after age 65 for both measures of risk. This may partly be attributed to diminished adiposity overall in the older age group. The implications of these results are that serum cholesterol level, smoking, hypertension, diabetes, and possibly alcohol consumption continue to be important predictors for CHD when measured after age 65, and that the public health impact of these risk factors, in terms of excess risk, may be more important in the elderly.

Will lowering population levels of serum cholesterol affect total mortality? Expectations from the Honolulu Heart Program.

Major campaigns now underway to reduce the serum cholesterol levels of entire national populations have not given serious consideration to the high rates of noncardiovascular disease and death associated with low cholesterol levels (less than 190 mg/dl). To explore this problem, the relationships between serum cholesterol levels, measured in 1965-1968 in 7478 Japanese American men in Hawaii, and subsequent total and cause-specific mortality through 1985, were analyzed by multivariate Cox regression to control for potential confounders. Total mortality rates for 1648 deaths showed a U-shaped curve by baseline cholesterol level, with significant inverse trends (p less than 0.03) for deaths due to hemorrhagic stroke, all cancer, benign liver disease, chronic obstructive lung disease and "unknown cause". Only the inverse trends for cancer and benign liver disease showed flattening when 227 deaths in the first 5 years of follow-up were deleted from the analysis. Simulation models using three different strategies of cholesterol reduction in this cohort revealed that none of these approaches had any substantial impact on predicted total mortality over 15 years. However, the population-based approach might theoretically increase mortality for 60% of the cohort with baseline cholesterol levels less than 225 mg/dl.

Purification and characterization of a proteolytic active fragment of DNA topoisomerase I from the brine shrimp Artemia franciscana (Crustacea Anostraca).

The ATP-independent type I topoisomerase from the crustacean Artemia franciscana was purified to near-homogeneity. Its activity was measured by an assay that uses the formation of an enzyme-cleaved DNA complex in the presence of the specific inhibitor camptothecin. The purification procedure is reported. Purified topoisomerase is a single-subunit enzyme with a molecular mass of 63 kDa. Immunoblot performed on the different steps of purification shows that the purified 63 kDa peptide is a proteolytic fragment of a protein with a molecular mass of 110 kDa. Similarly to the other purified eukaryotic topoisomerases, the crustacean enzyme does not require a bivalent cation for activity, but is stimulated in the presence of 10 mM-MgCl2; moreover, it can relax both negative and positive superhelical turns. The enzyme activity is strongly inhibited by the antitumour drug camptothecin. The enzyme inhibition is related to the stabilization of the cleavable complex between topoisomerase I and DNA.

Lipid and lipoprotein predictors of coronary heart disease in elderly men in the Honolulu Heart Program.

Data from the baseline and follow-up examinations of the Honolulu Heart Program (HHP) cohort of 8006 men of Japanese ancestry were used to examine several questions concerning the predictive role of lipids and lipoproteins for incident CHD. For the question "Do serum cholesterol levels measured in middle age predict incident CHD in elderly men 65 years old or older?" the answer was clearly yes. Multivariate relative risks and attributable risks for early and late onset of CHD were similar and statistically significant. For the question "Do serum cholesterol levels measured in the elderly predict subsequent CHD?" the answer again was clearly yes. Multivariate relative risks for elderly men were similar to those for middle-aged men, and attributable risks were consistently higher for elderly men. For the question "Do other lipid or lipoprotein levels measured in the elderly predict incident CHD better than serum cholesterol level?" the answer was no. Multivariate relative risks for low-density-lipoprotein (LDL) and non-high-density-lipoprotein cholesterol were similar to those for total cholesterol. HDL cholesterol was protective for incident CHD, but the patterns were not significant for the elderly. Serum triglyceride level was not a significant predictor of CHD for the elderly. The conclusion was that no matter at what age serum cholesterol was measured, it predicted subsequent CHD in the elderly men in this cohort.

Does cigarette smoking have an independent effect on coronary heart disease incidence in the elderly?

In order to evaluate the effects of cigarette smoking on coronary heart disease (CHD) in elderly persons in the Honolulu Heart Program, 1,394 men between ages 65 and 74 were followed during an average 12-year period for new cases of nonfatal myocardial infarction and fatal CHD. Incidence rates increased progressively in individuals classified at baseline as never, former, and current smokers, respectively. The absolute excess risk associated with cigarette smoking was nearly twice as high in elderly compared with middle-aged men.

Highly repetitive DNA sequence in parthenogenetic Artemia.

The study of the structural organization of the eukaryotic genome is one of the most important tools for disclosing the evolutionary relationships between species. Artemia (Crustacea, Phyllopoda) offers a very interesting model for speciation studies. The genus, distributed all over the world, comprises both bisexual sibling species and parthenogenetic populations, exhibiting different chromosome numbers (diploidy, polyploidy, and heteroploidy). Digestion of genomic DNA of the parthenogenetic Artemia sp. from Tsing-Tao (China) with the restriction enzymes Eco RI and Alu I reveals that a highly repetitive sequence of 133 bp is present. The Eco RI fragment has been cloned and characterized by genomic organization. The distribution of the Eco RI family of repeats was also studied in several bisexual and parthenogenetic Artemia populations and compared with an Alu I repetitive fragment previously identified in Artemia franciscana.

A binding protein (p82 protein) recognizes specifically the curved heterochromatic DNA in Artemia franciscana.

DNA bending has been suggested to play a role in the regulation of gene expression, initiation of DNA replication, site specific recombination and DNA packaging. In Artemia franciscana (Phillopoda anostraca) cells we have revealed that an AluI DNA family of repeats, 113-bp in length, is the major component of the constitutive heterochromatin found in the species. By analysis of cloned oligomeric (monomer to hexamer) heterochromatic fragments and electrophoretic experiments we verified that the repetitive DNA shows a stable curvature that confers a solenoidal geometry to the double helix. Using the cloned monomeric fragment, as molecular probe, we describe the detection in an A. franciscana cell extract of a protein of 82 kDa (p82) that preferentially binds to heterochromatic DNA. This protein, purified of the other DNA binding proteins present in the crude cell extract, shows a greater affinity with the tandem copies of the AluI DNA fragment than with the monomer sequence. The binding of p82 protein to heterochromatic DNA is also drastically reduced in the presence of the antibiotic distamycin A, suggesting a role of the DNA curvature in the formation of the nucleoproteic complex.