D2 dopamine receptor A1 allele in Alzheimer disease and aging.

BACKGROUND: The apolipoprotein E4 (APOE*4) allele is a major risk factor for the common forms of late-onset Alzheimer disease (AD), but does not account for all the genetic variation in late-onset AD; hence, other genetic markers must be examined. The D2 dopamine receptor (DRD2) A1 allele is associated with abnormal brain function and decreased DRD2s. These receptors are decreased in hippocampus and amygdala in AD, and allele frequencies may vary with age. OBJECTIVE: To study APOE and DRD2 genotypes in patients with AD and cognitively intact controls of varying ages. DESIGN: The DRD2 and APOE genotypes were examined in 832 unrelated white subjects, including 554 patients with AD (486 sporadic; 68 familial) and 278 controls. Logistic regressions tested A1 allele effects on disease status and age, and DRD2 linkage with AD was investigated in 60 families with late-onset AD. SETTING: University medical centers. SUBJECTS: Patients (mean +/- SD age, 74.6 +/- 8.1 years; range, 52-98 years) had probable AD, according to standard consensus diagnostic criteria; controls (mean +/- SD age, 69.2 +/- 8.6 years; range, 50-93 years) were cognitively intact. MAIN OUTCOME MEASURES: Disease status, age, and DRD2 linkage with AD. RESULTS: No association between the DRD2 and APOE alleles was found, and the presence of the A1 allele did not increase the risk for AD. There was also no evidence of linkage between DRD2 and AD. Age analyses, including both patients and controls, indicated a decrease in A1 allele frequency with age. CONCLUSIONS: The A1 allele does not contribute to AD risk, alone or in combination with the APOE*4 allele. The DRD2 A1 allele frequencies decrease with age in both patients and controls. Thus, studies of DRD2 disease association need to control for age.

No association between alpha 1-antichymotrypsin and familial Alzheimer's disease.

Alzheimer's disease (AD) is the most common mid to late age-of-onset neurodegenerative disorder. AD has a strong and complex genetic etiology, and multiple genes, acting independently and/or interacting, likely affect the risk of developing AD. Several genes involved with AD already have been described, but only the APOE gene on chromosome 19q has been shown to affect the risk of the most common form of AD, occurring with onset over the age of 65. Because a substantial portion of late-onset AD is not explained by APOE, other genes affecting late-onset AD likely occur. These could act either independently or perhaps interact with APOE. alpha 1-Antichymotrypsin (ACT) is a major component of the amyloid plaques found in the brains of AD patients and may play a role in the pathophysiology of AD. It has been proposed that a specific polymorphism within the ACT gene interacts with APOE to increase the risk of developing AD. Our results do not confirm this finding.

No association between very low density lipoprotein receptor (VLDL-R) and Alzheimer disease in American Caucasians.

The very low density lipoprotein receptor gene (VLDL-R) is a receptor for apolipoprotein-epsilon (APOE)-containing lipoproteins, and thus has been suggested as a possible risk factor for Alzheimer disease (AD). Recently, Okuizumi et al. [Nature Genet, II (1995) 207-209] reported an association between the 96 bp allele at the VLDL-R locus and AD in a Japanese population. The association resulted in a two-fold increase of risk that decreased with increasing age. We have examined this association in 316 Caucasian sporadic AD patients, comparing their findings to 160 Caucasian AD spouse controls. We also investigated 53 late-onset Caucasian AD families for association and linkage. Our data failed to confirm linkage and/or association to the VLDL-R locus. Stratification by age at onset or APOE genotype also failed to show significant results.

No genetic effect of alpha1-antichymotrypsin in Alzheimer disease.

Alzheimer disease (AD) is the most common neurodegenerative disorder for individuals over the age of 40. AD has a complex etiology, and it is likely that multiple genes, acting independently and/or interacting, affect the risk of developing AD. Several genes involved with AD have been described already, but only the APOE gene on chromosome 19q has been shown to affect the risk of the common late onset form of AD. alpha1-Antichymotrypsin (AACT) is a major component of the amyloid plaques found in the brains of AD patients, and an allele in its gene has been proposed to increase the risk of developing AD when also associated with the APOE-4 allele. We have examined the role of this AACT polymorphism in a large set of families and sporadic cases, and do not see any effect, either alone or in combination with the APOE-4 allele.

Physical activity and the risk of colorectal adenomas.

Physical activity has repeatedly been shown to protect against colorectal cancer. Since adenomas are precursors to most colorectal cancers, we examined the relation of physical activity to the risk for colorectal adenomas in a colonoscopy-based study of 200 adenoma cases and 384 adenoma-free controls. Physical activity was assessed by telephone using a modification of a validated questionnaire comprising 17 items concerning three activity dimensions: leisure, work, and sport. Dietary information was obtained with a validated semiquantitative food frequency questionnaire. We created indices for each dimension of physical activity, which we categorized by quartile. Using logistic regression analysis, we found that leisure activity protected women against colorectal adenomas. Women in the second through fourth quartiles were considerably less likely than those in the first quartile to develop adenomas [crude odds ratio = 0.48; 95% confidence interval (CI) = 0.28-0.81]. There was no protective effect of work activity among either men or women. Men who participated in no sport were at increased risk for adenomas (odds ratio = 1.68; 95% CI = 0.93-3.02). Physical activity appears to protect against both colorectal cancer and colorectal adenomas.

Comparison of two prognostic models predicting survival in patients with malignant melanoma.

Accurate predictions of prognosis are important in the clinical management of patients with malignant melanoma. Primary lesions from 55 patients with cutaneous melanoma, having 10 or more years of clinical follow-up, were evaluated by two models predicting patient survival. One model was simple and relied solely on tumor thickness. The other model was complex and considered stage of tumor progression, and six clinical and histological variables. Accuracy of the two models was determined by retrospective review of the medical record, and the predictive power of each model was compared by receiver operating characteristic (ROC) curve analysis. The area under the ROC curve (a measure of the "goodness" of the model) for the single variable model was 0.70 +/- 0.0775 standard error (SE), and the area under the ROC curve for the multiple variable model was 0.77 +/- 0.0779 SE. Although a modest improvement in predictive power is suggested for the multiple variable model, the SEs for the two models overlap, and the difference is not statistically significant. Further study using a larger database may be required to determine definitively if the multiple variable model significantly increases the ability to predict patient survival or death, and make better clinical decisions.

A truncated v-abl-derived tyrosine-specific tyrosine kinase expressed in Escherichia coli.

Several biochemical properties of a 43 kDa v-abl-encoded tyrosine-specific protein kinase (p43v-abl) expressed in Escherichia coli were examined. p43v-abl is a fragment of a 60 kDa v-abl-encoded precursor, p60v-abl, and could be generated by limited proteolysis of a purified p60v-abl with trypsin. Tryptic cleavage of p60v-abl was prevented in the presence of ATP. These results suggest that the catalytic kinase domain of v-abl-derived protein can be separated from other (regulatory) domains by limited proteolysis. p43v-abl readily phosphorylated tyrosine residues on several different protein and peptide substrates, including peptides containing only two amino acid residues. However, the local sequence of the tyrosine-containing peptide substrate significantly affected its rate of phosphorylation. Thus the primary structure and local conformation at the tyrosine acceptor site can play an important role in determining the substrate specificity of v-abl-derived kinase. Phosphorylation by p43v-abl requires Mn2+, Co2+ or Mg2+ and exhibits a strong preference for ATP as phosphate donor. Analogues of ATP and the thiol-reactive reagent N-ethylmaleimide inhibited p43v-abl kinase activity. Purified p43v-abl is intrinsically thermolabile (t1/2 = 5 min at 40 degrees C) and phosphorylates glycerol inefficiently (Km = 1.4 M).

Cytogenetics of an endometrial adenocarcinoma cell line and its implications.

Despite the fact that adenocarcinoma of the endometrium is currently the most common gynecologic malignancy in the United States, few chromosomal studies have been done to date characterizing this disease. HEC-1A, a cell line used by many laboratories as a reference cell line for endometrial carcinoma, has never been subjected to definitive karyotyping. For this reason, with the use of improved banding techniques, this has now been accomplished, and several consistent abnormalities have been identified. There was a marker chromosome formed from an insertion of 2q21, probably representing an insertion of the lacking chromosome 14. In addition, there was a translocation to the telomeric region of 1p; and trisomies of 3, 7, and 17. Many of these abnormalities are known to consistently be associated with other primary malignancies. In addition, the chromosomes in which trisomy is noted carry genes associated with epidermal growth factor and estrogen receptors, which also bear marked homology to known oncogenes. It would appear that further detailed studies of various grades and stages of endometrial carcinoma, as well as histologic types and "precursor lesions," may lead to an understanding of those chromosomal changes associated with disease initiation and progression.

Synthesis and evaluation of multisubstrate inhibitors of an oncogene-encoded tyrosine-specific protein kinase. 1.

The synthesis and testing of potential multisubstrate inhibitors of tyrosine-specific protein kinases are described. One of the substrates, ATP, was mimicked by the known kinase inhibitor 5'-[4-(fluorosulfonyl)benzoyl]adenosine, which was covalently linked via the sulfonyl moiety to tyrosine mimics. The resulting multisubstrate inhibitors were tested for their ability to inhibit the transfer of phosphate from ATP to a protein acceptor by p60v-abl, the tyrosine kinase encoded by the transforming gene (v-abl) of the Abelson murine leukemia virus (A-MuLV). Although the series of inhibitors displayed moderately potent activity (IC50 values as low as 19 microM), the absence of large effects produced by modification of the tyrosine mimic suggests that they do not behave as multisubstrate inhibitors but bind primarily through the adenosine moiety common to all the inhibitors. This interpretation is strengthened by the finding that the inhibitors lack specificity, inhibiting a serine kinase at comparable concentrations.

Synthesis and evaluation of multisubstrate inhibitors of an oncogene-encoded tyrosine-specific protein kinase. 2.

Tyrosine-specific protein kinases that transfer the terminal phosphate from ATP to protein acceptors are associated with certain transforming viruses and cell surface growth factor receptors. Here we describe the synthesis and testing of potential multisubstrate inhibitors of this class of enzymes. The inhibitors were prepared by covalent attachment of the terminal phosphate of ATP or its tetraphosphate analogue to tyrosine mimics. Testing against p60v-abl, the tyrosine kinase from the Abelson murine leukemia virus, showed that the series of inhibitors was moderately potent (IC50 values as low as 13 microM). However, structural modification of the tyrosine mimic, including replacement with a serine-like moiety, had little effect on potency. It is therefore concluded that the ATP moiety is largely responsible for binding and that the enzyme requires additional structural features for recognition of the tyrosine-containing substrate.

Characteristics of cell lines derived from normal and malignant endometrial tissue.

Five established cell lines of human endometrium, two of normal endometrium and three of proven tumorigenicity, have been compared in terms of morphology and chromosomal numbers. Each of the five cell lines was then analyzed using immunocytochemical techniques to show that the epithelial and stromal elements could be separately identified. Antibodies directed against cytokeratin and desmoplakins were used to identify epithelial elements and antibodies directed against fibronectin were used as a marker for stroma. These results were then confirmed using Western blot analysis. We conclude that cell lines of human endometrium in culture can be differentiated as being of epithelial or stromal origin. Cell lines derived from reportedly normal human endometrium exhibit a stromal phenotype with a normal karyotype, whereas cells of tumorigenic human endometrial cell lines exhibit an epithelial phenotype and abnormal karyologic characteristics.

Isolation and analysis of an Abelson murine leukemia virus-encoded tyrosine-specific kinase produced in Escherichia coli.

A segment of the coding sequence of the Abelson murine leukemia virus transforming gene (v-abl) has been inserted into a plasmid vector that allows its efficient and regulated expression in Escherichia coli. The product of the v-abl-derived coding sequence, designated p60v-abl, accumulated to a level of approximately 10% of total E. coli protein. A procedure is described for the isolation of p60v-abl from E. coli that yields about 50 micrograms of p60v-abl/g wet weight of E. coli. p60v-abl was capable of autophosphorylation and phosphorylating certain E. coli proteins specifically at tyrosine residues. The E. coli-expressed p60v-abl specifically phosphorylated tyrosine residues on casein and angiotensin II. The Km and Vmax values for ATP, casein, and angiotensin II in the p60v-abl kinase reaction have been determined and compared to values reported for other tyrosine-specific kinases. The expression system and isolation procedure described here permit the preparation of functional p60v-abl in quantities sufficient for detailed physical and biochemical characterization and examination of its biological action(s).

Formation of putrescine in rat liver.

Two pathways exist for the formation of putrescine in rat liver. Putrescine can be produced by the action of L-ornithine decarboxylase, an inducible enzyme which can be irreversibly inhibited by the drug, alpha-difluoromethylornithine. A method for quantitating the amount of active ornithine decarboxylase protein present in the liver under various conditions by measuring the binding of [5-14C]alpha-difluoromethylornithine is described. The results indicated that, even when maximally induced, less than 0.0002% of the liver cytosol protein is ornithine decarboxylase. A second pathway for the production of putrescine occurs in the liver by means of the acetylation of spermidine to N1-acetylspermidine and its oxidation to putrescine and N-acetyl-3-aminopropionaldehyde by polyamine oxidase. This pathway is controlled by the activity of spermidine N1-acetyltransferase which is induced by hepatotoxins. Both ornithine decarboxylase and spermidine N1-acetyltransferase turn over rapidly as indicated by the loss of activity in response to cycloheximide. Following treatment with either carbon tetrachloride or thioacetamide, changes in spermidine N1-acetyltransferase activity precede those in ornithine decarboxylase and experiments with appropriate inhibitors indicate that the early enhancement of hepatic putrescine levels if brought about by the acetylase/oxidase pathway. Subsequently, enhanced ornithine decarboxylase activity maintains the putrescine levels and restores the depleted spermidine content of the liver.