Midlife predictors of Alzheimer's disease.

Factors contributing to increased risk for Alzheimer's disease (AD) include age, sex, genes, and family history of AD. Several risk factors for AD are endogenous; however, accumulating evidence implicates modifiable risk factors in the pathogenesis of AD. Although the continued task of identifying new genes will be critical to learning more about the disease, several research findings suggest that potentially alterable environmental factors influence genetic contributions, providing targets for disease prevention and treatment. Here, we review midlife risk factors for AD, and address the potential for therapeutic intervention in midlife.

Acyl-coenzyme A: cholesterol acyltransferase modulates the generation of the amyloid beta-peptide.

The pathogenic event common to all forms of Alzheimer's disease is the abnormal accumulation of the amyloid beta-peptide (Abeta). Here we provide strong evidence that intracellular cholesterol compartmentation modulates the generation of Abeta. Using genetic, biochemical and metabolic approaches, we found that cholesteryl-ester levels are directly correlated with Abeta production. Acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyses the formation of cholesteryl esters, modulates the generation of Abeta through the tight control of the equilibrium between free cholesterol and cholesteryl esters. We also show that pharmacological inhibitors of ACAT, developed for the treatment of atherosclerosis, are potent modulators of Abeta generation, indicating their potential for use in the treatment of Alzheimer's disease.

Alzheimer disease: 100 years later.

Almost 100 years since the first clinical report of a case of Alzheimer disease (AD), three early-onset and two late-onset AD genes have been identified. While rare mutations in the early-onset genes (amyloid precursor protein, and presenilins 1 and 2) lead to increased generation of specific forms of the amyloid beta protein (A,beta), common polymorphisms in the late-onset genes (apolipoprotein E and alpha 2-macroglobulin) are thought to alter the clearance and degradation of A,beta in brain. Although definite proof for a direct link between altered A beta generation/clearance and neurodegeneration has not yet been attained, mechanism-based approaches for the therapeutic treatment of AD based on lowering levels of the potentially pathogenic A beta are currently underway. The recent discovery of the enzymes (secretases) responsible for generating A beta have paved the way for the development of such drugs and increase the prospects for successful therapeutic intervention to arrest AD neuropathogenesis.

Impairment of the Golgi GDP-L-fucose transport and unresponsiveness to fucose replacement therapy in LAD II patients.

Leukocyte adhesion deficiency type II is an autosomal recessive syndrome characterized by generalized reduction of L-fucose in glycoconjugates; the specific molecular defect is still undefined. The most important clinical symptoms include severe growth and mental retardation and severe immunodeficiency. Patients from two ethnic groups have been reported, i.e. Arab and Turkish. We have observed that GDP-L-fucose transport into Golgi vesicles was specifically impaired in an Arab patient, with a significant reduction of the V:(max) but no significant differences in the K:(m) from control and parents. GDP-L-fucose transport showed simple saturation kinetics in all samples. Transport of UDP-galactose, UDP-N:-acetylglucosamine, and CMP-sialic acid was comparable into vesicles from the Arab patient, parents, and control. These kinetic parameters probably account for the failure to obtain any clinical and biochemical response to fucose therapy in Arab patients. This contrasts both with the distinctive kinetic properties of GDP-L-fucose transport and with the success of fucose therapy, which have been recently reported in one patient of Turkish origin. Accordingly, the biochemical properties of GDP-L-fucose transport into the Golgi are consistent with different variants of leukocyte adhesion deficiency type II that are probably the result of different molecular defects.

Reconstitution, identification, and purification of the rat liver golgi membrane GDP-fucose transporter.

Glycosylation of glycoproteins, proteoglycans, and glycolipids occurring in the Golgi apparatus requires the translocation of nucleotide sugars from the cytosol into the lumen of the Golgi. Translocation is mediated by specific nucleotide sugar transporters, integral Golgi membrane proteins that regulate the above glycosylation reactions. A defect in GDP-fucose transport into the lumen of the Golgi apparatus has been recently identified in a patient affected by leukocyte adhesion deficiency type II syndrome (Lubke, T., Marquardt, T., von Figura, K., and Korner, C. (1999) J. Biol. Chem. 274, 25986-25989). We have now identified and purified the rat liver Golgi membrane GDP-fucose transporter, a protein with an apparent molecular mass of 39 kDa, by a combination of column chromatography, native functional size determination on a glycerol gradient, and photoaffinity labeling with 8-azidoguanosine-5'-[alpha-(32)P] triphosphate, an analog of GDP-fucose. The purified transporter appears to exist as a homodimer within the Golgi membrane. When reconstituted into phosphatidylcholine liposomes, it was active in GDP-fucose transport and was specifically photolabeled with 8-azidoguanosine-5'-[alpha-(32)P]triphosphate. Transport was also stimulated 2-3-fold after preloading proteoliposomes with GMP, the putative antiporter.

Identification, purification, and characterization of the rat liver golgi membrane ATP transporter.

Phosphorylation of secretory and integral membrane proteins and of proteoglycans also occurs in the lumen of the Golgi apparatus. ATP, the phosphate donor in these reactions, must first cross the Golgi membrane before it can serve as substrate. The existence of a specific ATP transporter in the Golgi membrane has been previously demonstrated in vitro using intact Golgi membrane vesicles from rat liver and mammary gland. We have now identified and purified the rat liver Golgi membrane ATP transporter. The transporter was purified to apparent homogeneity by a combination of conventional ion exchange, dye color, and affinity chromatography. An approximately 70,000-fold purification (2% yield) was achieved starting from crude rat liver Golgi membranes. A protein with an apparent molecular mass of 60 kDa was identified as the putative transporter by a combination of column chromatography, photoaffinity labeling with an analog of ATP, and native functional size determination on a glycerol gradient. The purified transporter appears to exist as a homodimer within the Golgi membrane, and when reconstituted into phosphatidylcholine liposomes, was active in ATP but not nucleotide sugar or adenosine 3'-phosphate 5'-phosphosulfate transport. The transport activity was saturable with an apparent Km very similar to that of intact Golgi vesicles.

Identification and purification of the rat liver Golgi membrane UDP-N-acetylgalactosamine transporter.

Glycosylation of glycoproteins, proteoglycans, and glycosphingolipids occurs mainly in the lumen of the endoplasmic reticulum and the Golgi apparatus. Nucleotide sugars, donors of all the sugars involved in Golgi glycosylation reactions, are synthesized in the cytoplasm and require specialized transporters to be translocated into the lumen of the Golgi apparatus. By controlling the supply of sugar nucleotides in the lumen of the Golgi apparatus, these transporters directly regulate the glycosylation of macromolecules transiting the Golgi. We have identified and purified the rat liver Golgi membrane UDP-N-acetylgalactosamine transporter. The transporter was purified to apparent homogeneity by a combination of conventional and dye color chromatography. An approximately 63,000-fold purification (6% yield) was achieved starting from crude rat liver Golgi membranes and resulting in a protein with an apparent molecular mass of 43 kDa. The transporter was active when reconstituted into phosphatidylcholine vesicles and could be specifically photolabeled with P3-(4-azidoanilido)-uridine-5'-[P1-32P]triphosphate, an analog of UDP-N-acetylgalactosamine. Native functional size determination on a glycerol gradient suggested that the transporter exists as a homodimer within the Golgi membrane.

Genetic epidemiology of cholesterol cholelithiasis among Chilean Hispanics, Amerindians, and Maoris.

BACKGROUND & AIMS: The etiology of cholesterol gallstones is multifactorial, with interactions of genes and the environment. The hypothesis that aborigine cholesterol lithogenic genes are widely spread among Chileans, a population with a high prevalence of gallstones, was tested. METHODS: Medical history and anthropometric measurements were obtained and abdominal ultrasonography was performed in 182 Mapuche Indians, 225 Maoris of Easter Island, and 1584 Hispanics. Blood groups, DNA, lipids, and glucose were analyzed. The Amerindian Admixture Index and mitochondrial DNA (mtDNA) assessed the ethnicity and degree of racial admixture. RESULTS: Amerindian Admixture Index was 0.8 in Mapuches and 0.4 in Hispanics. All Mapuches, 88% of Hispanics, but none of Maoris had Amerindian mtDNA haplotypes. Age- and sex-adjusted global prevalence of gallstone disease was higher in Mapuches (35%) than in Hispanics (27%) and Maoris (21%). Compared with Hispanics, the youngest group of Mapuches had the greatest corrected risk of gallstones: odds ratios of 6.0 in women and 2.3 in men. In contrast, the gallstone risk in Maoris was lower compared with Hispanics: odds ratios of 0.6 for women and 0.5 for men. CONCLUSIONS: Cholesterol lithogenic genes appear widely spread among Chilean Indians and Hispanics. They could determine the early formation of gallstones and explain the high prevalence of gallbladder diseases among some South American populations.

Uptake of dodecanedioic acid by isolated rat liver.

The uptake of dodecanedioic acid (C12); a dicarboxylic acid with 12 carbon atoms, was studied in the isolated perfused rat liver. Fifty mumol of C12 were injected as a bolus into the perfusing liver solution. The concentration of C12 in perfusate samples taken over 2 h from the beginning of the experiments were analyzed by high performance liquid chromatography. An in vitro experimental session was performed to determine the binding curve of C12 to defatted bovine serum albumin. These data were then used to compute the perfusate C12 free fraction. The number of binding sites on the albumin molecule was equal to 4.29 +/- 0.21 (S.E.), while the affinity constant was 6.33 +/- 0.87 x 10(3). M-1. Experimental values of perfusate C12 concentration versus time were individually plotted and fitted to a monoexponential decay for each liver perfused. The predicted C12 concentration at time zero averaged 0.354 +/- 0.0375 mumol/ml. Prom this value the apparent volume of distribution of C12 was obtained and corresponded to 153.02 +/- 14.56 ml. The disappearance rate constant from the perfusate was 0.0278 +/- 0.0030 min-1. The C12 half life was 26.6 +/- 2.3 min. The mean hepatic clearance from the perfusate was 4.08 +/- 0.38 ml/min. In conclusion, C12 is quickly taken up by the liver so that in about 100 min it was completely cleared from the perfusate.

Modulation of intrahepatic cholesterol trafficking: evidence by in vivo antisense treatment for the involvement of sterol carrier protein-2 in newly synthesized cholesterol transport into rat bile.

Biliary cholesterol represents one of the two major excretory pathways for sterol elimination from the body and plays a central role in cholesterol gallstone formation. Biliary cholesterol originates from a precursor pool of preformed and newly synthesized free cholesterol. Although it has been suggested that newly synthesized and preformed biliary cholesterol are secreted by independent pathways, the specific cellular and molecular mechanisms are unknown. We used male Wistar rats to study the time-course of the appearance of newly synthesized cholesterol, phosphatidylcholine and protein into bile. The specific role of sterol carrier protein-2 (SCP-2) in the transport of newly synthesized biliary cholesterol was evaluated by an in vivo antisense oligonucleotide approach. In contrast to [14C]phosphatidylcholine and [35S]proteins, the time-course of [14C]cholesterol appearance into bile was rapid, and microtubule- and Golgi-independent. In vivo SCP-2 antisense treatment reduced and delayed the appearance of biliary [14C]cholesterol. Furthermore, hepatic SCP-2 expression increased more than 3-fold over control values in rats that had been treated with diosgenin to increase biliary secretion of newly synthesized cholesterol. These results suggest that SCP-2 is necessary for the rapid transport of newly synthesized cholesterol into bile and that hepatocytes can induce SCP-2 expression according to the rate of biliary secretion of newly synthesized cholesterol.

Biliary aminopeptidase-N and the cholesterol crystallisation defect in cholelithiasis.

Several biliary proteins have cholesterol crystallisation promoting activity. One of these glycoproteins is aminopeptidase-N, a canalicular ectoenzyme. This study attempted to localise aminopeptidase-N along the biliary tree, to assess its concentration in a series of 98 patients subjected to abdominal surgery, 40 of them without gall stones, and to correlate its concentration with cholesterol crystal formation time of gall bladder bile. Aminopeptidase-N was isolated from purified native biliary vesicles. A specific polyclonal rabbit anti-aminopeptidase-N antibody was prepared for quantitative immunoblotting and for immunolocalisation. Tissue was obtained from liver biopsy specimens and from gall bladders removed at surgery because of gall stone disease. Aminopeptidase-N was immunolocalised to the apical membranes of hepatocytes and to the apical pole of ductular and gall bladder mucosal cells. The nucleation time of gall bladder bile was mean (SD) 4 (3) days in the gall stone group, compared with 21 (18) days in the control group (p < 0.001). Total absolute biliary protein and aminopeptidase-N concentrations were similar in both the control and gall stone patients. There was a reciprocal significant correlation, however, between the nucleation time and the relative aminopeptidase-N concentration (r = -0.35, p < 0.01) only in the gall stone group of patients. This study shows that this apical transmembrane ectoenzyme with cholesterol crystallisation promoting activity is present along the biliary tree and the hepatocyte. These findings support the concept that high concentrations or qualitative changes of biliary aminopeptidase-N contribute to cholesterol gall stone formation.

Sterol carrier protein-2 is involved in cholesterol transfer from the endoplasmic reticulum to the plasma membrane in human fibroblasts.

The cellular mechanism of cholesterol transport from the endoplasmic reticulum to the plasma membrane is currently unknown. To assess the possibility that sterol carrier protein-2 (SCP-2) is involved in this transport, we studied the time course of newly synthesized cholesterol incorporation in the plasma membrane of normal and SCP-2-deficient (Zellweger syndrome) human fibroblasts. Cholesterol transfer was rapid, cytoskeleton-independent, and Golgi-independent in normal cells, but it was slower, cytoskeleton-dependent, and Golgi-dependent in SCP-2-deficient cells. After SCP-2 antisense oligonucleotides treatment of normal fibroblasts, the rapid transport was reduced by 81% with a simultaneous increase of the slower one. These results suggest that in normal fibroblasts the major fraction of newly synthesized cholesterol is transported to the plasma membrane by a SCP-2-dependent mechanism. In contrast, in SCP-2-deficient cells, newly synthesized cholesterol leaves the endoplasmic reticulum by a cytoskeleton/Golgi-dependent mechanism.

Protective role of biliary cholesterol and phospholipid lamellae against bile acid-induced cell damage.

BACKGROUND/AIMS: Bile salts (BS) are cytotoxic agents, but cell damage is not observed in the hepatobiliary system. We hypothesized that biliary lipid vesicles (unilamellae and multilamellae) could have a protective role against BS-induced cytotoxicity. METHODS: Biliary lipid lamellar secretion was induced by feeding rats with 0.5% diosgenin. Cytoprotection was assessed in bile duct-obstructed rats and by incubating human erythrocytes with sodium taurocholate. RESULTS: Biliary cholesterol concentration increased > 300% in diosgenin-fed rats; electron microscopic examination showed a great abundance of lipid lamellar vesicles in bile and within the canaliculi. After bile duct obstruction, serum hepatic enzyme activities were significantly lower in diosgenin-fed rats. Histologically severe and confluent hepatocellular necrosis was only observed in control rats. Biliary lamellar lipid material significantly reduced the BS-induced hemolytic effect in vitro in a concentration-dependent manner. This protective effect correlated to a progressive decrease in the intermicellar BS concentration. Phosphatidylcholine or cholesterol, alone or as lamellar structures, also showed cytoprotective effect in vitro but always less than native biliary lamellae. CONCLUSIONS: These results support the concept that native biliary cholesterol phospholipid lamellae represent an important cytoprotective factor for hepatocytes and biliary epithelial cells against BS-induced damage.

Biliary lipid secretion: immunolocalization and identification of a protein associated with lamellar cholesterol carriers in supersaturated rat and human bile.

Feeding a 0.5% diosgenin plus 0.02% simvastatin diet to rats increases biliary cholesterol concentration and saturation to levels generally found in human native supersaturated bile. By using preparative ultracentrifugation, gel filtration chromatography, and electron microscopy, we isolated, purified, and identified lamellar structures (unilamellar vesicles and multilamellae) as a major biliary cholesterol transport in supersaturated human and rat bile. It was estimated that more than 60% of biliary cholesterol is transported in these lamellar carriers, which were identified by transmission electron microscopy as unilamellar vesicles and multilamellar bodies within bile canaliculi of rats with cholesterol supersaturated bile. By SDS-PAGE, a characteristic and constant protein profile was found associated to the purified lamellar carriers. One of these proteins, a 130-kDa protein, was isolated from human biliary lamellae and used for preparation of a rabbit polyclonal antibody, which cross-reacted with the homologous rat protein. By Western blotting, it was established that the purified low density fraction of bile-Metrizamide gradients, containing lamellae, was enriched with the 130-kDa protein. The 130-kDa protein was characteristically detected at the canalicular membrane by Western blotting of hepatic subcellular fractions and by immunohistochemistry of rat and human liver biopsies. Amino acid sequencing of the amino terminus of the 130-kDa protein demonstrated a complete identity with aminopeptidase N, a canalicular transmembrane hydrophobic glycoprotein. These studies show that biliary lipids may acquire an ordered multilamellar structure that is present in the canaliculi of rats with supersaturated bile. These biliary lamellae are similar to lamellar bodies and surfactant-like material frequently found in other epithelia, suggesting common biogenetic, structural, and functional properties. The identification of aminopeptidase N associated with biliary lamellae is consistent with the involvement of the canalicular membrane in the secretory mechanism of biliary lipids.

Cholesterol crystallization-promoting activity of aminopeptidase-N isolated from the vesicular carrier of biliary lipids.

Different hydrophobic glycoproteins are associated to native biliary vesicles, which are the major carrier of biliary cholesterol. Some of these proteins promote cholesterol crystallization, a key step in cholesterol gallstone formation. This study was specifically conducted to identify the 130 kDa biliary vesicle-associated glycoprotein and to determine its in vitro effect on the cholesterol crystal formation time. The 130 kDa vesicular glycoprotein was identified as aminopeptidase-N by amino acid sequencing and specific enzymatic assay. Polyclonal antibodies raised against aminopeptidase-N allowed us to determine its concentration in human hepatic bile, which varied from 17.3 to 57.6 micrograms/ml. Aminopeptidase-N showed a concentration-dependent cholesterol crystallization activity when it was added to supersaturated model bile at a concentration range usually found in native bile. Because of this promoting effect on in vitro cholesterol crystal formation, we suggest that biliary aminopeptidase-N may play a critical role in the pathogenesis of cholesterol gallstone disease.

[Verification of the prognostic significance of a left-ventricular aneurysm after a first myocardial infarct]. / Verifica del significato prognostico dell'aneurisma del ventricolo sinistro dopo un primo infarto miocardico.

The prognostic implication of a left ventricular aneurysm after a first myocardial infarction has been assessed on a series of 64 patients (mean age 65 +/- 10 years; 55 males and 9 females) having a diagnosis of a left ventricular aneurysms made by the equilibrium gated radionuclide angiocardiography. The control group was composed by 80 patients (mean age 63 +/- 10 years; 65 males and 15 females) with first myocardial infarction and comparable clinical characteristics but without left ventricular aneurysm. Aneurysm was defined as a ventricular segment in phase with atria in the phase parametric imaging. A left ventricular ejection fraction less than 52% was diagnosed in 83% and in 49% of the patients with and without aneurysm respectively (p less than 0.0005). The study group also showed a higher use of digoxin (39% vs 21%; p less than 0.05) and a higher prevalence of ventricular arrhythmias (31% vs 12%; p less than 0.05). After 36 months, mortality was 34% and 17% in patients with and without left ventricular aneurysm, respectively (p less than 0.05). According to the logistic regression analysis, mortality was predicted by a left ventricular ejection fraction less than 52% (odd ratio = 1.91; confidence limits = 1.03-3.48) while neither left ventricular aneurysm nor any of the remaining variables (age, sex, site of the myocardial infarction, peak filling rate, congestive heart failure, ventricular arrhythmias and their Lown class) could affect survival. In conclusion, a left ventricular aneurysm has no prognostic implication after a first myocardial infarction provided that the patients are stratified for the left ventricular ejection fraction.

[Goiter recurrences after surgical resection]. / Le recidive del gozzo dopo resezione chirurgica.

A personal experience about 135 pz. with non-toxic goitre treated by mono o bilateral thyroidectomy and followed during 5 years (2-8) to evaluate the incidence of recurrences is reported. The recurrences were 10.5%, especially after monolateral thyroidectomy (9 pz.). Therefore the authors stress the importance of a more accurate surgical indication, a wider resection of the gland and a careful follow-up to undertake opotherapy when hypofunction appears.

Right ventricular aneurysm: a new prognostic indicator after a first acute myocardial infarction.

The prognostic implication of a right ventricular aneurysm after a first acute myocardial infarction (AMI) was assessed on a series of 137 AMI patients 12 of whom had a right ventricular aneurysm detected at radionuclide angiocardiography. The follow-up lasted 36 months. Mortality was 50 and 18.4% in patients with and without right ventricular aneurysm, respectively (p less than 0.02). Groups did not differ in age, male-to-female ratio, AMI site, left ventricular ejection fraction (LVEF), peak filling rate (PFR), left ventricular size. A multivariate logistic analysis showed that only three out of ten clinical and functional variables qualified to be independent predictors of death: right ventricular aneurysm (odd ratio = 2.48, confidence limits = 1.21-4.98), LVEF less than 52% (odd ratio = 1.91, confidence limits = 1.03-3.48), abnormal terminal P wave forces (odd ratio = 1.72, confidence limits = 1.07-2.75). The analysis of single case histories did not provide a clue to clarify the reasons accounting for the negative prognostic implication of a right ventricular aneurysm. In conclusion, a significant positive relationship between right ventricular aneurysm and mortality after AMI has been demonstrated; further study is needed to clarify the relevant mechanisms.