Statins cause intracellular accumulation of amyloid precursor protein, beta-secretase-cleaved fragments, and amyloid beta-peptide via an isoprenoid-dependent mechanism

The use of statins, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors that block the synthesis of mevalonate (and downstream products such as cholesterol and nonsterol isoprenoids), as a therapy for Alzheimer disease is currently the subject of intense debate. It has been reported that statins reduce the risk of developing the disorder, and a link between cholesterol and Alzheimer disease pathophysiology has been proposed. Moreover, experimental studies focusing on the cholesterol-dependent effects of statins have demonstrated a close association between cellular cholesterol levels and amyloid production. However, evidence suggests that statins are pleiotropic, and the potential cholesterol-independent effects of statins on amyloid precursor protein (APP) metabolism and amyloid beta-peptide (A beta) genesis are unknown. In this study, we developed a novel in vitro system that enabled the discrete analysis of cholesterol-dependent and -independent (i.e. isoprenoid-dependent) statin effects on APP cleavage and A beta formation. Given the recent interest in the role that intracellular A beta may play in Alzheimer disease, we analyzed statin effects on both secreted and cell-associated A beta. As reported previously, low cellular cholesterol levels favored the alpha-secretase pathway and decreased A beta secretion presumably within the endocytic pathway. In contrast, low isoprenoid levels resulted in the accumulation of APP, amyloidogenic fragments, and A beta likely within biosynthetic compartments. Importantly, low cholesterol and low isoprenoid levels appeared to have completely independent effects on APP metabolism and A beta formation. Although the implications of these effects for Alzheimer disease pathophysiology have yet to be investigated, to our knowledge, these results provide the first evidence that isoprenylation is involved in determining levels of intracellular A beta.     

Plasma levels of vitamin A and its protein vectors (RBP and PA), carotene, total cholesterol and cholesterol-HDL in patients with dysplasia of the uterine cervix

In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol were determined in 19 female subjects with cervical dysplasia and compared to those of the healthy female subjects described in our previous research. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion and HDL- and total cholesterol by enzymatic colorimetry. Plasma mean values of vitamin A and HDL-cholesterol were lower (P less than 0.05 and P less than 0.01, respectively) than in the control group. On the contrary total cholesterol was higher (P less than 0.01) in the patients than in the control group. Vitamin A plasma levels were significantly related (P less than 0.01) to RBP and PA. No significant statistical correlation was found between the severity of the dysplasia and vitamin A plasma levels.     

Plasma levels of vitamin A and its protein vectors (RBP and PA), carotene, total cholesterol and HDL-cholesterol in patients with carcinoma of the cervix

In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol of 40 subjects with carcinoma of cervix uteri were determined and compared to those of the healthy female subjects described in our previous research. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion, and HDL-and total cholesterol by enzymatic colorimetry. Plasma mean values of carotene, vitamin A, RBP and HDL-cholesterol were lower (P less than 0.01) than in the control group, and the same applies to the PA mean plasma level (P less than 0.05). On the contrary, total cholesterol mean value of the patients resulted to be higher (P less than 0.01) than in the control group. Vitamin A plasma levels were significantly related (P less than 0.01) to RBP and PA. No significant statistical correlation was found between the clinical stage and the histological grading and vitamin A plasma levels.     

Modulation of DNA modification (I-compound) levels in rat liver and kidney by dietary carbohydrate, protein, fat, vitamin, and mineral content.

I-compounds are DNA modifications detected by 32P-postlabeling that increase with age in rodents without known carcinogen exposure. Diet type (natural ingredient versus purified) greatly influences patterns and levels of I-compounds. To test the hypothesis that I-compound formation is affected, also, by dietary macro- and micronutrients, effects of carbohydrate, protein, fat, vitamin, and mineral content on rat liver and kidney I-compounds were determined. Female Sprague-Dawley rats were fed basic or modified AIN-76A purified diets for 3-6 months. High protein (HP) diet (50%, w/w) increased I-compound levels in liver but not kidney. High carbohydrate (HC) diet (78%) produced a significant increase in the polar as well as total I-compound levels in both tissues. High fat diets (20%) elicited significantly lower levels of liver I-compounds than HC, HP, and basic diets. There were few significant differences between high polyunsaturated (safflower oil) and saturated fat (lard) diet groups. No qualitative differences in I-compound profiles were observed in either tissue. In rats fed basic diet supplemented with vitamins and/or minerals, increased vitamin content reduced the levels of polar I-compounds in liver. No extra diet-induced adducts were observed; all effects were of a quantitative nature. These data provide direct evidence that nutrients significantly influence I-compound levels and support the hypothesis that normal metabolism of nutrients leads to the production of small amounts of DNA-reactive electrophiles. These observations suggest a novel mechanism where nutrient composition of the diet may play a role in development of neoplasia and other adverse health effects.     

Application of an allosteric model to describe the interactions among retinol binding protein 4, transthyretin, and small molecule retinol binding protein 4 ligands

Retinol binding protein 4 (RBP4) is a serum protein that serves as the major transport protein for retinol (vitamin A). Recent reports suggest that elevated levels of RBP4 are associated with insulin resistance and that insulin sensitivity may be improved by reducing serum RBP4 levels. This can be accomplished by administration of small molecules, such as fenretinide, that compete with retinol for binding to RBP4 and disrupt the protein-protein interaction between RBP4 and transthyretin (TTR), another serum protein that protects RBP4 from renal clearance. We developed a fluorescence resonance energy transfer (FRET) assay that measures the interaction between RBP4 and TTR and can be used to determine the binding affinities of RBP4 ligands. We present an allosteric model that describes the pharmacology of interaction among RBP4, TTR, retinol, and fenretinide, and we show data that support the model. We show that retinol increases the affinity of RBP4 for TTR by a factor of 4 and determine the affinity constants of fenretinide and retinyl acetate. The assay may be useful for characterizing small molecule ligands that bind to RBP4 and disrupt its interaction with TTR. In addition, such a model could be used to describe other protein-protein interactions that are modulated by small molecules.     

Protein protein interactions, evolutionary rate, abundance and age

Background

Does a relationship exist between a protein's evolutionary rate and its number of interactions? This relationship has been put forward many times, based on a biological premise that a highly interacting protein will be more restricted in its sequence changes. However, to date several studies have voiced conflicting views on the presence or absence of such a relationship.

Results

Here we perform a large scale study over multiple data sets in order to demonstrate that the major reason for conflict between previous studies is the use of different but overlapping datasets. We show that lack of correlation, between evolutionary rate and number of interactions in a data set is related to the error rate. We also demonstrate that the correlation is not an artifact of the underlying distributions of evolutionary distance and interactions and is therefore likely to be biologically relevant. Further to this, we consider the claim that the dependence is due to gene expression levels and find some supporting evidence. A strong and positive correlation between the number of interactions and the age of a protein is also observed and we show this relationship is independent of expression levels.

Conclusion

A correlation between number of interactions and evolutionary rate is observed but is dependent on the accuracy of the dataset being used. However it appears that the number of interactions a protein participates in depends more on the age of the protein than the rate at which it changes.     

Cholesteryl ester transfer protein gene haplotypes, plasma high-density lipoprotein levels and the risk of coronary heart disease

High-density lipoprotein cholesterol (HDL-C) is a known inverse predictor of coronary heart disease (CHD) and is thus a potential therapeutic target. Cholesteryl ester transfer protein (CETP) is a key protein in HDL-C metabolism such that elevated CETP activity is associated with lower HDL-C. Currently available HDL-C raising drugs are relatively ineffective and evidence suggesting the role of CETP in HDL-C levels has promoted the development of CETP inhibitors as potential therapeutic agents for CHD. We investigated three SNPs in the CETP gene in two cross-sectional community-based populations (n = 1,574 and 1,109) and a population of 556 CHD patients to determine if reduced CETP activity due to genetic variations in the CETP gene would increase HDL-C levels and reduce the risk of CHD. CETP genotypes and haplotypes were tested for association with lipid levels, CETP activity and risk of CHD. Multivariate analysis showed the common AAB2 haplotype defined by the G-2708A, C-629A and TaqIB polymorphisms, was consistently associated with reduced CETP activity and increased HDL-C levels. A mean increase in HDL-C levels of 0.16–0.24 mmol/l was observed in individuals with two copies of the AAB2 haplotype relative to non AAB2 carriers across all three populations (P < 0.001). A case-control study of males indicated no association between single SNPs or haplotypes and the risk of CHD. These results suggest that raising HDL-C via CETP inhibition may not alter risk of CHD. Randomized control trials are needed to determine whether CETP inhibition will in reality reduce risk of CHD by raising HDL-C. Pamela A. McCaskie and John P. Beilby contributed equally to this work.     

A comparison of ghrelin, glucose, alpha-amylase and protein levels in saliva from diabetics

During the past decade, many salivary parameters have been used to characterize disease states. Ghrelin (GAH) is recently-discovered peptide hormone secreted mainly from the stomach but also produced in a number of other tissues including salivary glands. The aim of this work was to examine the relationship between active (aGAH) and inactive (dGAH) ghrelin in the saliva and other salivary parameters in type II diabetic patients and healthy controls. Salivary parameters were assessed in a single measurement of unstimulated whole saliva from 20 obese and 20 non-obese type II diabetes patients, and in 22 healthy controls. Total protein and alpha-amylase were determined by colorimetric methods, and glucose by the glucose-oxidase method. Saliva aGAH and dGAH levels were measured using a commercial radioimmunoassay (RIA) kit. Salivary concentrations of aGAH and dGAH ghrelin were more markedly decreased in obese diabetic subjects than in the two other groups. Glucose and alpha-amylase levels were higher in diabetic subjects than in controls. Furthermore, there were correlations between GAH levels and BMI, and between GAH and blood pressure. However, there was no marked variability in saliva flow rates among the groups. These results indicate that measurement of salivary GAH and its relationship to other salivary parameters might help to provide insight into the role of ghrelin in diabetes.     

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.     

Critical roles for the COOH terminus of the Cu-ATPase ATP7B in protein stability, trans-Golgi network retention, copper sensing, and retrograde trafficking

ATP7A and ATP7B are copper-transporting P-type ATPases that are essential to eukaryotic copper homeostasis and must traffic between intracellular compartments to carry out their functions. Previously, we identified a nine-amino acid sequence (F37-E45) in the NH(2) terminus of ATP7B that is required to retain the protein in the Golgi when copper levels are low and target it apically in polarized hepatic cells when copper levels rise. To understand further the mechanisms regulating the intracellular dynamics of ATP7B, using multiple functional assays, we characterized the protein phenotypes of 10 engineered and Wilson disease-associated mutations in the ATP7B COOH terminus in polarized hepatic cells and fibroblasts. We also examined the behavior of a chimera between ATP7B and ATP7A. Our results clearly demonstrate the importance of the COOH terminus of ATP7B in the protein's copper-responsive apical trafficking. L1373 at the end of transmembrane domain 8 is required for protein stability and Golgi retention in low copper, the trileucine motif (L1454-L1456) is required for retrograde trafficking, and the COOH terminus of ATP7B exhibits a higher sensitivity to copper than does ATP7A. Importantly, our results demonstrating that four Wilson disease-associated missense mutations behaved in a wild-type manner in all our assays, together with current information in the literature, raise the possibility that several may not be disease-causing mutations.     

The effects of age,energy and protein intake on protein turnover and the expression of proteolysis-related genes in the broiler breeder hen

A study was conducted to determine the changes that occur to proteolysis and related genes due to age, protein, and energy intake in high-yield broiler breeder hens (Gallus gallus). Cobb 700 broiler breeders were randomly assigned to one of six diets in a 2 × 3 factorial fashion. Two levels of energy (390 and 450 kcal/day) and three levels of protein (22, 24, and 26 g CP/day) were utilized. Protein turnover was determined in the left pectoralis at 22, 26, 31 and 44 weeks. Relative mRNA expression of calpain 2 (CAPN2), proteasome C2 subunit (PSMA1), and F box protein 32 (FBXO32) were determined via RT-PCR at 20, 25, and 44 weeks. Contrasts indicate fractional synthesis rate (FSR) and FBXO32 increase to a maximum at 25–26 weeks and a decrease thereafter. A significant drop in PSMA1 and FBXO32 was observed between 25 and 44 weeks and matched the decrease observed in FBR. No differences were detected in the levels of fractional synthesis and degradation, or the expression of CAPN2, PSMA1, and FBXO32, due to protein or energy intake. In summary, protein turnover was upregulated during the transition into sexual maturity and decreased thereafter. The observed changes in degradation appeared to be mediated by the ubiquitin–proteasome pathway.     

Retinol-binding protein 4, visceral fat, and the metabolic syndrome: effects of weight loss

Retinol-binding protein 4 (RBP-4) has been reported to be associated with visceral-fat accumulation and parameters of the metabolic syndrome (MetS). In this study, we investigated the relationship between RBP-4, visceral fat, and the MetS during pronounced weight loss after bariatric surgery. Thirty-six subjects were examined before and 2 years after surgery. Abdominal-fat distribution was determined by ultrasound, metabolic parameters, and serum RBP-4 levels by standard methods. After surgery BMI decreased by 9.07 kg/m(2), visceral-fat diameter (VFD) decreased by 60.6%, and RBP-4 serum levels by 16.6%. Change of RBP-4 levels was associated with reductions of waist (r = 0.364, P = 0.037), waist-to-hip ratio (WHR) (r = 0.415, P = 0.016), and VFD (r = 0.425, P = 0.010). MetS, as defined by International Diabetes Federation (IDF), was present in 19 patients at baseline and in nine patients at follow-up. Change in RBP-4 levels was the best predictor for the diagnosis of MetS at follow-up. In the subgroup without MetS at baseline, the decrease in RBP-4 levels (-28.1% vs. -6.3%, P = 0.020) and reduction in VFD (-66.9% vs. -55.0%, P = 0.038) were significantly greater compared to the subgroup with MetS. We demonstrate a marked decrease of RBP-4 levels after bariatric surgery, which correlates with reduction in visceral-fat mass. Furthermore, the extent of changes in RBP-4 levels differs according to the severity of the MetS.     

Clinical association of metabolic syndrome,C‐reactive protein and testosterone levels with clinically significant prostate cancer

Recently, the influence that metabolic syndrome (MetS), hormonal alterations and inflammation might have on prostate cancer (PCa) risk has been a subject of controversial debate. Herein, we aimed to investigate the association between MetS‐components, C‐reactive protein (CRP) and testosterone levels, and the risk of clinically significant PCa (Sig‐PCa) at the time of prostate biopsy. For that, men scheduled for transrectal ultrasound guided biopsy of the prostate were studied. Clinical, laboratory parameters and criteria for MetS characterization just before the biopsy were collected. A total of 524 patients were analysed, being 195 (37.2%) subsequently diagnosed with PCa and 240 (45.8%) meet the diagnostic criteria for MetS. Among patients with PCa, MetS‐diagnosis was present in 94 (48.2%). Remarkably, a higher risk of Sig‐PCa was associated to MetS, greater number of MetS‐components and higher CRP levels (odds‐ratio: 1.83, 1.30 and 2.00, respectively; P < 0.05). Moreover, higher circulating CRP levels were also associated with a more aggressive Gleason score in PCa patients. Altogether, our data reveal a clear association between the presence of MetS, a greater number of MetS‐components or CRP levels >2.5 mg/L with an increased Sig‐PCa diagnosis and/or with aggressive features, suggesting that MetS and/or CRP levels might influence PCa pathophysiology.     

Correlation between calmodulin-like protein, phospholipids, and growth in glucose-grown Mycobacterium phlei.

In Mycobacterium phlei TMC 1548 supplementation of growth medium containing 2% v/v glycerol with glucose (up to 5% w/v) resulted in an increase in growth (yield of cells), in amount of total phospholipids, and in each of the individual phospholipids (cardiolipin, phosphatidylethanolamine, phosphatidylinositol and its mannosides, and phosphatidylglycerol). However, when the medium was supplemented with a higher concentration (7.5% w/v) of glucose, both growth and phospholipid levels decreased to near control values (2% v/v glycerol alone). Cyclic AMP levels, which decreased at all concentrations of glucose, had no relation to phospholipid content or growth. The presence of a protein that possesses the property of stimulating c-AMP phosphodiesterase activity was recently demonstrated in Mycobacterium smegmatis (Falah et al. 1988. FEMS Microbiol. Lett. 56: 89-93). In M. phlei the level of this calmodulin-like protein (assayed by radioimmunoassay) changed with different concentrations of glucose in the growth medium in a manner identical with that of phospholipids. We suggest that in mycobacteria (i) intracellular calmodulin-like protein levels are affected by glucose concentration in the growth medium and (ii) there is a positive correlation between the levels of calmodulin-like protein, total and individual phospholipids, and growth (yield of cells) in glucose-grown M. phlei.     

Evaluation of C-reactive protein, Haptoglobin and cardiac Troponin 1 levels in brachycephalic dogs with upper airway obstructive syndrome

ABSTRACT: BACKGROUND: Brachycephalic dogs have unique upper respiratory anatomy with abnormal breathing patterns similar to those in humans with obstructive sleep apnea syndrome (OSAS). The objective of this study was to evaluate the correlation between anatomical components, clinical signs and several biomarkers, used to determine systemic inflammation and myocardial damage (C-reactive protein, CRP; Haptoglobin, Hp; cardiac troponin I, cTnI), in dogs with brachycephalic upper airway obstructive syndrome (BAOS). RESULTS: Fifty brachycephalic dogs were included in the study and the following information was studied: signalment, clinical signs, thoracic radiographs, blood work, ECG, components of BAOS, and CRP, Hp and cTnI levels. A high proportion of dogs with BAOS (88%) had gastrointestinal signs. The prevalence of anatomic components of BAOS was: elongated soft palate (100%), stenotic nares (96%), everted laryngeal saccules (32%) and tracheal hypoplasia (29.1%). Increased serum levels of biomarkers were found in a variable proportion of dogs: 14% (7/50) had values of CRP > 20 mg/L, 22.9% (11/48) had values of Hp > 3 g/L and 47.8% (22/46) had levels of cTnI > 0.05 ng/dl. Dogs with everted laryngeal saccules had more severe respiratory signs (p<0.02) and higher values of CRP (p<0.044). No other statistical association between biomarkers levels and severity of clinical signs was found. CONCLUSIONS: According to the low percentage of patients with elevated levels of CRP and Hp, BAOS does not seem to cause an evident systemic inflammatory status. Some degree of myocardial damage may occur in dogs with BAOS that can be detected by cTnI concentration.     

Matrix GLA protein, a regulatory protein for bone morphogenetic protein-2.

Matrix GLA protein (MGP) has been identified as a calcification inhibitor in cartilage and vasculature. Part of this effect may be attributed to its influence on osteoinductive activity of bone morphogenetic protein-2 (BMP-2). To detect binding between MGP and BMP-2, we performed immunoprecipitation using MGP and BMP-2 tagged with FLAG and c-Myc. The results showed co-precipitation of BMP-2 with MGP. To quantify the effect of MGP on BMP-2 activity, we assayed for alkaline phosphatase activity and showed a dose-dependent effect. Low levels of MGP relative to BMP-2 (<1-fold excess) resulted in mild enhancement of osteoinduction, whereas intermediate levels (1-15-fold excess) resulted in strong inhibition. High levels of MGP (>15-fold excess), however, resulted in pronounced enhancement of the osteoinductive effect of BMP-2. Cross-linking studies showed that inhibitory levels of MGP abolished BMP-2 receptor binding. Immunoblotting showed a corresponding decrease in activation of Smad1, part of the BMP signaling system. Enhancing levels of MGP resulted in increased Smad1 activation. To determine the cellular localization of BMP-2 in the presence of MGP, binding assays were performed on whole cells and cell-synthesized matrix. Inhibitory levels of MGP yielded increased matrix binding of BMP-2, suggesting that MGP inhibits BMP-2 in part via matrix association. These results suggest that MGP is a BMP-2 regulatory protein.     

Effect of age on cholinesterase activity and protein of unfed larval ticks

The cholinesterase (AChE) activity and total protein in homogenates of unfed larvae of Amblyomma americanum (L.), A. cajennense (Fabricius), A. maculatum Koch, Anocentor nitens Neumann, and Boophilus microplus (Canestrini) were determined weekly in ticks 1-6 wk of age. There was a considerable variation in total protein, AChE, and the ratio of AChE to protein as the ticks aged. However, AChE activities were constant or increased with age and total protein levels were constant or decreased. No direct relationship was detected between AChE activity and total protein levels. The ratio of AChE to protein increased as the unfed larval ticks aged.