Study of the insulin signaling pathways in the regulation of ACAT1 expression in cultured macrophages

Objective

To determine the signaling pathways and components involved in insulin-mediated regulation of Acyl-CoA: cholesterol acyltransferase1 (ACAT1).

Methods

THP-1 cells were cultured in RPMI 1640 medium and were induced into macrophages in the presence of 160 nM phorbol 12-myristate 13-acetate (PMA). Before insulin was added in, macrophages were preincubated with the inhibitors of the insulin signaling pathway, including wortmannin, phosphatidylinositol 3-kinase (PI3 K) inhibitor; PD98059, extracellular signal-regulated kinase (ERK) inhibitor; SB203580, p38 mitogen-activated protein kinase (p38MAPK) inhibitor; SP600125, c-Jun N-terminal kinase (JNK) inhibitor and U73122, phospholipase C-γ (PLC-γ) inhibitor. ACAT1 mRNA and protein expression level in macrophages were determined by real-time quantitative polymerase chain reaction and western blotting, respectively.

Results

Real-time quantitative polymerase chain reaction and western blotting demonstrated that PD98059, SB203580 or SP600125 down-regulated the expression of ACAT1 in a dose-dependent manner. However, no obvious alteration was found in wortmannin and U73122 groups.

Conclusion

These results suggest that the ERK, p38MAPK and JNK signaling pathways may be involved in insulin-mediated regulation of ACAT1, but no PI3K and PLC-γ signaling pathways were involved in the present study.     

Localization of the Sodium-Taurocholate cotransporting polypeptide in membrane rafts and modulation of its activity by cholesterol in vitro

Background

The relevance of discrete localization of hepatobiliary transporters in specific membrane microdomains is not well known.

Aim

To determine whether the Na⁺/taurocholate cotransporting polypeptide (Ntcp), the main hepatic sinusoidal bile salt transporter, is localized in specific membrane microdomains.

Methods

Presence of Ntcp in membrane rafts obtained from mouse liver was studied by immunoblotting and immunofluorescence. HEK-293 cells stably transfected with rat Ntcp were used for in vitro studies. Expression, localization and function of Ntcp in these cells were assessed by immunoblotting, immunofluorescence and biotinylation studies and Na⁺-dependent taurocholate uptake assays, respectively. The effect of cholesterol depletion/repletion assays on Ntcp function was also investigated.

Results

Ntcp localized primarily to membrane rafts in in vivo studies and localized partially in membrane rafts in transfected HEK-293 cells. In these cells, membrane cholesterol depletion resulted in a shift of Ntcp localization into non-membrane rafts, which correlated with a 2.5-fold increase in taurocholate transport. Cholesterol repletion shifted back part of Ntcp into membrane rafts, and normalized taurocholate transport to values similar to control cells.

Conclusion

Ntcp localizes in membrane rafts and its localization and function are regulated by membrane cholesterol content. This may serve as a novel regulatory mechanism of bile salt transport in liver.     

MALDI-typing of infectious algae of the genus Prototheca using SOM portraits

Background

MALDI-typing has become a frequently used approach for the identification of microorganisms and recently also of invertebrates. Similarity-comparisons are usually based on single-spectral data. We apply self-organizing maps (SOM) to portray the MS-spectral data with individual resolution and to improve the typing of Prototheca algae by using meta-spectra representing prototypes of groups of similar-behaving single spectra.

Results

The MALDI-TOF peaklists of more than 300 algae extracts referring to five Prototheca species were transformed into colored mosaic images serving as molecular portraits of the individual samples. The portraits visualize the algae-specific distribution of high- and low-amplitude peaks in two dimensions. Species-specific pattern of MS intensities were readily discernable in terms of unique single spots of high amplitude MS-peaks which collect characteristic fingerprint spectra. The spot patterns allow the visual identification of groups of samples referring to different species, genotypes or isolates. The use of meta-peaks instead of single-peaks reduces the dimension of the data and leads to an increased discriminating power in downstream analysis.

Conclusions

We expect that our SOM portray method improves MS-based classifications and feature selection in upcoming applications of MALDI-typing based species identifications especially of closely related species.     

Large enhancement of skeletal muscle cell glucose uptake and suppression of hepatocyte glucose-6-phosphatase activity by weak uncouplers of oxidative phosphorylation

Background

Perturbation of energy homeostasis in skeletal muscle and liver resulting from a transient inhibition of mitochondrial energy transduction can produce effects of relevance for the control of hyperglycemia through activation of the AMP-activated protein kinase, as exemplified by the antidiabetic drug metformin. The present study focuses on uncoupling of oxidative phosphorylation rather than its inhibition as a trigger for such effects.

Methods

The reference weak uncoupler 2,4-dinitrophenol, fourteen naturally-occurring phenolic compounds identified as uncouplers in isolated rat liver mitochondria, and fourteen related compounds with little or no uncoupling activity were tested for enhancement of glucose uptake in differentiated C2C12 skeletal muscle cells following 18 h of treatment at 25-100 μM. A subset of compounds were tested for suppression of glucose-6-phosphatase (G6Pase) activity in H4IIE hepatocytes following 16 h at 12.5-25 μM. Metformin (400 μM) was used as a standard in both assays.

Results

Dinitrophenol and nine of eleven compounds that induced 50% or more uncoupling at 100 μM in isolated mitochondria enhanced basal glucose uptake by 53 to 269%; the effect of the 4′-hydroxychalcone butein was more than 6-fold that of metformin; negative control compounds increased uptake by no more than 25%. Dinitrophenol and four 4′-hydroxychalconoids also suppressed hepatocyte G6Pase as well as, or more effectively than metformin, whereas the unsubstituted parent compound chalcone, devoid of uncoupling activity, had no effect.

Conclusions

Activities key to glycemic control can be induced by a wide range of weak uncouplers, including compounds free of difficult-to-metabolize groups typically associated with uncouplers.

General significance

Uncoupling represents a valid and possibly more efficient alternative to inhibition for triggering cytoprotective effects of therapeutic relevance to insulin resistance in both muscle and liver. Identification of actives of natural origin and the insights into their structure-activity relationship reported herein may lead to alternatives to metformin.     

Changes in iron-regulatory gene expression occur in human cell culture models of Parkinson's disease

Background

Neuronal iron accumulation is thought to be relevant to the pathogenesis of Parkinson’s disease (PD), although the mechanism remains elusive. We hypothesized that neuronal iron uptake may be stimulated by functional mitochondrial iron deficiency.

Objective

To determine firstly whether the mitochondrial toxin, 1-methyl-4-phenylpyridinium iodide (MPP⁺), results in upregulation of iron-import proteins and transporters of iron into the mitochondria, and secondly whether similar changes in expression are induced by toxins with different mechanisms of action.

Methods

We used quantitative PCR and Western blotting to investigate expression of the iron importers, divalent metal transporter, transferrin receptor 1 and 2 (TfR1 and TfR2) and mitoferrin-2 and the iron exporter ferroportin in differentiated SH-SY5Y cells exposed to three different toxins relevant to PD, MPP⁺, paraquat (a free radical generator) and lactacystin (an inhibitor of the ubiquitin-proteasome system (UPS)).

Results

MPP⁺ resulted in increased mRNA and protein levels of genes involved in cellular iron import and transport into the mitochondria. Similar changes occurred following exposure to paraquat, another inducer of oxidative stress. Lactacystin also resulted in increased TfR1 mRNA levels, although the other changes were not found.

Conclusion

Our results support the hypothesis of a functional mitochondrial iron deficit driving neuronal iron uptake but also suggest that differences exist in neuronal iron handling induced by different toxins.     

Factores asociados a la polifarmacia en población anciana no institucionalizada. Análisis de la submuestra de la Encuesta Nacional de Salud 2006 para personas mayores de Castilla y León

Background

The increase in chronic and degenerative diseases in the elderly leads to increased and multiple drug usage, which in turn leads to problems associated with adverse reactions and drug interactions.

Material and methods

We analysed the subsample of the National Health Survey 2006, for adults over 65 living in Castile-León (n = 458). Using a logistic regression model and correlation analysis the variables having more influence on polypharmacy were evaluated.

Results

A total of 86% of those interviewed claimed to be taking drugs and 93.9% had a chronic illness. The most common health problems included arthrosis, arthritis or rheumatism (53.5%) and hypertension (48.3%), and most frequently used drugs were hypotensives (45%), pain medications (37.1%) and those for rheumatism (21.4%). Both the mean number of illnesses suffered and the drugs consumed are significantly higher in those who claimed to have, “or or fair health, used the health services, had impaired eyesight and hearing, dependent for personal care and domestic tasks, and mobility (P < .05). The variables associated with polypharmacy are three or more chronic diseases (OR = 18.3), regular-poor self perceived health (OR = 3.4) and females (OR = 1.9).

Conclusions

Given the magnitude of the problem it would be appropriate to include a review of the medications in health examinations of the elderly, particularly in women older than 75 years, with regular or self-perceived poor health and who have 3 or more diseases.     

Protective effect of tetramethylpyrazine isolated from Ligusticum chuanxiong on nephropathy in rats with streptozotocin-induced diabetes

Purpose

This study was designed to investigate the protective effect of tetramethylpyrazine isolated from Ligusticum chuanxiong, a traditional Chinese medicine, on diabetic nephropathy in a rat model, and to explore the possible mechanism involved in a protective function.

Materials

Diabetes was induced in male Sprague-Dawley rats by a single intraperitoneal injection of 70 mg/kg of streptozotocin. One week later, 200 mg/kg/day of tetramethylpyrazine was administered intragastric gavage daily for 8 weeks. Renal functions and expression of vascular endothelial growth factor were examined at 4 and 8 weeks after tetramethylpyrazine administration.

Results

Blood glucose and renal function were significantly improved in the tetramethylpyrazine-treated group compared to the untreated diabetic rats. Diabetic nephropathy resulted in an increase in the expression of vascular endothelial growth factor, while tetramethylpyrazine administration greatly decreased the expression.

Conclusions

Our results suggest that administration of tetramethylpyrazine may reduce kidney damage caused by diabetes. This protective effect may be mediated, in part, by downregulated expression of vascular endothelial growth factor in the kidney.     

Calidad de vida percibida por las personas mayores. Consecuencias de un programa de estimulación cognitiva a través de la motricidad «Memoria en movimiento»

Introduction

This article presents the results of evaluation of health related quality of life in a group of elderly people who were engaged in the program of cognitive stimulation «Memory in movement».

Materials and methods

A content analysis was conducted to determine the type of experience that the subjects stated in the context of an interview. The statements were classified into categories created ad hoc. The sample consisted of 32 participants with a mean age of 69.92 ± 4.98 years. The software QSR NVivo-8 was used for data processing.

Results

The results show that the 48% of the elderly perceive some improvements in their memorisation abilities, 47% in attention and concentration, and 10% in the satisfaction with life after being in the program. They are especially worried about the loss of memory -stated by the 65.62%- and they came to the cognitive stimulation program with the aim of improving this capacity. The feelings that participants reported having experienced during the program have to do with fun, vitality and energy.

Conclusions

Programs on cognitive stimulation that incorporate some physical activities, together with a clear playful and social component, improve the psychic and social capacities and they open an important line of intervention due to their good acceptance by the elderly.     

Relationships among the concentrations of 25 inflammation-associated proteins during the first postnatal weeks in the blood of infants born before the 28th week of gestation

Background

Inflammation appears to be involved in processes leading to organ damage in preterm newborns, yet little is known about the relationships among elevated concentrations of inflammation-associated proteins in the blood of preterm newborns.

Methods

In this exploratory study, we used an electrochemiluminescence method to measure 25 proteins in blood obtained on postnatal day 1 (range 1-3), day 7 (range 5-8), and day 14 (range 12-15) from 939 children born before the 28th week of gestation and evaluated to what extent those whose concentration of each protein was elevated (defined as in the highest quartile for gestational age and day the specimen was obtained) also had an elevated concentration of every other protein the same day or on a day 1 or 2 weeks later (p < .0001).

Results

On each of the 3 days assessed, elevated concentrations of 17 proteins were associated with elevated concentrations of 15 or more of the other 24 proteins. VEGF, VEGF-R1, VEGF-R2 were among these proteins, while IGFBP-1 was associated with 13 other proteins on day 7. An elevated concentration of eight proteins on day 1 predicted an elevated concentration of 10 or more proteins on day 7, while an elevated concentration of only two proteins on day 7 were associated with elevated concentrations of 10 or more proteins on day-14. Few associations were seen between days 1 and 14.

Conclusions/inferences

Inflammation is a diffuse process in ELGANs, with elevated concentrations of cytokines, chemokines, adhesion molecules, matrix metalloproteinases, a growth factor and its receptors, as well as a growth factor binding protein associated with each other the same day, as well as on subsequent days.     

Regulatory T cells (CD4CD25FoxP3) expansion in systemic sclerosis correlates with disease activity and severity

Background

The role and function of T regulatory (Treg) cells have not been fully investigated in patients with systemic sclerosis (SSc).

Methods

Ten patients with SSc donated 20 ml of peripheral blood. Activity (Valentini) and severity (Medsger) scores for SSc were calculated for all patients. Healthy volunteers (controls) were matched to each patient by gender and age. CD4⁺ cells were separated using the MACS system. The numbers of Treg cells were estimated by flow cytometry after staining for CD4, CD25, and FoxP3 and calculated as patient-to-control ratio separately for each experiment. Correlations with activity and severity indices of the disease were performed. Twenty-four-hour production of TGF-β and IL-10 by activated CD4⁺ cells was measured by ELISA in culture supernatants.

Results

The numbers of Treg cells, expressed as patient-to-control ratio, correlated significantly with both activity and severity indices (r = 0.71, p = 0.034 and r = 0.67, p = 0.044, respectively). ELISA-measured production of TGF-β and IL-10 by CD4⁺ cells was similar in patients and controls.

Conclusions

Increased numbers of Treg cells are present in patients with SSc, correlating with activity and severity of the disease. This expansion of Treg cells was not accompanied, however, by heightened TGF-β or IL-10 production. Further studies to elaborate the causes and functional significance of Treg cell expansion in SSc are needed.     

Application of novel peptide (Pp1) improving the half-life of exendin-4 in vivo

Aims

The multiple physiological characterizations of exendin-4 make it as a promising drug candidate for the therapy of type 2 diabetes. Although the longer biological half-life offered the exendin-4 with excellent therapeutic potentials for the clinical utility of type 2 diabetes than glucagon-like peptide-1, the exendin-4 still did not free from the inconveniently frequent injections. Therefore, there are increasing requirements for the long-acting exendin-4.

Methods

Pp1 regard as a novel exendin-4 protecting peptide, which are predicted to have the ability of increasing the stabilization of exendin-4 in vivo. Protecting peptide is able to form stable complex by non-covalent interaction with human exendin-4.

Results

In this study, the stability of the exendin-4/Pp1 complex was investigated, and the physiological functions of it were analyzed. Results indicated that exendin-4/Pp1 complex remarkably raised the stabilization of exendin-4 in vivo; it also showed better glucose tolerance and higher HbA_1c reduction than exendin-4 which was utilized chronically in rodents.

Conclusion

Based upon these results, it is suggested that an exendin-4/Pp1 complex might be utilized as a potent anti-diabetic drug in the treatment of type 2 diabetes mellitus.     

A comparison of the LDL-cholesterol lowering efficacy of plant stanols and plant sterols over a continuous dose range: results of a meta-analysis of randomized, placebo-controlled trials

Purpose

To determine if plant stanols and plant sterols differ with respect to their low-density lipoprotein cholesterol (LDL-CH) lowering efficacies across a continuous dose range.

Methods

Dose-response relationships were evaluated separately for plant stanols and plant sterols and reductions in LDL-CH, using a first-order elimination function.

Results

Altogether, 113 publications and 1 unpublished study report (representing 182 strata) complied with the pre-defined inclusion and exclusion criteria and were included in the assessment. The maximal LDL-CH reductions for plant stanols (16.4%) and plant stanol ester (17.1%) were significantly greater than the maximal LDL-CH reductions for plant sterols (8.3%) and plant sterol ester (8.4%). These findings persisted in several additional analyses.

Discussion and conclusions

Intakes of plant stanols in excess of the recommended 2 g/day dose are associated with additional and dose-dependent reductions in LDL-CH, possibly resulting in further reductions in the risk of coronary heart disease (CHD).     

La transición hacia el retiro: adaptación en una muestra de prejubilados españoles de la escala Retirement Satisfaction Inventory

Objectives

This study provides psychometric data for the Spanish adaptation of the scale of Retirement Satisfaction Inventory (RSI). We explore the factor structure and psychometric properties a sample of Spanish early retirees. RSI measures the motivation to retirement, satisfaction with life in retirement and leisure activities.

Material and methods

The RSI scale (Floyd et al, 1992) was applied to a sample of 638 Spanish early-pensioners with a mean age of 61.28 (SD: 5.77) years and a mean age at early retirement of 53.89 years (SD: 2.71). An exploratory factorial analysis was performed using the SPSS statistics package, and a confirmatory factorial using the AMOS 6.0 module. The data collection process was carried out by means of the questionnaires by post method.

Results

The adaptation of the RSI to Spanish showed a three-scale structure:: the reasons for retirement, satisfaction with life in retirement, and leisure activities. These scales showed a structure with four, two and three main factors, respectively, which differ slightly from those reported in the original instrument.The confirmatory factorial analysis demonstrated the optimum fit of the model for the three scales.

Conclusions

The RSI adaptation to Spanish provides a tool that could asses the motivation towards retirement during the leaving work process, satisfaction with retirement life, and the leisure sources in the Spanish early retired population.     

Evaluation of nitrate reduction assay, resazurin microtiter assay and microscopic observation drug susceptibility assay for first line antitubercular drug susceptibility testing of clinical isolates of M. tuberculosis

Background

Drug resistant tuberculosis (TB) is a growing concern worldwide. Early detection of multidrug-resistant Mycobacterium tuberculosis is of primary importance for both patient management and infection control. Optimal method for identifying drug-resistant M. tuberculosis in a timely and affordable way in resource-limited settings is not yet available.

Aim

This study evaluated; nitrate reductase assay (NRA), resazurin microtiter assay (REMA) and microscopic observation drug susceptibility assay (MODS) against the conventional 1% proportion method (PM) for the detection of resistance to first line antitubercular drugs, in M. tuberculosis clinical isolates.

Methods

A total of one hundred and five clinical isolates of M. tuberculosis; 50 pan sensitive and 55 pan resistant were tested with NRA, REMA and MODS. The 1% proportion method on Lowenstein-Jensen medium was used as reference test.

Results

Of all three methods which were tested NRA was found to be most sensitive and specific. Sensitivity for rifampicin resistance detection was 100%, 94.55% and 92.73% by NRA, REMA and MODS respectively. NRA and REMA were found to be 100% specific, while the MODS was 98% specific for detection of rifampicin resistance. Test results with all these methods were obtained within 8-14 days.

Conclusion

Rapid non-conventional and inexpensive methods may serve as a replacement for 1% proportion method in resource limited settings.     

Évaluation de méthodes automatiques de segmentation des volumes tumoraux en tomographie par émission de positons par comparaison avec des contours manuels réalisés par un groupe d’experts

Objective

Set up a framework for evaluating automatic segmentation methods of tumour volumes on PET images.

Patient and methods

This study was performed with PET images of 18 patients with non-Hodgkin's lymphoma. One target lesion per patient was pointed out. Each lesion was then three times manually delineated by five experts. Four automatic methods (the application of a threshold of 42% of the maximum SUV, the MIP-based method, the Daisne et al. method and the Nestle et al. method) were evaluated by comparison with the set of manual delineations.

Results

From the manual delineations, we have concluded to a moderate intra-operator variability and to a reduced interoperator reproducibility. From statistical tests performed on various quantitative criteria, there was no significant difference between the MIP-based method, the Daisne et al. method and the Nestle et al. one. The application of a threshold of 42% of the maximum SUV appears to be less efficient.

Conclusion

This work proposes a comparison and an evaluation protocol for segmentation methods. The generated data set will be distributed online for the community to simplify the evaluation of any new method of segmentation.