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.     

Phosphorylation of the arginine/serine repeats of lamin B receptor by SRPK1—Insights from molecular dynamics simulations

Background

Arginine/serine (RS) repeats are found in several proteins in metazoans with a wide variety of functions, many of which are regulated by SR protein kinase 1 (SRPK1)-mediated phosphorylation. Lamin B receptor (LBR) is such a protein implicated in chromatin anchorage to the nuclear envelope.

Methods

Molecular dynamics simulations were used to investigate the conformation of two LBR peptides containing four (human-) and five (turkey-orthologue) consecutive RS dipeptides, in their unphosphorylated and phosphorylated forms and of a conserved peptide, in isolation and in complex with SRPK1. GST pull-down assays were employed to study LBR interactions.

Results

Unphosphorylated RS repeats adopt short, transient helical conformations, whereas serine phosphorylation induces Arginine-claw-like structures. The SRSRSRSPGR peptide, overlapping with the LBR RS repeats, docks into the known, acidic docking groove of SRPK1, in an extended conformation. Phosphorylation by SRPK1 is necessary for the association of LBR with histone H3.

Conclusions

The C-terminal region of the LBR RS domain constitutes a recognition platform for SRPK1, which uses the same recognition mechanism for LBR as for substrates with long RS domains. This docking may promote unfolding of the RS repeats destined to be phosphorylated. Phosphorylation induces Arginine-claw-like conformations, irrespective of the RS-repeat length, that may facilitate interactions with basic partners.

General significance

Our results shed light on the conformational preferences of an important class of repeats before and after their phosphorylation and support the idea that even short RS domains may be constituents of recognition platforms for SRPK1, thus adding to knowledge towards a full understanding of their phosphorylation mechanism.     

Eficiencia de las unidades geriátricas de agudos: metaanálisis de estudios controlados

Objective

After analysing the effectiveness in the reduction in the incidence of functional impairment and a higher probability of returning home between elderly patients hospitalised due to an acute medical illness cared for in acute geriatric units (AGU) compared to conventional care units, we propose to assess the efficiency of this care.

Material and methods

A systematic review and meta-analysis was made of controlled studies (randomised, no randomised and case-control) that compared care in UGA with care in conventional hospital units of patients of 65 years and over with an acute medical illness. Studies on administrative data bases, those that evaluated care of a single disease, and those that assessed units with care in the acute and sub-acute phase were excluded. A literature review was performed on articles published up to 31st of August 2008 in Medline, Embase, Cochrane Library, and references of systematic reviews and reviewed articles. The selection of the studies and the extraction of data on the hospital stay and care costs was made independently by two different researchers.

Results

A total of 11 studies were included, of which 5 were randomised, 4 were non-randomised, and 2 case control, all of them providing data on hospital stay, with 7 of them providing data on hospital costs (4 clinical trials, 2 non-randomised and 1 case-control). The overall analysis of all the studies showed that those admitted to UGA had a statistically significant reduction in hospital length of stay compared to the elderly hospitalised in conventional units (mean difference -1.01 days; 95% CI, -1.66 to -0.36) and hospital care costs (mean difference of -330 US dollars; 95% CI, -540 to -120).

Conclusions

Care in AGU is more efficient than that provided in conventional units, since, as well as achieving a reduction in the incidence of functional impairment at discharge and increasing the probability of returning home, they reduce mean hospital stay and the hospital care costs.     

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).     

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.     

Tissue-specific knockout of TSHr in white adipose tissue increases adipocyte size and decreases TSH-induced lipolysis

Background

The primary function of TSH is to activate TSH receptors (TSHr) in the thyroid gland and thereby stimulate thyroid hormone synthesis and secretion. TSHr are also expressed in other organs, but their physiological importance is still unclear. We have previously shown that TSHr, expressed in adipocytes, are of potential importance for lipolysis and extrauterine adaptation of the neonate.

Methodology

To further study the role of TSHr in adipocytes we selectively removed the TSHr gene in mice adipocytes by using the Cre-loxP recombination system (B6.Cg-Tg (Fabp4-Cre) 1Rev/J. TSHr knockout (KO) newborn mice were phenotypically characterized. Isolated adipocytes from 8-week-old male mice were studied in term of adipocyte size and metabolism.

Results

Mice lacking TSHr in adipocytes were apparently normal at birth and no differences in thyroid gland function or histology were observed. Sensitivity to TSH-induced lipolysis was ten times lower in adipocytes from targeted animals compared to wild-type. This indicates that adipocytes from targeted animals are refractory to stimulation of physiological concentrations of TSH. Catecholamine-induced lipolysis and insulin-induced inhibition of lipolysis were unaltered. Adipocyte size was increased in the targeted animals. Basal lipolysis was increased as an effect of the increased adipocyte size.

Conclusion

Our results indicate that adipocyte TSHr under normal conditions affects adipocyte growth and development.     

Increased ribosomal biogenesis induces pancreatic β cell failure in mice model of type 2 diabetes

Aim

To study the changes in gene expression by pancreatic β cells under insulin resistance conditions.

Method

An exhaustive gene expression analysis was performed, using isolated pancreatic islets of obese diabetic model Lepr^−/− mice. Overexpression of cyclin D2 was induced in cells from the pancreatic β cell line, namely, INS-1.

Results

Through a gene expression analysis using islets isolated from db/db mice, we found a significant increase in the expression of ribosome-related molecules. In addition, increased expression of cyclin D2 was found at certain protein levels. As INS-1 cells were induced to overexpress cyclin D2, we found an increase in the expression of ribosome-related molecules. Concurrently, an increase in the expression of endoplasmic reticulum stress (ER stress)-related molecules was also found.

Conclusion

In cases of pancreatic β cell hyperplasia associated with insulin resistance, ribosomal biogenesis is increased, and ER stress is induced.     

Glutathione-S-transferase M1 and T1 genes and gastric cancer: a case control study in North Indian population

Background

Difference in the capacity of xenobiotic metabolising enzymes might be an important factor in genetic susceptibility to cancer.

Methods

A case control study involving forty one gastric cancer patients and one hundred and thirty controls was carried out to determine the frequency of GSTM1 and GSTT1 null genotypes. The frequency of GSTM1 and GSTT1 null genotype was observed by carrying out multiplex PCR.

Results

There was no difference in the frequencies of the GSTM1 and GSTT1 null and the combined GSTM1 and GSTT1 null genotype between patients and control.

Conclusions

Our data suggest that GSTM1 and GSTT1 status may not influence the risk of developing gastric cancer.     

Oxidized low density lipoprotein impairs endothelial progenitor cell function by downregulation of E-selectin and integrin alpha(v)beta5

Background

Oxidized low density lipoprotein (oxLDL) has been shown to induce apoptosis and senescence of endothelial progenitor cells (EPC). In the present study, we hypothesized that even sub-apoptotic concentrations of oxLDL impair the angiogenic potential of EPC and investigated if this effect is mediated by affecting adhesion and incorporation.

Methods

A co-culture system of human microvascular endothelial cells and EPC was used to study the effect of sub-apoptotic concentrations of native (nLDL) and oxLDL on cell-cell interaction. The expression and the functional role of angiogenic adhesion molecules and integrins was monitored by FACS and neutralizing assay, respectively.

Results

We observed an inhibition of tube formation and impairment of EPC integration into the vascular network of mature endothelial cells by oxLDL. In contrast, nLDL did not affect angiogenic properties of EPC. Incubation of EPC with sub-apoptotic oxLDL concentrations significantly decreased E-selectin and integrin α_vβ₅ expression (37.6% positive events vs. 71.5% and 24.3% vs. 49.9% compared to control culture media without oxLDL). Interestingly, expression of α_vβ₃, VE-cadherin and CD31 remained unchanged. Blocking of E-selectin and integrin α_vβ₅ by neutralizing antibody effectively inhibited adhesion of EPC to differentiated endothelial cells (56.5% and 41.9% of control; p < 0.001).

Conclusion

In conclusion, oxidative alteration of LDL impairs angiogenic properties of EPC at sub-apoptotic levels by downregulation of E-selectin and integrin α_vβ₅, both substantial mediators of EPC-endothelial cell interaction.     

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.     

The challenge of continuous exogenous glucocorticoid administration in mice

Background

Chronic administration of exogenous glucocorticoids is often required in experimental research. We compared the efficacy and reliability of three different methods of continuous glucocorticoid administration in mice.

Materials and methods

Male CD1 Swiss White mice aged 7-9 weeks received corticosterone (CS) or carrier by either subcutaneous (s.c.) injection (n = 15), s.c. implantation of micro-osmotic pumps (n = 20) or s.c. implantation of slow-release pellets (n = 20). Serial blood samples were taken for the measurement of plasma CS and osteocalcin (OC). Bone structural parameters were analysed by micro-computed tomography (μ-CT) in animals treated via slow-release pellets for 4 weeks.

Results

Injection of CS (10 mg/kg) resulted in peak plasma CS levels of up to 2600 μg/L after 1 h, with levels returning to baseline within 4 h post-injection. Micro-osmotic pumps failed to consistently alter plasma CS levels and had variable effects on plasma OC levels. Implantation of 10 mg CS pellets induced hypercorticosteronemia within 24 h but levels returned to baseline within 7 days. Plasma OC levels fell rapidly on day 1 and remained suppressed until day 7. Weekly replacement of pellets maintained elevated plasma CS and suppressed plasma OC concentrations, and resulted in significant bone loss at the tibia and spine after 28 days.

Conclusion

Once-weekly s.c. implantation of slow-release pellets to mice appears to result in relatively consistent plasma CS and OC levels with significant biological effects. However, at least in our hands, no method delivered CS at a constant rate and variability in plasma CS levels was pronounced.     

Paraneoplastic syndrome in mice bearing high-angiogenic variant of Lewis lung carcinoma: relations with tumor derived VEGF

Background

It is well-known that tumor exerts nonmetastatic systemic effect on organism caused the development of paraneoplastic syndrome (PNS). Recent findings point to relationships between development of PNS and tumor-derived vascular endothelial growth factor (VEGF).

Aim

Comparative study of PNS manifestations in mice with transplanted two variants of Lewis lung carcinoma with different angiogenic potential.

Methods

Plasma VEGF level was determined by immunoenzyme method, hematological indices were estimated with the use of hematological analyzer, the weight and cellularity of spleen and thymus were registered and histological analysis of tissue section of these organs was performed.

Results

Manifestations of anemia, extramedullary hemopoiesis and tumor-associated inflammatory disease was observed in animals with high angiogenic LLC/R9 variant and was not registered in low angiogenic LLC. The emergence of PNS symptoms correlated with elevated level of circulating VEGF at the early stages of LLC/R9 growth.

Conclusion

Manifestation of the paraneoplastic hematological syndrome most likely is conditioned on the ability of cancer cell to secrete VEGF in a high rate.     

Chemical composition and therapeutic effects of Lippia alba (Mill.) N. E. Brown leaves hydro-alcoholic extract in patients with migraine

Background

There is no universally accepted and effective prophylaxis of migraine headache episodes. Thus we aimed to investigate the effects of Lippia alba (Mill.) N. E. Brown, an herb with many effects on central nervous system, on pain frequency and intensity of migraine patients.

Methods

Patients were enrolled in a prospective, phase 2, non-controlled cohort study to orally receive hydro-alcoholic extract of L. alba leaves. Headache intensity and frequency of episodes were recorded before and after 30-60 days of treatment. We also studied the chemical composition of its essential oil by gas chromatography-mass spectrometry.

Results

We described for the first time a particular L. alba chemotype with geranial and carvenone as major compounds. With treatment, both frequency and intensity of pain episodes significantly decreased from baseline to first reassessment date. More than 80% of patients experienced a minimum 50% reduction on pain intensity and frequency. No side effects were reported.

Conclusions

Treatment with a geranial plus carvenone chemotype of L. alba hydro-alcoholic extract is a cheap, widely available, highly effective therapy to reduce both the intensity and the frequency of headache episodes of migraine patients with no side effects.     

Disnea y psicopatología en el paciente anciano con enfermedad pulmonar obstructiva crónica

Chronic Obstructive Pulmonary Disease (COPD) is a progressive disease with dyspnoea perception as a main symptom. In severe stages, dyspnoea can constitute a risk factor for depression, anxiety and somatization disorders.

Objective

The objective was to evaluate the presence of these psychopathologies based on dyspnoea and severity stages in patients with COPD.

Materials and methods

Patients (n=51) were evaluated by means of the Hospital Anxiety and Depression Scale, the dyspnoea scale (MRC), the General Health Questionnaire (GHQ-28) and spirometric criteria.

Results

The increase in dyspnoea level and disease severity lead to a progressive worsening of anxiety, depressive and somatic symptoms with clinical relevance (P<0.05). There was a significant correlation between those parameters (P<0.05).

Conclusions

The early detection and treatment of these psychopathologies associated with dyspnoea and progression of the disease must be taken into account in this complex pathology.     

Gaseous persufflation with carbon monoxide during ischemia protects the isolated liver and enhances energetic recovery

Background

The benefit of carbon monoxide as applied by controlled, continuous gaseous persufflation during liver preservation on postischemic graft recovery was investigated in an isolated rat liver model.

Methods

Livers from male Wistar rats were retrieved 30 min after cardiac arrest of the donor and subjected to 18 h of cold storage. Some grafts were subjected to gaseous persufflation with carbon monoxide (CO, dissolved in nitrogen) during static cold storage at a concentration of 50 ppm or 250 ppm. Graft viability was assessed thereafter upon warm reperfusion in vitro.

Results

CO-persufflation significantly reduced cellular enzyme loss (maximal at 50 ppm) and functional recovery (bile production and energy charge) upon reperfusion by about 50%. The effect was associated with a reduction of free radical-induced lipid peroxidation, lower vascular perfusion resistance, and improved mitochondrial ultrastructure.

Conclusion

Viability of cold stored liver grafts can be notably augmented by gaseous ex vivo application of low dose CO to the isolated organ.