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Institute
Background/Aims: In diabetic nephropathy (DN), the current angiotensin-II-blocking pharmacotherapy is frequently failing. For diabetic cardiomyopathy (DC), there is no specific remedy available. Relaxin-2 (Rlx) - an anti-fibrotic, anti-inflammatory, and vasoprotecting peptide - is a candidate drug for both. Methods: Low-dose (32 mu g/kg/day) and high-dose (320 mu g/kg/day) Rlx were tested against vehicle (n = 20 each) and non-diabetic controls (n = 14) for 12 weeks in a model of type-1 diabetes induced in endothelial nitric oxide synthase knock-out (eNOS-KO) mice by intraperitoneal injection of streptozotocin. Results: Diabetic animals showed normal plasma creatinine, markedly increased albuminuria and urinary malonyldialdehyde, elevated relative kidney weight, glomerulosclerosis, and increased glomerular size, but no relevant interstitial fibrosis. Neither dose of Rlx affected these changes although the drug was active and targeted plasma levels were achieved. Of note, we found no activation of the renal TGF-beta pathway in this model. In the hearts of diabetic animals, no fibrotic alterations indicative of DC could be determined which precluded testing of the initial hypothesis. Conclusions: We investigated a model showing early DN without overt tubulo-interstitial fibrosis and activation of the TGF-beta-Smad-2/3 pathway. In this model, Rlx proved ineffective; however, the same may not apply to other models and types of diabetes.
Loss of pdr-1/parkin influences Mn homeostasis through altered ferroportin expression in C-elegans
(2015)
cis-Diamminedichloroplatinum(II) (Cisplatin) is one of the most important and frequently used cytostatic drugs for the treatment of various solid tumors. Herein, a laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method incorporating a fast and simple sample preparation protocol was developed for the elemental mapping of Cisplatin in the model organism Caenorhabditis elegans (C. elegans). The method allows imaging of the spatially-resolved elemental distribution of platinum in the whole organism with respect to the anatomic structure in L4 stage worms at a lateral resolution of 5 mm. In addition, a dose- and time-dependent Cisplatin uptake was corroborated quantitatively by a total reflection X-ray fluorescence spectroscopy (TXRF) method, and the elemental mapping indicated that Cisplatin is located in the intestine and in the head of the worms. Better understanding of the distribution of Cisplatin in this well-established model organism will be instrumental in deciphering Cisplatin toxicity and pharmacokinetics. Since the cytostatic effect of Cisplatin is based on binding the DNA by forming intra- and interstrand crosslinks, the response of poly(ADP-ribose) metabolism enzyme 1 (pme-1) deletion mutants to Cisplatin was also examined. Loss of pme-1, which is the C. elegans ortholog of human poly(ADP-ribose) polymerase 1 (PARP-1) led to disturbed DNA damage response. With respect to survival and brood size, pme-1 deletion mutants were more sensitive to Cisplatin as compared to wildtype worms, while Cisplatin uptake was indistinguishable.
Arsenic-containing fatty acids are a group of fat-soluble arsenic species (arsenolipids) which are present in marine fish and other seafood. Recently, it has been shown that arsenic-containing hydrocarbons, another group of arsenolipids, exert toxicity in similar concentrations comparable to arsenite although the toxic modes of action differ. Hence, a risk assessment of arsenolipids is urgently needed. In this study the cellular toxicity of a saturated (AsFA 362) and an unsaturated (AsFA 388) arsenic-containing fatty acid and three of their proposed metabolites (DMA(V), DMAPr and thio-DMAPr) were investigated in human liver cells (HepG2). Even though both arsenic-containing fatty acids were less toxic as compared to arsenic-containing hydrocarbons and arsenite, significant effects were observable at mu M concentrations. DMA(V) causes effects in a similar concentration range and it could be seen that it is metabolised to its highly toxic thio analogue thio-DMA(V) in HepG2 cells. Nevertheless, DMAPr and thio-DMAPr did not exert any cytotoxicity. In summary, our data indicate that risks to human health related to the presence of arsenic-containing fatty acids in marine food cannot be excluded. This stresses the need for a full in vitro and in vivo toxicological characterisation of these arsenolipids.
Background: Previous studies have shown that BMP4 may play an important part in the development of auditory neurons (ANs), which are degenerated in sensorineural hearing loss. However, whether BMP4 can promote sensory fate specification from mesenchymal stromal cells (MSCs) is unknown so far.
Methods: MSCs isolated from Sprague-Dawley (SD) rats were confirmed by expression of MSC markers using flow cytometry and adipogenesis/osteogenesis using differentiation assays. MSCs treated with a complex of neurotrophic factors (BMP4 group and non-BMP4 group) were induced into auditory neuron-like cells, then the differences between the two groups were analyzed in morphological observation, cell growth curve, qRT-PCR, and immunofluorescence.
Results: Flow cytometric analysis showed that the isolated cells expressed typical MSC surface markers. After adipogenic and osteogenic induction, the cells were stained by oil red O and Alizarin Red. The neuronal induced cells were in the growth plateau and had special forms of neurons. In the presence of BMP4, the inner ear genes NF-M, Neurog1, GluR4, NeuroD, Calretinin, NeuN, Tau, and GATA3 were up-regulated in MSCs.
Conclusions: MSCs have the capacity to differentiate into auditory neuron-like cells in vitro. As an effective inducer, BMP4 may play a key role in transdifferentiation.
Exposure to organic mercury compounds promotes primarily neurological effects. Although methylmercury is recognized as a potent neurotoxicant, its transfer into the central nervous system (CNS) is not fully evaluated. While methylmercury and thiomersal pass the blood-brain barrier, limited data are available regarding the second brain regulating interface, the blood-cerebrospinal fluid (CSF) barrier. This novel study was designed to investigate the effects of organic as well as inorganic mercury compounds on, and their transfer across, a porcine in vitro model of the blood-CSF barrier for the first time. The barrier system is significantly more sensitive towards organic Hg compounds as compared to inorganic compounds regarding the endpoints cytotoxicity and barrier integrity. Whereas there are low transfer rates from the blood side to the CSF side, our results strongly indicate an active transfer of the organic mercury compounds out of the CSF. These results are the first to demonstrate an efflux of organic mercury compounds regarding the CNS and provide a completely new approach in the understanding of mercury compounds specific transport.
Organic mercury (Hg) species exert their toxicity primarily in the central nervous system. The food relevant Hg species methylmercury (MeHg) has been frequently studied regarding its neurotoxic effects in vitro and in vivo. Neurotoxicity of thiomersal, which is used as a preservative in medical preparations, is to date less characterised. Due to dealkylation of organic Hg or oxidation of elemental Hg, inorganic Hg is present in the brain albeit these species are not able to readily cross the blood brain barrier. This study compared for the first time toxic effects of organic MeHg chloride (MeHgCl) and thiomersal as well as inorganic mercury chloride (HgCl2) in differentiated human neurons (LUHMES) and human astrocytes (CCF-STTG1). The three Hg species differ in their degree and mechanism of toxicity in those two types of brain cells. Generally, neurons are more susceptible to Hg species induced cytotoxicity as compared to astrocytes. This might be due to the massive cellular mercury uptake in the differentiated neurons. The organic compounds exerted stronger cytotoxic effects as compared to inorganic HgCl2. In contrast to HgCl2 exposure, organic Hg compounds seem to induce the apoptotic cascade in neurons following low-level exposure. No indicators for apoptosis were identified for both inorganic and organic mercury species in astrocytes. Our studies clearly demonstrate species-specific toxic mechanisms. A mixed exposure towards all Hg species in the brain can be assumed. Thus, prospectively coexposure studies as well as cocultures of neurons and astrocytes could provide additional information in the investigation of Hg induced neurotoxicity.
Inflammation in Cachexia
(2015)
Background/Aims: To investigate the renal phenotype under conditions of an activated renal ET-1 system in the status of nitric oxide deficiency, we compared kidney function and morphology in wild-type, ET-1 transgenic (ET+/+), endothelial nitric oxide synthase knockout (eNOS-/-) and ET+/+eNOS-/- mice. Methods: We assessed blood pressure, parameters of renal morphology, plasma cystatin C, urinary protein excretion, expression of genes associated with glomerular filtration barrier and tissue remodeling, and plasma metabolites using metabolomics. Results: eNOS-/- and ET+/+eNOS-/- mice developed hypertension. Osteopontin, albumin and protein excretion were increased in eNOS-/- and restored in ET+/+eNOS-/- animals. All genetically modified mice developed renal interstitial fibrosis and glomerulosclerosis. Genes involved in tissue remodeling (serpinel, TIMP1, Collal, CCL2) were up-regulated in eNOS-/-, but not in ET+/+eNOS-/- mice. Plasma levels of free carnitine and acylcarnitines, amino acids, diacyl phosphatidylcholines, lysophosphatidylcholines and hexoses were descreased in eNOS-/- and were in the normal range in ET+/+eNOS-/- mice. Conclusion: eNOS-/- mice developed renal dysfunction, which was partially rescued by ET-1 overexpression in eNOS-/- mice. The metabolomics results suggest that ET-1 overexpression on top of eNOS knockout is associated with a functional recovery of mitochondria (rescue effect in 13-oxidation of fatty acids) and an increase in antioxidative properties (normalization of monounsaturated fatty acids levels). (C) 2015 The Author(s) Published by S. Karger AG, Basel
The matricellular protein connective tissue growth factor (CTGF/CCN2) is recognized as key player in the onset of fibrosis in various tissues, including skeletal muscle. In many circumstances, CTGF has been shown to be induced by transforming growth factor beta (TGF beta) and accounting, at least in part, for its biological action. In this study it was verified that in cultured myoblasts CTGF/CCN2 causes their transdifferentiation into myofibroblasts by up-regulating the expression of fibrosis marker proteins alpha-smooth muscle actin and transgelin. Interestingly, it was also found that the profibrotic effect exerted by CTGF/CCN2 was mediated by the sphingosine kinase (SK)-1/S1P(3) signaling axis specifically induced by the treatment with the profibrotic cue. Following CTGF/CCN2-induced up-regulation, S1P(3) became the SIP receptor subtype expressed at the highest degree, at least at mRNA level, and was thus capable of readdressing the sphingosine 1-phosphate signaling towards fibrosis rather than myogenic differentiation. Another interesting finding is that CTGF/CCN2 silencing prevented the TGF beta-dependent up-regulation of SKI/S1P(3) signaling axis and strongly reduced the profibrotic effect exerted by TGF beta, pointing at a crucial role of endogenous CTGF/CCN2 generated following TGF beta challenge in the transmission of at least part of its profibrotic effect These results provide new insights into the molecular mechanism by which CTGF/CCN2 drives its biological action and strengthen the concept that SK1/S1P(3) axis plays a critical role in the onset of fibrotic cell phenotype. (C) 2014 Elsevier B.V. All rights reserved.
2-Phenylethanol (2PE) and 3,5-dimethoxytoluene (DMT) are characteristic scent compounds in specific roses such as Rosa x hybrida cv. 'Yves Piaget'. We analyzed the endogenous concentrations and emission of 2PE and DMT during the unfurling process in different floral organs, as well as changes in transcript levels of the two key genes, PAR and OOMT2.
The emission of both 2PE and DMT increased during floral development to reach peaks at the fully unfurled stage. The relative transcripts of PAR and OOMT2 also increased during floral development. Whereas the maximum for OOMT2 was found at the fully unfurled stage (stage 4), similar expression levels of PAR were detected at stage 4 and the senescence stage (stage 6). The results demonstrate a positive correlation between the expression levels of PAR and OOMT2 and the emission of 2PE and DMT. In addition, endogenous volatiles and relative transcripts showed tissue- and development-specific patterns. (C) 2014 Elsevier Masson SAS. All rights reserved.
Pak Choi Fed to Mice: Formation of DNA Adducts and Influence on Xenobiotic-Metabolizing Enzymes
(2015)
Xenobiotics may interfere with the hypothalamic-pituitary-thyroid endocrine axis by inducing enzymes that inactivate thyroid hormones and thereby reduce the metabolic rate. This induction results from an activation of xeno-sensing nuclear receptors. The current study shows that benzo[a]pyrene, a frequent contaminant of processed food and activator of the arylhydrocarbon receptor (AhR) activated the promoter and induced the transcription of the nuclear receptor constitutive androstane receptor (CAR, NR1I3) in rat hepatocytes. Likewise, phenobarbital induced the AhR transcription. This mutual induction of the nuclear receptors enhanced the phenobarbital-dependent induction of the prototypic CAR target gene Cyp2b1 as well as the AhR-dependent induction of UDP-glucuronosyltransferases. In both cases, the induction by the combination of both xenobiotics was more than the sum of the induction by either substance alone. By inducing the AhR, phenobarbital enhanced the benzo[a]pyrene-dependent reduction of thyroid hormone half-life and the benzo[a]pyrene-dependent increase in the rate of thyroid hormone glucuronide formation in hepatocyte cultures. CAR ligands might thus augment the endocrine disrupting potential of AhR activators by an induction of the AhR. (C) 2014 Elsevier Ireland Ltd. All rights reserved.
A novel common variant in DCST2 is associated with length in early life and height in adulthood
(2015)
Common genetic variants have been identified for adult height, but not much is known about the genetics of skeletal growth in early life. To identify common genetic variants that influence fetal skeletal growth, we meta-analyzed 22 genome-wide association studies (Stage 1; N = 28 459). We identified seven independent top single nucleotide polymorphisms (SNPs) (P < 1 x 10(-6)) for birth length, of which three were novel and four were in or near loci known to be associated with adult height (LCORL, PTCH1, GPR126 and HMGA2). The three novel SNPs were followed-up in nine replication studies (Stage 2; N = 11 995), with rs905938 in DC-STAMP domain containing 2 (DCST2) genome-wide significantly associated with birth length in a joint analysis (Stages 1 + 2; beta = 0.046, SE = 0.008, P = 2.46 x 10(-8), explained variance = 0.05%). Rs905938 was also associated with infant length (N = 28 228; P = 5.54 x 10(-4)) and adult height (N = 127 513; P = 1.45 x 10(-5)). DCST2 is a DC-STAMP-like protein family member and DC-STAMP is an osteoclast cell-fusion regulator. Polygenic scores based on 180 SNPs previously associated with human adult stature explained 0.13% of variance in birth length. The same SNPs explained 2.95% of the variance of infant length. Of the 180 known adult height loci, 11 were genome-wide significantly associated with infant length (SF3B4, LCORL, SPAG17, C6orf173, PTCH1, GDF5, ZNFX1, HHIP, ACAN, HLA locus and HMGA2). This study highlights that common variation in DCST2 influences variation in early growth and adult height.
Characterization of freeze-fractured epithelial plasma membranes on nanometer scale with ToF-SIMS
(2015)
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to characterize the freeze-fracturing process of human epithelial PANC-1 and UROtsa cells. For this purpose, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine standard samples were investigated to find specific signals with both high specificity and signal intensity. The results were used to investigate single cells of subconfluent cell layers prepared with a special silicon wafer sandwich preparation technique. This freeze-fracturing technique strips cell membranes off the cells, isolating them on opposing silicon wafer substrates. Criteria were found for defining regions with stripped off cell membranes and, on the opposing wafer, complementary regions with the remaining cells. Measured ethanolamine/choline and serine/choline ratios in these regions clearly showed that in the freeze-fracturing process, the lipid bilayer of the plasma membrane is split along its central zone. Accordingly, only the outer lipid monolayer is stripped off the cell, while the inner lipid monolayer remains attached to the cell on the opposing wafer, thus allowing detailed analysis of a single lipid monolayer. Furthermore, it could be shown that using different washing procedures did not influence the transmembrane lipid distribution. Under optimized preparation conditions, it became feasible to detect lipids with a lateral resolution of approximately 100 nm. The data indicate that ToF-SIMS would be a very useful technique to study with very high lateral resolution changes in lipid composition caused, for example, by lipid storage diseases or pharmaceuticals that interfere with the lipid metabolism.
All components of the endothelin (ET) system are present in renal tubular cells. In this review, we summarize current knowledge about ET and the most common tubular diseases: acute kidney injury (AKI) and polycystic kidney disease. AKI originally was called acute tubular necrosis, pointing to the most prominent morphologic findings. Similarly, cysts in polycystic kidney disease, and especially in autosomal-dominant polycystic kidney disease, are of tubular origin. Preclinical studies have indicated that the ET system and particularly ETA receptors are involved in the pathogenesis of ischemia-reperfusion injury, although these findings have not been translated to clinical studies. The ET system also has been implicated in radiocontrast-dye-induced AKI, however, ET-receptor blockade in a large human study was not successful. The ET system is activated in sepsis models of AKI; the effectiveness of ET blocking agents in preclinical studies is variable depending on the model and the ET-receptor antagonist used. Numerous studies have shown that the ET system plays an important role in the complex pathophysiology associated with cyst formation and disease progression in polycystic kidney disease. However, results from selective targeting of ET-receptor subtypes in animal models of polycystic kidney disease have proved disappointing and do not support clinical trials. These studies have shown that a critical balance between ETA and ETB receptor action is necessary to maintain structure and function in the cystic kidney. In summary, ETs have been implicated in the pathogenesis of several renal tubulointerstitial diseases, however, experimental animal findings have not yet led to use of ET blockers in human beings. (C) 2015 Elsevier Inc. All rights reserved.
Carotenoids accumulated in the egg yolk are of importance for two reasons. Firstly they are important pigments influencing customer acceptance and secondly they are essential components with positive health effects either as antioxidants or as precursor of vitamin A. Different analytical methods are available to quantitatively identify carotenoids from egg yolk such as spectrophotometric methods described by AOAC (Association of Official Analytical Chemists) and HPLC (High Performance Liquid Chromatography). Both methods have in common that they are time consuming, need a laboratory environment and well trained technical operators. Recently, a rapid lab-independent spectrophotometric method (iCheck, BioAnalyt GmbH, Germany) has been introduced that claims to be less time consuming and easy to operate. The aim of the current study was therefore to compare the novel method with the two standard methods. Yolks of 80 eggs were analysed as aliquots by the three methods in parallel. While both spectrometric methods are only able measure total carotenoids as total beta-carotene, HPLC enables the determination of individual carotenoids such lutein, zeaxanthin, canthaxanthin, beta-carotene and beta-apocarotenoic ester. In general, total carotenoids levels as obtained by AOAC were in average 27% higher than those obtained by HPLC. Carotenoid values obtained by the reference methods AOAC and HPLC are highly correlated with the iCheck method with r(2) of 0.99 and 0.94 for iCheck vs. AOAC and iCheck vs. HPLC, respectively (both p < 0.001). Bland Altman analysis showed that the novel iCheck method is comparable to the reference methods. In conclusion, the novel rapid and portable iCheck method is a valid and effective tool to determine total carotenoid of egg yolk under laboratory-independent conditions with little trained personal. (C) 2014 Elsevier Ltd. All rights reserved.
Camellia sinensis synthesizes and emits a large variety of volatile phenylpropanoids and benzenoids (VPB). To investigate the enzymes involved in the formation of these VPB compounds, a new C. sinensis short-chain dehydrogenase/reductase (CsSDR) was isolated, cloned, sequenced, and functionally characterized. The complete open reading frame of CsSDR contains 996 nucleotides with a calculated protein molecular mass of 34.5 kDa. The CsSDR recombinant protein produced in Escherichia coli exhibited dehydrogenase-reductase activity towards several major VPB compounds in C. sinensis flowers with a strong preference for NADP/NADPH co-factors, and showed affinity for (R)/(S)-1-phenylethanol (1PE), phenylacetaldehyde, benzaldehyde, and benzyl alcohol, and no affinity for acetophenone (AP) and 2-phenylethanol. CsSDR showed the highest catalytic efficiency towards (R)/(S)-1PE. Furthermore, the transient expression analysis in Nicotiana benthamiana plants validated that CsSDR could convert 1PE to AP in plants. CsSDR transcript level was not significantly affected by floral development and some jasmonic acid-related environmental stress, and CsSDR transcript accumulation was detected in most floral tissues such as receptacle and anther, which were main storage locations of VPB compounds. Our results indicate that CsSDR is expressed in C. sinensis flowers and is likely to contribute to a number of floral VPB compounds including the 1PE derivative AP.
Scope: Flavan-3-ols are abundant polyphenols in human nutrition and are associated with beneficial health effects. The aim of this study was to comparatively investigate the metabolic fate of (-)-epicatechin, procyanidin B1, and polymeric procyanidins in a randomized cross-over study in humans.
Methods and results: Parent compounds, conjugates, and microbial metabolites were determined in plasma, urine, and faeces by HPLC-MS and GC-MS/MS. Glucuronidated, sulfated, and methylated (-)-epicatechin and 5-(3',4'-dihydroxyphenyl)-valerolactone were the dominant metabolites in blood and urine. In addition, minor amounts of procyanidin B1 and 4-hydroxy-5-(3',4'-dihydroxyphenyl) valeric acid and their conjugated metabolites were detected. The formation of 5-(3',4'-dihydroxyphenyl)-valerolactone and 4-hydroxy-5-(3',4'-dihydroxyphenyl) valeric acid varied largely between individuals as well as with the degree of polymerization of flavan-3-ols. Monomer units were not detectable in plasma or urine after procyanidin B1 and polymeric procyanidin intake. No correlation was found between the intake of flavan-3-ols and the occurrence of phenolic acids in blood and urine or the phenolic compound profiles in faeces.
Conclusion: In addition to conjugated metabolites derived from the absorption of monomeric flavan-3-ols, 5-(3',4' -dihydroxyphenyl)-valerolactone represents an important in vivo metabolite of (-)-epicatechin and procyanidin B1 produced by the gut microbiota.
Direct stimulation of soluble guanylate cyclase (sGC) is emerging as a potential new approach for the treatment of renal disorders. sGC catalyzes the formation of cyclic guanosine monophosphate (cGMP), deficiency of which is implicated in the pathogenesis of chronic kidney disease (CKD). Therefore, new classes of drugs sGC stimulators and activators are being investigated in preclinical models under conditions where nitric oxide is deficient. In preclinical models with different etiologies of CKD, the sGC stimulators BAY 41-2272, BAY 41-8543, BAY 60-4552, riociguat and vericiguat and the sGC activators cinaciguat, ataciguat, BI 703704 and GSK2181236A have shown consistently renoprotective effects. Clinical trials are required to confirm these findings in humans, and to ascertain whether these agents could provide a future alternative to guideline-recommended treatments.
Continuing advances in 'omics methodologies and instrumentation is enhancing the understanding of how plants cope with the dynamic nature of their growing environment. 'Omics platforms have been only recently extended to cover horticultural crop species. Many of the most widely cultivated vegetable crops belong to the genus Brassica: these include plants grown for their root (turnip, rutabaga/swede), their swollen stem base (kohlrabi), their leaves (cabbage, kale, pak choi) and their inflorescence (cauliflower, broccoli). Characterization at the genome, transcript, protein and metabolite levels has illustrated the complexity of the cellular response to a whole series of environmental stresses, including nutrient deficiency, pathogen attack, heavy metal toxicity, cold acclimation, and excessive and sub optimal irradiation. This review covers recent applications of omics technologies to the brassicaceous vegetables, and discusses future scenarios in achieving improvements in crop end-use quality.
CXCL12-CXCR4 signaling controls multiple physiological processes and its dysregulation is associated with cancers and inflammatory diseases. To discover as-yet-unknown endogenous ligands of CXCR4, we screened a blood-derived peptide library for inhibitors of CXCR4-tropic HIV-1 strains. This approach identified a 16 amino acid fragment of serum albumin as an effective and highly specific CXCR4 antagonist. The endogenous peptide, termed EPI-X4, is evolutionarily conserved and generated from the highly abundant albumin precursor by pH-regulated proteases. EPI-X4 forms an unusual lasso-like structure and antagonizes CXCL12-induced tumor cell migration, mobilizes stem cells, and suppresses inflammatory responses in mice. Furthermore, the peptide is abundant in the urine of patients with inflammatory kidney diseases and may serve as a biomarker. Our results identify EPI-X4 as a key regulator of CXCR4 signaling and introduce proteolysis of an abundant precursor protein as an alternative concept for chemokine receptor regulation.
The role of serum amyloid A and sphingosine-1-phosphate on high-density lipoprotein functionality
(2015)
The high-density lipoprotein (HDL) is one of the most important endogenous cardiovascular protective markers. HDL is an attractive target in the search for new pharmaceutical therapies and in the prevention of cardiovascular events. Some of HDL's anti-atherogenic properties are related to the signaling molecule sphingosine-1-phosphate (S1P), which plays an important role in vascular homeostasis. However, for different patient populations it seems more complicated. Significant changes in HDL's protective potency are reduced under pathologic conditions and HDL might even serve as a proatherogenic particle. Under uremic conditions especially there is a change in the compounds associated with HDL. S1P is reduced and acute phase proteins such as serum amyloid A (SAA) are found to be elevated in HDL. The conversion of HDL in inflammation changes the functional properties of HDL. High amounts of SAA are associated with the occurrence of cardiovascular diseases such as atherosclerosis. SAA has potent pro-atherogenic properties, which may have impact on HDL's biological functions, including cholesterol efflux capacity, antioxidative and anti-inflammatory activities. This review focuses on two molecules that affect the functionality of HDL. The balance between functional and dysfunctional HDL is disturbed after the loss of the protective sphingolipid molecule S1P and the accumulation of the acute-phase protein SAA. This review also summarizes the biological activities of lipid-free and lipid-bound SAA and its impact on HDL function.
Objectives: This study aims to increase understanding of the connection between oxidative stress and inflammation in diabetes disease progression to provide a basis for investigating improved diagnostic possibilities, treatment and prevention of prediabetes.
Design and methods: Differences in the level of biochemical markers of oxidative stress (erythrocyte GSH/GSSG and urinary 8-isoprostane), inflammation (CRP, IL-6), endothelial dysfunction (plasma homocysteine, urinary 8-hydroxy-2-deoxy-guanosine) and coagulation/fibrinolysis (C5a, D-Dimer) were determined in prediabetes and control subjects.
Results: While no difference was found in the 8-isoprostane levels between the two groups, the erythrocyte GSH/GSSG ratio was significantly reduced in the prediabetes group compared to control, indicating increased oxidative stress in the prediabetic state. Both urinary 8-OHdG and surprisingly also plasma homocysteine were significantly elevated in the prediabetes group, indicating endothelial dysfunction. The inflammation markers were slightly elevated in the prediabetic subjects and the same trend was found for the coagulation/fibrinolysis markers C5a and D-Dimer. These results were however not significant.
Conclusions: The small elevation of blood glucose levels in the prediabetic state may have a detectable influence on endothelial function as indicated by changes to 8-OHdG, indicating an increased DNA-damage and homocysteine release from endothelial cells. Increased oxidative stress as indicated by the reduced GSH/GSSG ratio is likely to be the link between the moderate hyperglycaemia in prediabetes and pathological changes in endothelial function, which in the long-term may promote atherogenesis and result in the development of cardiovascular disease. Early detection of prediabetes is essential to avoid diabetes development and the associated complications like cardiovascular disease. The GSH/GSSG ratio and biomarkers like urinary 8-OHdG and plasma homocysteine offer a possible tool for the assessment of prediabetes in prevention screenings. (C) 2015 The Authors. The Canadian Society of Clinical Chemists. Published by Elsevier Inc.
Metastatic dissemination of cancer cells is the ultimate hallmark of malignancy and accounts for approximately 90% of human cancer deaths. We investigated the role of acid sphingomyelinase (Asm) in the hematogenous metastasis of melanoma cells. Intravenous injection of B16F10 melanoma cells into wild-type mice resulted in multiple lung metastases, while Asm-deficient mice (Smpd1(-/-) mice) were protected from pulmonary tumor spread. Transplanting wild-type platelets into Asm-deficient mice reinstated tumor metastasis. Likewise, Asm-deficient mice were protected from hematogenous MT/ret melanoma metastasis to the spleen in a mouse model of spontaneous tumor metastasis. Human and mouse melanoma cells triggered activation and release of platelet secretory Asm, in turn leading to ceramide formation, clustering, and activation of 51 integrins on melanoma cells finally leading to adhesion of the tumor cells. Clustering of integrins by applying purified Asm or C-16 ceramide to B16F10 melanoma cells before intravenous injection restored trapping of tumor cells in the lung in Asm-deficient mice. This effect was revertable by arginine-glycine-aspartic acid peptides, which are known inhibitors of integrins, and by antibodies neutralizing 1 integrins. These findings indicate that melanoma cells employ platelet-derived Asm for adhesion and metastasis.
Iron deficiency anemia, zinc and vitamin A deficiencies are serious public health problems in Cameroon, as in many developing countries. Local vegetables which are sources of provitamin A carotenoids (PACs) can be used to improve vitamin A intakes. However, traditional meals are often unable to cover zinc and iron needs. The aim of this study was to determine the bioavailability of 3 PACs (alpha-carotene, beta-carotene, and beta-cryptoxanthin) in young men, who were fed with a vitamin A-free diet and received iron and zinc supplementation. Twelve healthy participants were divided into three groups and were supplemented with elemental iron (20 mg of iron fumarate), 20 mg of zinc sulfate or iron + zinc (20 mg of iron in the morning and 20 mg of zinc in the evening) for 11 d. They were given a vitamin A- and PAC-free diet from the 6th to the 11th day, followed by a test meal containing 0.55 kg of freshly peeled papaya as a source of PACs. Blood samples were collected four times successively on the 11th day (the test meal day), at TO (just after the test meal), after 2 h (T2), after 4 h (T4) and after 7 h (T7). Ultracentrifugation was used to isolate serum chylomicrons. Retinol appearance and PAC postprandial concentrations were determined. The supplementation with zinc, iron and iron+zinc influenced the chylomicron appearance of retinol and PACs differently as reflected by retention times and maximum absorption peaks. Iron led to highest retinol levels in the chylomicron. Zinc and iron+zinc supplements were best for optimal intact appearance of alpha-carotene, beta-carotene and beta-cryptoxanthin respectively. Supplementation with iron led to the greatest bioavailability of PACs from papaya and its conversion to retinol.
Background: Sub-Saharan Africa is facing a double burden of malnutrition: vitamin A deficiency (VAD) prevails, whereas the nutrition-related chronic conditions type 2 diabetes (T2D) and hypertension are emerging. Serum retinol a VAD marker increases in kidney disease and decreases in inflammation, which can partly be attributed to alterations in the vitamin A transport proteins retinol-binding protein 4 (RBP4) and prealbumin. Kidney dysfunction and inflammation commonly accompany T2D and hypertension.
Objective: Among urban Ghanaians, we investigated the associations of T2D and hypertension with serum retinol as well as the importance of kidney function and inflammation in this regard.
Design: A hospital-based, case-control study in individuals for risk factors of T2D, hypertension, or both was conducted in Kumasi, Ghana (328 controls, 197 with T2D, 354 with hypertension, and 340 with T2D plus hypertension). In 1219 blood samples, serum retinol, RBP4, and prealbumin were measured. Urinary albumin and estimated glomerular filtration rate (eGFR) defined kidney function. C-reactive protein (CRP) >5 mg/L indicated inflammation. We identified associations of T2D and hypertension with retinol by linear regression and calculated the contribution of RBP4, prealbumin, urinary albumin, eGFR, and CRP to these associations as the percentages of the explained variance of retinol.
Results: VAD (retinol <1.05 mu mol/L) was present in 10% of this predominantly female, middle-aged, overweight, and deprived population. Hypertension, but not T2D, was positively associated with retinol (beta: 0.12; 95% CI: 0.08, 0,17), adjusted for age, sex, socioeconomic factors, anthropometric measurements, and lifestyle. In addition to RBP4 (72%) and prealbumin (22%), the effect of increased retinol on individuals with hypertension was mainly attributed to impaired kidney function (eGFR: 30%; urinary albumin: 5%) but not to inflammation.
Conclusions: In patients with hypertension, VAD might be underestimated because of increased serum retinol in the context of kidney dysfunction. Thus, the interpretation of serum retinol in sub-Saharan Africa should account for hypertension status.
Glucolipotoxic stress has been identified as a key player in the progression of pancreatic beta-cell dysfunction contributing to insulin resistance and the development of type 2 diabetes mellitus (T2D). It has been suggested that bioactive lipid intermediates, formed under lipotoxic conditions, are involved in these processes. Here, we show that sphingosine 1-phosphate (S1P) levels are not only increased in palmitate-stimulated pancreatic beta-cells but also regulate beta-cell homeostasis in a divergent manner. Although S1P possesses a prosurvival effect in beta-cells, an enhanced level of the sphingolipid antagonizes insulin-mediated cell growth and survival via the sphingosine 1-phosphate receptor subtype 2 (S1P(2)) followed by an inhibition of Akt-signaling. In an attempt to investigate the role of the S1P/S1P(2) axis in vivo, the New Zealand obese (NZO) diabetic mouse model, characterized by beta-cell loss under high-fat diet (HFD) conditions, was used. The occurrence of T2D was accompanied by an increase of plasma S1P levels. To examine whether S1P contributes to the morphologic changes of islets via S1P(2), the receptor antagonist JTE-013 was administered. Most interestingly, JTE-013 rescued beta-cell damage clearly indicating an important role of the S1P(2) in beta-cell homeostasis. Therefore, the present study provides a new therapeutic strategy to diminish beta-cell dysfunction and the development of T2D.
Background: The objective of the study was to investigate the relationship between first trimester maternal serum levels of the TTR-RBP4-ROH complex components and the later insurgence of an altered glucose metabolism during pregnancy.
Methods: Retrospective case control study including 96 patients between the 12th and 14th week of gestation, 32 that developed gestational diabetes mellitus (GDM), respectively, 21 non-insulin-treated (dGDM) and 11 insulin-treated (iGDM), 20 large for gestational age fetuses (LGA) without GDM and 44 patients with normal outcome as control. Serum concentrations of RBP4 and TTR were assessed by ELISA; serum concentration of ROH by reverse-phase high performance liquid chromatography (rpHPLC). The molecular heterogeneity of TTR and RBP4 was analyzed after immunoprecipitation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS).
Results: iGDM patients were characterized by reduced TTR, RBP4 and ROH compared to controls (respectively, iGDM vs. controls, mean +/- SD: TTR 3.96 +/- 0.89 mu mol/L vs. 4.68 +/- 1.21 mu mol/L, RBP4 1.13 +/- 0.25 mu mol/L vs. 1.33 +/- 0.38 mu mol/L and ROH 1.33 +/- 0.17 mu mol/L vs. 1.62 +/- 0.29 mu mol/L, p < 0.05). TTR containing Gly10 in place of Cys10 was lower in the iGDM group (p < 0.05) compared to controls. In the final logistic regression model ROH significantly predicted the diagnosis of iGDM (OR 0.93, 95% CI 0.87-0.98, p < 0.05).
Conclusions: First trimester maternal serum ROH, RBP4 and TTR represent potential biomarkers associated with the development of iGDM.
Exposure to high manganese (Mn) levels may damage the basal ganglia, leading to a syndrome analogous to Parkinson's disease, with motor and cognitive impairments. The molecular mechanisms underlying Mn neurotoxicity, particularly during development, still deserve further investigation. Herein, we addressed whether early-life Mn exposure affects motor coordination and cognitive function in adulthood and potential underlying mechanisms. Male Wistar rats were exposed intraperitoneally to saline (control) or MnCl2 (5, 10 or 20 mg/kg/day) from post-natal day (PND) 8-12. Behavioral tests were performed on PND 60-65 and biochemical analysis in the striatum and hippocampus were performed on PND14 or PND70. Rats exposed to Mn (10 and 20 mg/kg) performed significantly worse on the rotarod test than controls indicating motor coordination and balance impairments. The object and social recognition tasks were used to evaluate short-term memory. Rats exposed to the highest Mn dose failed to recognize a familiar object when replaced by a novel object as well as to recognize a familiar juvenile rat after a short period of time. However, Mn did not alter olfactory discrimination ability. In addition, Mn-treated rats displayed decreased levels of non-protein thiols (e.g. glutathione) and increased levels of glial fibrillary acidic protein (GFAP) in the striatum. Moreover, Mn significantly increased hippocampal glutathione peroxidase (GPx) activity. These findings demonstrate that acute low-level exposure to Mn during a critical neurodevelopmental period causes cognitive and motor dysfunctions that last into adulthood, that are accompanied by alterations in antioxidant defense system in both the hippocampus and striatum. (C) 2015 Elsevier Inc. All rights reserved.
The stems of Abrus precatorius were used to extract a beta-amylase enriched fraction. A three phase partitioning method and a Doehlert design with 3 variables (ratio of crude extract/t-butanol, the ammonium sulphate saturation and pH) were used. The data was fitted in a second-order polynomial model and the parameters were optimized to enrich beta-amylase. Experimental responses for the modulation were recovery of activity and the purification factor. The optimal conditions were: a ratio of crude extract/t-butanol of 0.87 (v/v), saturation in ammonium sulphate of 49.46% (w/v) and a pH of 5.2. An activity recovery of 156.2% and a purification factor of 10.17 were found. The enriched enzyme was identified as a beta-amylase and its molecular weight was 60.1 kDa. K-m and V-max values were 79.37 mg/ml and 5.13 U/ml, respectively and the highest activity was registered at a temperature of 70 degrees C and a pH between 6 and 6.5. A significant stabilization of the beta-amylase was observed up to 65 degrees C. (C) 2015 Elsevier Ltd. All rights reserved.
Pulmonary arterial hypertension (PAH) is a fatal disease that eventually results in right heart failure and death. Current pharmacologic therapies for PAH are limited, and there are no drugs that could completely cure PAH. Enhanced activity of endothelin system has been implicated in PAH severity and endothelin receptor antagonists have been used clinically to treat PAH. However, there is limited experimental evidence on the direct role of enhanced endothelin system activity in PAL-I. Here, we investigated the correlation between endothelin-1 (ET-1) and PAH using ET-1 transgenic (ETTG) mice. Exposure to chronic hypoxia increased right ventricular pressure and pulmonary arterial wall thickness in ETTG mice compared to those in wild type mice. Of note, ETTG mice exhibited modest but significant increase in right ventricular pressure and vessel wall thickness relative to wild type mice even under normoxic conditions. To induce severe PAH, we administered SU5416, a vascular endothelial growth factor receptor inhibitor, combined with exposure to chronic hypoxia. Treatment with SU5416 modestly aggravated hypoxia-induced pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial vessel wall thickening in ETTG mice in association with increased interleukin-6 expression in blood vessels. However, there was no sign of obliterative endothelial cell proliferation and plexiform lesion formation in the lungs. These results demonstrated that enhanced endothelin system activity could be a causative factor in the development of PAH and provided rationale for the inhibition of endothelin system to treat PAH. (C) 2015 Elsevier Inc. All rights reserved.
1,2-unsaturated pyrrolizidine alkaloids (PA) are plant metabolites predominantly occurring in the plant families Asteraceae and Boraginaceae. Acute and chronic PA poisoning causes severe hepatotoxicity. So far, the molecular mechanisms of PA toxicity are not well understood. To analyze its mode of action, primary human hepatocytes were exposed to a non-cytotoxic dose of 100 mu M of four structurally different PA: echimidine, heliotrine, senecionine, senkirkine. Changes in mRNA expression were analyzed by a whole genome microarray. Employing cut-off values with a vertical bar fold change vertical bar of 2 and a q-value of 0.01, data analysis revealed numerous changes in gene expression. In total, 4556, 1806, 3406 and 8623 genes were regulated by echimidine, heliotrine, senecione and senkirkine, respectively. 1304 genes were identified as commonly regulated. PA affected pathways related to cell cycle regulation, cell death and cancer development. The transcription factors TP53, MYC, NF kappa B and NUPR1 were predicted to be activated upon PA treatment. Furthermore, gene expression data showed a considerable interference with lipid metabolism and bile acid flow. The associated transcription factors FXR, LXR, SREBF1/2, and PPAR alpha/gamma/delta were predicted to be inhibited.
In conclusion, though structurally different, all four PA significantly regulated a great number of genes in common. This proposes similar molecular mechanisms, although the extent seems to differ between the analyzed PA as reflected by the potential hepatotoxicity and individual PA structure. (C) 2015 Elsevier Ltd. All rights reserved.
Generally, pea proteins are extracted at comparatively acidic or basic pH values to provide a basis for protein isolate production. Such processing steps result in partial denaturation of the proteins rendering them in most cases insoluble at food processing pH conditions and limiting their application in food products. Here, the comparison of the solubility properties of pea proteins in protein enriched fractions deriving from air classification is reported. Protein content, solubility, and physicochemical parameters of different fractions of the pea (Pisum sativum) variety 'Salamanca' were investigated as a function of pH using SDS-PAGE and surface hydrophobicity. Whole pea flour (20% protein), air classified, protein-enriched pea flour (48% protein), pea flour made from hulls (2.8% protein), and pea protein isolate (81% protein) served as test materials. Fractionation and pH value affected the composition and surface hydrophobicity of the proteins as well as the content of trypsin inhibitors. All samples showed a high buffering capacity in the range of pH 4 to 10. The direct comparison documents the comparatively better protein quality of the air classified, protein enriched pea fraction. The solubility of the pea protein isolate can be improved by using selected additives, giving new possibilities for plant protein application. Relevant technofunctional properties were determined and compared with two commercially available pea-based products (whole pea flour and an isolate). Water binding capacity was highest for the commercially available pea flour followed by the pea hull flour. Fat binding capacity remained more or less unchanged. (C) 2015 Elsevier Ltd. All rights reserved.
Scope: Arsenic-containing hydrocarbons (AsHCs) and arsenic-containing fatty acids (AsFAs) represent two classes of arsenolipids occurring naturally in marine food. Toxicological data are yet scarce and an assessment regarding the risk to human health has not been possible. Here, we investigated the transfer and presystemic metabolism of five arsenolipids in an intestinal barrier model.
Methods and results: Three AsHCs and two AsFAs were applied to the Caco-2 intestinal barrier model. Thereby, the short-chain AsHCs reached up to 50% permeability. Transport is likely to occur via passive diffusion. The AsFAs showed lower intestinal bioavailability, but respective permeabilities were still two to five times higher as compared to arsenobetaine or arsenosugars. Interestingly, AsFAs were effectively biotransformed while passing the in vitro intestinal barrier, whereas AsHCs were transported to the blood-facing compartment essentially unchanged.
Conclusion: AsFAs can be presystemically metabolised and the amount of transferred arsenic is lower than that for AsHCs. In contrast, AsHCs are likely to be highly intestinally bioavailable to humans. Since AsHCs exert strong toxicity in vitro and in vivo, toxicity studies with experimental animals as well as a human exposure assessment are needed to assess the risk to human health related to the presence of AsHCs in seafood.
The interaction between endothelial cells and pericytes is crucial for the stabilization of newly formed vessels in angiogenesis. The comprehension of the mechanisms regulating peiicyte recruitment might open therapeutical perspectives on vascular-related pathologies. Sphingosine 1phosphate (SIP) is a bioactive sphingolipid that derives from sphingomyelin catabolism and regulates biological functions in cell survival, proliferation, and differentiation. In this study, we aimed to identify the role of SIP axis in the intercellular communication between human mesenchymal progenitor mesoangioblasts (MAB) and endothelial cells (human microvascular endothelial cells (HMVEC)) in the formation of capillary-like structures. We demonstrated that the SIP biosynthetic pathway brought about by sphingosine kinases (SK) SKI and SK2 as well as spinster homolog 2 (SPNS2) transporter in H-MVEC is crucial for MAB migration measured by Boyden chambers and for the formation and stabilization of capillary-like structures in a 3D Matrigel culture. Moreover, the conditioned medium (CM) harvested from HMVEC, where SKI, 5K2, and SPNS2 were down-regulated, exerted a significantly diminished effect on MAB capillary morphogenesis and migration. Notably, we demonstrated that S I Pi and Si p3 receptors were positively involved in CM-induced capillary-like formation and migration, while S I P2 exerted a negative role on CM-induced migratory action of MAB. Finally, SK inhibition as well as MAB SlPi and S1P3 down-regulation impaired HMVEC-MAB cross-talk significantly reducing in vivo angiogenesis evaluated by Matrigel plug assay. These findings individuate novel targets for the employment of MAB in vascular-related pathologic conditions.
Background: A transient endothelial hyperpermeability is a hallmark of severe dengue infections. Sphingosine-1-phosphate (S1P) maintains vascular integrity and protects against plasma leakage. We related plasma S1P levels to dengue-induced plasma leakage and studied mechanisms that may underlie the decrease in S1P levels in dengue.
Methods: We determined circulating levels of S1P in 44 Indonesian adults with acute dengue and related levels to plasma leakage, as determined by daily ultrasonography, and to levels of its chaperone apolipoprotein M, other lipoproteins and platelets.
Results: Plasma S1P levels were decreased during dengue and patients with plasma leakage had lower median levels compared to those without (638 vs. 745 nM; p < 0.01). ApoM and other lipoprotein levels were also decreased during dengue, but did not correlate to S1P levels. Platelet counts correlated positively with S1P levels, but S1P levels were not higher in frozen-thawed platelet rich plasma, arguing against platelets as an important cellular source of S1P in dengue.
Conclusions: Decreased plasma S1P levels during dengue are associated with plasma leakage. We speculate that decreased levels of ApoM underlies the lower S1P levels. Modulation of S1P levels and its receptors may be a novel therapeutic intervention to prevent plasma leakage in dengue. (C) 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
Reporter gene assays are widely used for the assessment of transcription factor activation following xenobiotic exposure of cells. A critical issue with such assays is the possibility of interference of test compounds with the test system, for example, by direct inhibition of the reporter enzyme. Here we show that the pyrrolizidine alkaloid heliotrine interferes with reporter signals derived from GAL4-based nuclear receptor transactivation assays by a mechanism independent of luciferase enzyme inhibition. These data highlight the necessity to conduct proper control experiments in order to avoid perturbation of reporter assays by test chemicals. (C) 2015 Elsevier Inc. All rights reserved.
Quantitative Bioimaging to Investigate the Uptake of Mercury Species in Drosophila melanogaster
(2015)
The uptake of mercury species in the model organism Drosophila melanogaster was investigated by elemental bioimaging using laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS). The mercury distribution in Drosophila melanogaster was analyzed for the three species mercury(II) chloride, methylmercury chloride, and thimerosal after intoxication. A respective analytical method was developed and applied to the analysis of the entire Drosophila melanogaster first, before a particular focus was directed to the cerebral areas of larvae and adult flies. For quantification of mercury, matrix-matched standards based on gelatin were prepared. Challenges of spatially dissolved mercury determination, namely, strong evaporation issues of the analytes and an inhomogeneous distribution of mercury in the standards due to interactions with cysteine containing proteins of the gelatin were successfully addressed by complexation with meso-2,3-dimercaptosuccinic acid (DMSA). No mercury was detected in the cerebral region for mercury(II) chloride, whereas both organic species showed the ability to cross the blood brain barrier. Quantitatively, the mercury level in the brain exceeded the fed concentration indicating mercury enrichment, which was approximately 3 times higher for methylmercury chloride than for thimerosal.
Background: Alcohol abuse is a major risk factor for somatic and neuropsychiatric diseases. Despite their potential clinical importance, little is known about the alterations of plasma glycerophospholipid (GPL) and sphingolipid (SPL) species associated with alcohol abuse.
Methods: Plasma GPL and SPL species were quantified using electrospray ionization tandem mass spectrometry in samples from 23 male alcohol-dependent patients before and after detoxification, as well as from 20 healthy male controls.
Results: A comparison of alcohol-dependent patients with controls revealed higher phosphatidylcholine (PC; P-value = 0.008) and phosphatidylinositol (PI; P-value = 0.001) concentrations in patients before detoxification, and higher PI (P-value = 0.001) and phosphatidylethanolamine (PE)-based plasmalogen (PEP; P-value = 0.003) concentrations after detoxification. Lysophosphatidylcholines (LPC) were increased by acute intoxication (P-value = 0.002). Sphingomyelin (SM) concentration increased during detoxification (P-value = 0.011). The concentration of SM 23:0 was lower in patients (P-value = 2.79 x 10(-5)), and the concentrations of ceramide Cer d18:1/16:0 and Cer d18:1/18:0 were higher in patients (P-value = 2.45 x 10(-5) and 3.73 x 10(-5)). Activity of lysosomal acid sphingomyelinase (ASM) in patients correlated positively with the concentrations of eight LPC species, while activity of secreted ASM was inversely correlated with several PE, PI and PC species, and positively correlated with the molar ratio of PC to SM (Pearson's r = 0.432; P-value = 0.039).
Conclusion: Plasma concentrations of numerous GPL and SPL species were altered in alcohol-dependent patients. These molecules might serve as potential biomarkers to improve the diagnosis of patients and to indicate health risks associated with alcohol abuse. Our study further indicates that there are strong interactions between plasma GPL concentrations and SPL metabolism. (C) 2015 Elsevier B.V. All rights reserved.
Efficacy, Safety & Modification of Albuminuria in Type 2 Diabetes Subjects with Renal Disease with LINAgliptin (MARLINA-T2D), a multicentre, multinational, randomized, double-blind, placebo-controlled, parallel-group, phase 3b clinical trial, aims to further define the potential renal effects of dipeptidyl peptidase-4 inhibition beyond glycaemic control. A total of 350 eligible individuals with inadequately controlled type 2 diabetes and evidence of renal disease are planned to be randomized in a 1:1 ratio to receive either linagliptin 5mg or placebo in addition to their stable glucose-lowering background therapy for 24weeks. Two predefined main endpoints will be tested in a hierarchical manner: (1) change from baseline in glycated haemoglobin and (2) time-weighted average of percentage change from baseline in urinary albumin-to-creatinine ratio. Both endpoints are sufficiently powered to test for superiority versus placebo after 24weeks with =0.05. MARLINA-T2D is the first of its class to prospectively explore both the glucose- and albuminuria-lowering potential of a dipeptidyl peptidase-4 inhibitor in patients with type 2 diabetes and evidence of renal disease.
Dopamine (DA) and serotonin (SRT) are monoamine neurotransmitters that play a key role in regulating the central and peripheral nervous system. Their impaired metabolism has been implicated in several neurological disorders, such as Parkinson's disease and depression. Consequently, it is imperative to monitor changes in levels of these low-abundant neurotransmitters and their role in mediating disease. For the first time, a rapid, specific and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of DA and SRT in the nematode Caenorhabditis elegans (C. elegans). This model organism offers a unique approach for studying the effect of various drugs and environmental conditions on neurotransmitter levels, given by the conserved DA and SRT biology, including synaptic release, trafficking and formation. We introduce a novel sample preparation protocol incorporating the usage of sodium thiosulfate in perchloric acid as extraction medium that assures high recovery of the relatively unstable neurotransmitters monitored. Moreover, the use of both deuterated internal standards and the multiple reaction monitoring (MRM) technique allows for unequivocal quantification. Thereby, to the best of our knowledge, we achieve a detection sensitivity that clearly exceeds those of published DA and SRT quantification methods in various matrices. We are the first to show that exposure of C elegans to the monoamine oxidase B (MAOB) inhibitor selegiline or the catechol-O-methyltransferase (COMT) inhibitor tolcapone, in order to block DA and SRT degradation, resulted in accumulation of the respective neurotransmitter. Assessment of a behavioral output of the dopaminergic system (basal slowing response) corroborated the analytical LC-MS/MS data. Thus, utilization of the C elegans model system in conjunction with our analytical method is well-suited to investigate drug-mediated modulation of the DA and SRT system in order to identify compounds with neuroprotective or regenerative properties. (C) 2015 Elsevier B.V. All rights reserved.
In this study, structural changes in micellar caseins and whey proteins due to high pressure - low temperature treatments (HPLT) were investigated and compared to changes caused by high pressure treatments at room temperature. Whey protein isolate (WPI) solutions as well as micellar casein (MC) dispersions and mixtures were treated at 500 MPa (pH 7.0 and 5.8) at room temperature, -15 degrees C and -35 degrees C. Surface hydrophobicity and accessible thiol groups remained nearly unchanged after HPLT treatments whereas HP treatments at room temperature caused an unfolding of the WPI, resulting in an increase in surface hydrophobicity and exposure of the thiol groups. For HPLT treatments, distinct changes in the secondary structure (increase in the amount of beta-sheets) were observed while the tertiary structure remained unchanged. Large flocs, stabilized by hydrophobic interactions and hydrogen bonds, were formed in casein containing samples due to HPLT treatments. Depending on the pH and the applied HPLT treatment parameters, these interactions differed significantly from the interactions determined in native micelles. (C) 2015 Elsevier Ltd. All rights reserved.