Synthesis of two carboxylic haptens for raising antibodies to 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3

Hapten derivatives of 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) were synthesized using the Wittig-Horner approach. Both haptens bearing a carboxylic group at the side chain that can be linked to a protein for raising antibodies of potential utility for the determination of 25-hydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxylated vitamin D(3) analogues.     

Loss of MT1‐MMP causes cell senescence and nuclear defects which can be reversed by retinoic acid

MT1-MMP (MMP14) is a collagenolytic enzyme located at the cell surface and implicated in extracellular matrix (ECM) remodeling. Mmp14^−/− mice present dwarfism, bone abnormalities, and premature death. We demonstrate herein that the loss of MT1-MMP also causes cardiac defects and severe metabolic changes, and alters the cytoskeleton and the nuclear lamina structure. Moreover, the absence of MT1-MMP induces a senescent phenotype characterized by up-regulation of p16^INK4a and p21^CIP1/WAF1, increased activity of senescence-associated β-galactosidase, generation of a senescence-associated secretory phenotype, and somatotroph axis alterations. Consistent with the role of retinoic acid signaling in nuclear lamina stabilization, treatment of Mmp14^−/− mice with all-trans retinoic acid reversed the nuclear lamina alterations, partially rescued the cell senescence phenotypes, ameliorated the pathological defects in bone, skin, and heart, and extended their life span. These results demonstrate that nuclear architecture and cell senescence can be modulated by a membrane protease, in a process involving the ECM as a key regulator of nuclear stiffness under cell stress conditions.     

Metazoans of redoxcline sediments in Mediterranean deep-sea hypersaline anoxic basins

Background

The deep-sea hypersaline anoxic basins (DHABs) of the Mediterranean (water depth ~3500 m) are some of the most extreme oceanic habitats known. Brines of DHABs are nearly saturated with salt, leading many to suspect they are uninhabitable for eukaryotes. While diverse bacterial and protistan communities are reported from some DHAB haloclines and brines, loriciferans are the only metazoan reported to inhabit the anoxic DHAB brines. Our goal was to further investigate metazoan communities in DHAB haloclines and brines.

Results

We report observations from sediments of three DHAB (Urania, Discovery, L’Atalante) haloclines, comparing these to observations from sediments underlying normoxic waters of typical Mediterranean salinity. Due to technical difficulties, sampling of the brines was not possible. Morphotype analysis indicates nematodes are the most abundant taxon; crustaceans, loriciferans and bryozoans were also noted. Among nematodes, Daptonema was the most abundant genus; three morphotypes were noted with a degree of endemicity. The majority of rRNA sequences were from planktonic taxa, suggesting that at least some individual metazoans were preserved and inactive. Nematode abundance data, in some cases determined from direct counts of sediments incubated in situ with CellTracker^TM Green, was patchy but generally indicates the highest abundances in either normoxic control samples or in upper halocline samples; nematodes were absent or very rare in lower halocline samples. Ultrastructural analysis indicates the nematodes in L’Atalante normoxic control sediments were fit, while specimens from L’Atalante upper halocline were healthy or had only recently died and those from the lower halocline had no identifiable organelles. Loriciferans, which were only rarely encountered, were found in both normoxic control samples as well as in Discovery and L’Atalante haloclines. It is not clear how a metazoan taxon could remain viable under this wide range of conditions.

Conclusions

We document a community of living nematodes in normoxic, normal saline deep-sea Mediterranean sediments and in the upper halocline portions of the DHABs. Occurrences of nematodes in mid-halocline and lower halocline samples did not provide compelling evidence of a living community in those zones. The possibility of a viable metazoan community in brines of DHABs is not supported by our data at this time.

     

Deep subcutaneous adipose tissue: a distinct abdominal adipose depot

Objective: Abdominal visceral (VAT) and subcutaneous adipose tissue (SAT) display significant metabolic differences, with VAT showing a functional association to metabolic/cardiovascular disorders. A third abdominal adipose layer, derived by the division of SAT and identified as deep subcutaneous adipose tissue (dSAT), may play a significant and independent metabolic role. The aim of this study was to evaluate depot‐specific differences in the expression of proteins key to adipocyte metabolism in a lean population to establish a potential physiologic role for dSAT. Research Methods and Procedures: Adipocytes and preadipocytes were isolated from whole biopsies taken from superficial SAT (sSAT), dSAT, and VAT samples obtained from 10 healthy normal weight patients (7 women and 3 men), with a mean age of 56.4 ± 4.04 years and a mean BMI of 23.1 ± 0.5 kg/m². Samples were evaluated for depot‐specific differences in insulin sensitivity using adiponectin, glucose transport protein 4 (GLUT4), and resistin mRNA and protein expression, glucocorticoid metabolism by 11β‐hydroxysteroid dehydrogenase type‐1 (11β‐HSD1) expression, and alterations in the adipokines leptin and tumor necrosis factor‐α (TNF‐α). Results: Although no regional differences in expression were observed for adiponectin or TNF‐α, dSAT whole biopsies and adipocytes, while intermediary to both sSAT and VAT, reflected more of the VAT expression profile of 11β‐HSD1, leptin, and resistin. Only in the case of the intracellular pool of GLUT4 proteins in whole biopsies was an independent pattern of expression observed for dSAT. In an evaluation of the homeostatic model, dSAT 11β‐HSD1 protein (r = 0.9573, p = 0.0002) and TNF‐α mRNA (r = 0.8210, p = 0.0236) correlated positively to the homeostatic model. Discussion: Overall, dSAT seems to be a distinct abdominal adipose depot supporting an independent metabolic function that may have a potential role in the development of obesity‐associated complications.     

Hypertriglyceridemia Is Independently Associated with Renal,but Not Retinal Complications in Subjects with Type 2 Diabetes: A Cross-Sectional Analysis of the Renal Insufficiency And Cardiovascular Events (RIACE) Italian Multicenter Study

Objective

Atherogenic dyslipidemia seems to play a major role in microvascular complications and in residual microvascular risk after statin therapy, which reduces triglycerides up to 40%. We assessed whether raised TG levels are associated with an increased burden from microvascular complications in patients with type 2 diabetes.

Methods

Subjects from the Renal Insufficiency And Cardiovascular Events (RIACE) Italian Multicentre Study (n=15,773) were divided in 4 groups depending on whether they had plasma triglycerides below (NTG, 67.8%) or above (HTG, 32.2%) 1.7 mmol/L and were (42.4%) or not on (57.6%) statin therapy. Estimated GFR (eGFR) was calculated from serum creatinine, albuminuria was measured by immunonephelometry or immunoturbidimetry, and retinopathy was evaluated by fundus examination.

Results

HTG subjects, either with or without statin, had higher prevalence of albuminuria, reduced eGFR and chronic kidney disease (CKD), especially the albuminuric forms, but not of retinopathy, than NTG subjects. In contrast, cardiovascular disease and advanced DR were more prevalent in subjects on statin than in those not, independently of triglyceride levels. Logistic regression analysis confirmed that HTG, without or with statin, was independently associated with micro and macroalbuminuria, mildly to severely reduced eGFR, and all CKD phenotypes, but not with retinopathy. The adjusted odd ratios for CKD increased linearly for every 0.26 mmol/L increase (approximately one decile) in triglyceride levels. The increase was higher with increasing severity of albuminuria, eGFR loss and CKD phenotype as well as in subjects receiving than in those not receiving statin treatment.

Conclusions

Triglycerides are associated with CKD, but not retinopathy in subjects with type 2 diabetes, independently of statin treatment. These data point to a possible role of hypertriglyceridemia in the development of CKD, though it remains to be demonstrated that diabetic individuals might benefit from triglyceride reduction with statins and eventually with combination therapy with fibrates.

Trial Registration

www.ClinicalTrials.gov NCT00715481     

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Cannabinoid CB₁ receptors peripherally modulate energy metabolism. Here, we investigated the role of CB₁ receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB₁ receptor antagonist AM251 (3 mg kg^-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB₁ receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB₁ receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB₁ receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB ₁ ^-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C₂C₁₂ myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB₁ receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.     

Trinuclear and tetranuclear adduct formation between sodium perchlorate and copper(II) complexes of salicylaldimine type ligands: Structural characterization and theoretical investigation

The synthesis of two new sodium perchlorate adducts (1:2 and 1:3) with copper(II) “ligand-complexes” is reported. One adduct is trinuclear [(CuL¹)₂NaClO₄] (1) and the other is tetranuclear [(CuL²)₃Na]ClO₄·EtOH (2). The ligands are the tetradentate di-Schiff base of 1,3-propanediamines and salicylaldehyde (H₂L¹) or 2-hydroxyacetophenone (H₂L²). Both complexes have been characterized by X-ray single crystal structure analyses. In both structures, the sodium cation has a six-coordinate distorted octahedral environment being bonded to four oxygen atoms from two Schiff-base complexes in addition to a chelated perchlorate anion in 1 and to six oxygen atoms from three Schiff-base complexes in 2. We have carried out a DFT theoretical study (RI-B97-D/def2-SVP level of theory) to compute and compare the formation energies of 1:2 and 1:3 adducts. The DFT study reveals that the latter is more stabilized than the former. The X-ray crystal structure of 1 shows that the packing of the trinuclear unit is controlled by unconventional C-H···O H-bonds and Cu²⁺-π non-covalent interactions. These interactions explain the formation of 1 which is a priori disfavored with respect to 2.     

Pharmacological activation/inhibition of the cannabinoid system affects alcohol withdrawal-induced neuronal hypersensitivity to excitotoxic insults

Cessation of chronic ethanol consumption can increase the sensitivity of the brain to excitotoxic damages. Cannabinoids have been proposed as neuroprotectants in different models of neuronal injury, but their effect have never been investigated in a context of excitotoxicity after alcohol cessation. Here we examined the effects of the pharmacological activation/inhibition of the endocannabinoid system in an in vitro model of chronic ethanol exposure and withdrawal followed by an excitotoxic challenge. Ethanol withdrawal increased N-methyl-D-aspartate (NMDA)-evoked neuronal death, probably by altering the ratio between GluN2A and GluN2B NMDA receptor subunits. The stimulation of the endocannabinoid system with the cannabinoid agonist HU-210 decreased NMDA-induced neuronal death exclusively in ethanol-withdrawn neurons. This neuroprotection could be explained by a decrease in NMDA-stimulated calcium influx after the administration of HU-210, found exclusively in ethanol-withdrawn neurons. By contrast, the inhibition of the cannabinoid system with the CB1 receptor antagonist rimonabant (SR141716) during ethanol withdrawal increased death of ethanol-withdrawn neurons without any modification of NMDA-stimulated calcium influx. Moreover, chronic administration of rimonabant increased NMDA-stimulated toxicity not only in withdrawn neurons, but also in control neurons. In summary, we show for the first time that the stimulation of the endocannabinoid system is protective against the hyperexcitability developed during alcohol withdrawal. By contrast, the blockade of the endocannabinoid system is highly counterproductive during alcohol withdrawal.     

The crystal structure of necrosis- and ethylene-inducing protein 2 from the causal agent of cacao's Witches' Broom disease reveals key elements for its activity

The necrosis- and ethylene-inducing peptide 1 (NEP1)-like proteins (NLPs) are proteins secreted from bacteria, fungi and oomycetes, triggering immune responses and cell death in dicotyledonous plants. Genomic-scale studies of Moniliophthora perniciosa, the fungus that causes the Witches' Broom disease in cacao, which is a serious economic concern for South and Central American crops, have identified five members of this family (termed MpNEP1-5). Here, we show by RNA-seq that MpNEP2 is virtually the only NLP expressed during the fungus infection. The quantitative real-time polymerase chain reaction results revealed that MpNEP2 has an expression pattern that positively correlates with the necrotic symptoms, with MpNEP2 reaching its highest level of expression at the advanced necrotic stage. To improve our understanding of MpNEP2's molecular mechanism of action, we determined the crystallographic structure of MpNEP2 at 1.8 ? resolution, unveiling some key structural features. The implications of a cation coordination found in the crystal structure were explored, and we show that MpNEP2, in contrast to another previously described member of the NLP family, NLP(Pya) from Pythium aphanidermatum, does not depend on an ion to accomplish its necrosis- and electrolyte leakage-promoting activities. Results of site-directed mutagenesis experiments confirmed the importance of a negatively charged cavity and an unforeseen hydrophobic β-hairpin loop for MpNEP2 activity, thus offering a platform for compound design with implications for disease control. Electron paramagnetic resonance and fluorescence assays with MpNEP2 performed in the presence of lipid vesicles of different compositions showed no sign of interaction between the protein and the lipids, implying that MpNEP2 likely requires other anchoring elements from the membrane to promote cytolysis or send death signals.     

Reduced expression of SIRT1 is associated with diminished glucose-induced insulin secretion in islets from calorie-restricted rats

Alterations in food intake such as caloric restriction modulate the expression of SIRT1 and SIRT4 proteins that are involved in pancreatic β-cell function. Here, we search for a possible relationship between insulin secretion and the expression of SIRT1, SIRT4, PKC and PKA in islets from adult rats submitted to CR for 21 days. Rats were fed with an isocaloric diet (CTL) or received 60% (CR) of the food ingested by CTL. The dose-response curve of insulin secretion to glucose was shifted to the right in the CR compared with CTL islets (EC₅₀ of 15.1±0.17 and 10.5±0.11 mmol/L glucose). Insulin release by the depolarizing agents arginine and KCl was reduced in CR compared with CTL islets. Total islet insulin content and glucose oxidation were also reduced in CR islets. Leucine-stimulated secretion was similar in both groups, slightly reduced in CR islets stimulated by leucine plus glutamine but higher in CR islets stimulated by ketoisocaproate (KIC). Insulin secretion was also higher in CR islets stimulated by carbachol, compared with CTL islets. No differences in the rise of cytosolic Ca²⁺ concentrations stimulated by either glucose or KCl were observed between groups of islets. Finally, SIRT1, but not SIRT4, protein expression was lower in CR compared with CTL islets, whereas no differences in the expression of PKC and PKA proteins were observed. In conclusion, the lower insulin secretion in islets from CR rats was, at least in part, due to an imbalance between the expression of SIRT1 and SIRT4.