Overexpression of the dual-specificity phosphatase MKP-4/DUSP-9 protects against stress-induced insulin resistance

Insulin resistance, a hallmark of type 2 diabetes and obesity, is associated with increased activity of MAP and stress-activated protein (SAP) kinases, which results in decreased insulin signaling. Our goal was to investigate the role of MAP kinase phosphatase-4 (MKP-4) in modulating this process. We found that MKP-4 expression is up-regulated during adipocyte and myocyte differentiation in vitro and up-regulated during fasting in white adipose tissue in vivo. Overexpression of MKP-4 in 3T3-L1 cells inhibited ERK and JNK phosphorylation and, to a lesser extent, p38MAPK phosphorylation. As a result, the phosphorylation of IRS-1 serine 307 induced by anisomycin was abolished, leading to a sensitization of insulin signaling with recovery of insulin-stimulated IRS-1 tyrosine phosphorylation, IRS-1 docking with phosphatidylinositol 3-kinase, and Akt phosphorylation. MKP-4 also reversed the effect of TNF-alpha to inhibit insulin signaling; alter IL-6, Glut1 and Glut4 expression; and inhibit insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Overexpression of MKP-4 in the liver of ob/ob mice decreased ERK and JNK phosphorylation, leading to a reduction in fed and fasted glycemia, improved glucose intolerance, decreased expression of gluconeogenic and lipogenic genes, and reduced hepatic steatosis. Thus, MKP-4 has a protective effect against the development of insulin resistance through its ability to dephosphorylate and inactivate crucial mediators of stress-induced insulin resistance, such as ERK and JNK, and increasing MKP-4 activity might provide a therapy for insulin-resistant disorders.     

A second observation of the rare frameshift mutation in the β-globin gene: codon 46 (+A) (Hbb:c.138_139insA)

The preparation of a prenatal diagnosis in a family of North-African origin in which a child received a bone marrow transplant for β-thalassemia major (β-TM), prompted us to make the molecular diagnosis in the parents and siblings. Molecular and phenotype assays were carried on blood samples from the parents and the proband's sister. The father, a 45-year-old man, was found to be heterozygous for a rare mutation in exon 2 [codon 46 (+A), HBB:c.138_139insA] creating a frameshift, while the mother and sister were found to be carriers of the common codon 39 (C>T) stop mutation (HBB:c.118C>T). Because of the bone marrow transplant, proband genotyping was done from a buccal swab and revealed that he is a compound heterozygote for both the codon 46 and codon 39 mutations. In the parents and sister, hematological parameters were those of a thalassemia minor in agreement with the two β(0) mutations found in the family.     

Risk, determinants, and outcome implications of progression of mitral regurgitation after diagnosis of mitral valve prolapse in a single community

To analyze the evolution of mitral regurgitation (MR) after the diagnosis of mitral valve prolapse in community patients, 285 residents of Olmsted County, Minnesota, diagnosed with mitral valve prolapse without severe MR were studied. MR grade was assessed at diagnosis and at follow-up 1,663 +/- 1,079 days later using Doppler echocardiography. The progression of MR was defined as an increase of > or =1 MR grade. The patients' mean age was 56 +/- 22 years, 57% were women, and the mean ejection fraction was 60 +/- 9%. Between diagnostic and follow-up echocardiography, 108 patients showed progression of MR, 39 of whom had progression > or =1 grade. The mean overall MR grade increased from 0.4 +/- 0.7 to 0.9 +/- 1.1 (p <0.01). The progression of MR was observed in all subsets, irrespective of age, gender, prolapse localization, leaflet thickening, and initial MR grade. However, multivariate analysis identified age (p <0.01) and initial MR grade (p = 0.01) as independent predictors of progression. In addition, MR progression was associated with greater left atrial enlargement (p <0.001), ventricular dilatation (p = 0.02 for increase in end-diastolic and end-systolic diameters), and a worse outcome (adjusted p = 0.001). In conclusion, in patients with mitral valve prolapse, MR progression was observed over time in all clinical and anatomic subsets and was associated with more severe ventricular and atrial remodeling and worse outcome.     

5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside reduces glucose uptake via the inhibition of Na+/H+ exchanger 1 in isolated rat ventricular cardiomyocytes

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is activated by an increased AMP/ATP ratio. AMPK is now well recognized to induce glucose uptake in skeletal muscle and heart. 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) is phosphorylated to form the AMP analog ZMP, which activates AMPK. Its effects on glucose transport appear to be tissue specific. The purpose of our study was to examine the effect of AICAR on insulin-induced glucose uptake in adult rat ventricular cardiomyocytes. We studied isolated adult rat ventricular cardiomyocytes treated or not with the AMPK activators AICAR and metformin and, subsequently, with insulin or not. Insulin action was investigated by determining deoxyglucose uptake, insulin receptor substrate-1- or -2-associated phosphatidylinositol 3-kinase activity and protein kinase B (PKB) cascade using antibodies to PKB, glycogen synthase kinase-3, and Akt substrate of 160 kDa. Intracellular pH was evaluated using the fluorescent pH-sensitive dye 2',7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Na(+)/H(+) exchanger 1 (NHE1) activity was assessed using the NH(4)(+) prepulse method. Our key findings are as follows. AICAR and metformin enhance insulin signaling downstream of PKB. Metformin potentiates insulin-induced glucose uptake, but surprisingly, AICAR inhibits both basal and insulin-induced glucose uptake. Moreover, we found that AICAR decreases intracellular pH, via inhibition of NHE1. In conclusion, AMPK potentiates insulin signaling downstream of PKB in isolated cardiac myocytes, consistent with findings in the heart in vivo. Furthermore, AICAR inhibits basal and insulin-induced glucose uptake in isolated cardiac myocytes via the inhibition of NHE1 and the subsequent reduction of intracellular pH. Importantly, AICAR exerts these effects in a manner independent of AMPK activation.     

Development of a recombinant antithrombin variant as a potent antidote to fondaparinux and other heparin derivatives

Heparin derivative-based therapy has evolved from unfractionated heparin (UFH) to low-molecular-weight heparins (LMWHs) and now fondaparinux, a synthetic pentasaccharide. Contrary to UFH or LMWHs, fondaparinux is not neutralized by protamine sulfate, and no antidote is available to counteract bleeding disorders associated with overdosing. To make the use of fondaparinux safer, we developed an antithrombin (AT) variant as a potent antidote to heparin derivatives. This variant (AT-N135Q-Pro394) combines 2 mutations: substitution of Asn135 by a Gln to remove a glycosylation site and increase affinity for heparins, and the insertion of a Pro between Arg393 and Ser394 to abolish its anticoagulant activity. As expected, AT-N135Q-Pro394 anticoagulant activity was almost abolished, and it exhibited a 3-fold increase in fondaparinux affinity. AT-N135Q-Pro394 was shown to reverse fondaparinux overdosing in vitro in a dose-dependent manner through a competitive process with plasma AT for fondaparinux binding. This antidote effect was also observed in vivo: administration of AT-N135Q-Pro394 in 2.5-fold molar excess versus plasma AT neutralized 86% of the anti-Xa activity within 5 minutes in mice treated with fondaparinux. These results clearly demonstrate that AT-N135Q-Pro394 can reverse the anticoagulant activity of fondaparinux and thus could be used as an antidote for this drug.     

Kinetically inhibited order in a diamond-lattice antiferromagnet

Frustrated magnetic systems exhibit highly degenerate ground states and strong fluctuations, often leading to new physics. An intriguing example of current interest is the antiferromagnet on a diamond lattice, realized physically in A-site spinel materials. This is a prototypical system in three dimensions where frustration arises from competing interactions rather than purely geometric constraints, and theory suggests the possibility of unusual magnetic order at low temperature. Here, we present a comprehensive single-crystal neutron scattering study of CoAl₂O₄, a highly frustrated A-site spinel. We observe strong diffuse scattering that peaks at wavevectors associated with Néel ordering. Below the temperature T^∗ = 6.5 K, there is a dramatic change in the elastic scattering lineshape accompanied by the emergence of well-defined spin-wave excitations. T^∗ had previously been associated with the onset of glassy behavior. Our new results suggest instead that T^∗ signifies a first-order phase transition, but with true long-range order inhibited by the kinetic freezing of domain walls. This scenario might be expected to occur widely in frustrated systems containing first-order phase transitions and is a natural explanation for existing reports of anomalous glassy behavior in other materials.Frustration occurs in spin systems when constraints prevent the formation of a ground state satisfying all of the pairwise interactions. The defining characteristics of frustration are massive ground-state degeneracy and concomitant strong fluctuations. The latter suppress magnetic order and lead to spin-liquid regimes extending to low temperature. There has been great interest in such spin liquids because of the plethora of emergent phenomena (1–3) resulting from an extreme sensitivity to small, often neglected degeneracy-breaking effects. Noteworthy examples include unusual short-range correlations (4, 5), apparent spin-glass behavior in the absence of disorder (6–8), collective ring excitations in pyrochlore spinels (9–11), spontaneously reduced dimensionality in rare-earth titanates (12), and topological excitations like magnetic monopoles in spin-ice materials (13–15).The degeneracy-breaking effects can be small terms in the interaction Hamiltonian that enter the problem in a nonperturbative way. Examples include dipolar interactions in the pyrochlores (16) or spin-lattice interactions in multiferroics (17). Interestingly, degeneracy can also be broken in the complete absence of such interaction terms, through a mechanism known as “order-by-disorder” (18). Order-by-disorder refers to the scenario where the entropy associated with thermal or quantum fluctuations lowers the free energy of one ordered state compared to others of equal energy, thereby stabilizing order. Order-by-disorder was first predicted in studies of the diluted Ising model on a rectangular lattice (18). It has since emerged as a central feature in theories of various frustrated antiferromagnets, including the face-centered cubic (FCC) (19, 20) as well as some pyrochlore (21–24) and two-dimensional lattices (25–27). Despite the long history and ubiquity in theoretical models, few, if any, real materials have been found that exhibit this important phenomenon clearly.The classical (i.e., thermal-fluctuation induced) order-by-disorder mechanism often results in a first-order phase transition (4, 19–21). It is interesting to consider the possible implications of a first-order transition for the low-temperature properties of a frustrated magnetic system. Near and below the transition temperature, T_N, such a system is necessarily in a limit where the scale of thermal energies is small compared to that of the microscopic magnetic interactions. Below T_N, a possible scenario is nucleation at many sites into various near-degenerate ground states, resulting in multiple domains separated by antiphase domain walls. For the domains to grow, wall motion must be driven by thermal energy that is sufficient to overcome the magnetic interactions that dominate the surface energy of the walls. Because this condition is not met, the system is kinetically frozen in a state with small domains, and even though it is ordered, it can seem to exhibit glassy behavior regardless of the level of chemical disorder. To our knowledge, this scenario has not been proposed in the literature on frustrated magnets. However, here we present evidence that it occurs in the system CoAl₂O₄ and, moreover, explains anomalous behavior observed previously in other frustrated materials.CoAl₂O₄ is an “A-site spinel” material, where the S = 3/2 Co²⁺ ions occupy the tetrahedrally coordinated sites of the spinel structure, forming a diamond lattice. Theoretical investigations (4, 28, 29) have revealed particularly interesting physics in the diamond-lattice antiferromagnets arising from the frustrating effects of competing exchange interactions. The diamond-lattice antiferromagnet with only nearest-neighbor exchange (J₁) is not frustrated and orders with a collinear ground state as illustrated in Fig. 1. However, the lattice has only 4 nearest-neighbors versus 12 next-nearest-neighbors coupled via second neighbor exchange, J₂. Thus, even moderate J₂ is predicted to suppress the collinear order, leading to highly degenerate spin-liquid ground states (4) with order-by-disorder type transitions at low temperatures driven by thermal (4, 28) or quantum fluctuations (29). In particular, theory predicts that the point is both a tricritical point, where the Néel transition switches from second to first-order, and a Lifshitz point, where the low temperature ordered state changes from collinear antiferromagnetism to spin-spiral (4). In a limited range of J₂/J₁ just above , both collinear and spiral spin ordering transitions are predicted, with the upper Néel transition driven by the classical order-by-disorder mechanism (4).Open in a separate windowFig. 1.Structure of CoAl₂O₄. (A) Magnetic cobalt ions of CoAl₂O₄ on the A-site diamond-lattice. This can be deconstructed into two interpenetrating FCC sublattices (colored here blue and orange, respectively). Arrows represent the ordered state observed to exist in diamond-lattice antiferromagnets when nearest-neighbor exchange dominates. (B) The (H H L) plane of reciprocal space for the diamond-structure using cubic notation. Blue diamonds denote allowed structural Bragg peaks. Red circles denote allowed magnetic Bragg peaks for the ordered state shown in A. At reciprocal points indexed by three odd integers, both nuclear and magnetic Bragg scattering is expected. Green lines illustrate directions in reciprocal space for which spin-wave dispersions are plotted in Fig. 4.High-temperature magnetization measurements show strong antiferromagnetic interactions in CoAl₂O₄, with a Curie–Weiss temperature around -109 K. Despite this, only glass-like transitions have been reported at a greatly reduced T^∗ < 9 K (30–32), implying strong frustration. Powder neutron diffraction has revealed the emergence of short-range spin correlations at low temperatures (33), which was reproduced within the spiral-spin-liquid model of Bergman et al. by assuming . A further analysis of inelastic neutron powder data by Krimmel et al. (34) concluded —well into the regime where spin-spiral physics is expected. Contrarily, comparison of diffraction data and Monte Carlo calculations led Zaharko et al. (35) to conclude that , suggesting that the system might instead undergo a continuous transition to classical Néel order. To date, very little data exists on single crystals, although it has been emphasized on several occasions (4, 28, 34) that such data is essential to properly compare experiment and theory.To elucidate the nature of the low-temperature state and the significance of T^∗ in CoAl₂O₄, we have grown single crystals and studied them extensively with neutron scattering. X-ray diffraction showed the crystals to be of high purity with no discernable disorder and minimal Co–Al inversion, refining to 2% with an uncertainty of 4%. The magnetization data shows a cusp at T^∗ ≈ 6.5 K, in the neighborhood of a broad peak in the heat capacity, consistent with results reported for powders. Characterization of the samples is further discussed in SI Text. Neutron studies of both static magnetic correlations and spin excitations were performed using the High Flux Isotope Reactor (HFIR) and Spallation Neutron Source (SNS) facilities of Oak Ridge National Laboratory (ORNL).     

How can probiotics and prebiotics impact mucosal immunity?

The study of probiotics and prebiotics is an expanding field of interest and scientific research that has resulted in insights related to the host immune response. Recent advances have naturally led to key questions. What are the specific probiotic components that mediate immunomodulation? Can we extrapolate the results of in vitro studies in animal and human trials? Which biomarkers and immune parameters should be measured in probiotic and prebiotic intervention studies? These questions were part of a discussion entitled “How Can Probiotics and Prebiotics Impact Mucosal Immunity” at the 2009 Annual Meeting of the International Scientific Association for Probiotics and Prebiotics (ISAPP). This review highlights recent knowledge about the modulation of mucosal immunity by probiotics and prebiotics, as well as considerations for measuring their effects on mucosal immunity. A list of biomarkers and immune parameters to be measured in human clinical trials is included.Key words: probiotic, prebiotic, immunomodulation, immune, human trials, biomarkers, gastrointestinal tract, microbiota     

Delayed diagnosis of congenital adrenal hyperplasia with salt wasting due to type II 3beta-hydroxysteroid dehydrogenase deficiency

Classical 3beta-hydroxysteroid dehydrogenase (3beta-HSD) deficiency is a rare cause of congenital adrenal hyperplasia. We report two sisters presenting with delayed diagnoses of classical 3beta-HSD, despite salt wasting (SW) episodes in infancy. Sibling 1 was referred for premature pubarche, slight growth acceleration, and advanced bone age, whereas sibling 2 had no signs of virilization. At referral, increased 17alpha-hydroxyprogesterone associated with premature pubarche at first suggested a nonclassical 21-hydroxylase deficiency. Sequencing of the CYP21 gene showed both girls only heterozygotes (V281L mutation). This result, combined with SW in infancy, suggested a 3beta-HSD deficiency because of increased dehydroepiandrosterone sulfate levels. Further hormonal studies showed markedly elevated Delta5-steroids, in particular 17alpha-hydroxypregnenolone greater than 100 nmol/liter (the clue to the diagnosis) and elevated Delta5-/Delta4-steroid ratios. Sequencing of the type II 3beta-HSD gene documented that both girls were compound heterozygotes for T181I and 1105delA mutations. Retrospectively, elevated levels of 17alpha-hydroxyprogesterone were found on blood spots from Guthrie's test. There is no previous report of the combination of SW and premature pubarche due to mutations in the type II 3beta-HSD gene. Because neonatal diagnosis could have prevented life-threatening crises in these girls, this report further supports the benefits for neonatal screening for congenital adrenal hyperplasia whatever the etiology.     

Prevalence of Electrocardiographic and Echocardiographic Abnormalities in Ambulatory Ischemic Colitis

The aim of this study was to evaluate the prevalence of cardiac arrhythmia and intracardiac embolic process in ambulatory ischemic colitis. From November 1994 to November 1997, 33 consecutive cases of ambulatory ischemic colitis were detected. This study included 21 women and 12 men with a mean age of 71 years. All patients underwent a cardiovascular investigation including questioning, electrocardiogram, 24-hr ambulatory electrocardiography and transthoracic echocardiography. A prior history of ischemic colitis was found in four cases (12%). Cardiac arrhythmia was detected in eight cases. Transthoracic echocardiography showed an intracardiac process, potentially responsible for a peripheral embolism, in four cases. In conclusion, the aggregate, in 33% of the patients, there was potential cardiac etiology. This suggests that when ambulatory ischemic colitis occurs, it is necessary to perform an exhaustive cardiovascular evaluation similar to those performed in other ischemic diseases.