Discovery of β-1,4-d-Mannosyl-N-acetyl-d-glucosamine Phosphorylase Involved in the Metabolism of N-Glycans

A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, β-N-acetylhexosaminidase, exo-α-sialidase, and endo-β-N-acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of β-1,4-d-mannosyl-N-acetyl-d-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where β-1,4-d-mannosyl-N-acetyl-d-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-d-mannose 1-phosphate and N-acetyl-d-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where β-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the d-mannose residue of β-1,4-d-mannosyl-N-acetyl-d-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a phosphorylase. We propose 4-O-β-d-mannopyranosyl-N-acetyl-d-glucosamine:phosphate α-d-mannosyltransferase as the systematic name and β-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase as the short name for BT1033.     

Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.     

Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

The stimulation by Mg²⁺, Na⁺, K⁺, NH₄ ⁺, and ATP of (Na⁺, K⁺)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na⁺, K⁺)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M _r. Under saturating Mg²⁺, Na⁺, and K⁺ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V _M = 115.0 ± 2.3 U mg^?1, K _0.5 = 0.10 ± 0.01 mmol L^?1. Stimulation by Na⁺ (V _M = 110.0 ± 3.3 U mg^?1, K _0.5 = 1.30 ± 0.03 mmol L^?1), Mg²⁺ (V _M = 115.0 ± 4.6 U mg^?1, K _0.5 = 0.96 ± 0.03 mmol L^?1), NH₄ ⁺ (V _M = 141.0 ± 5.6 U mg^?1, K _0.5 = 1.90 ± 0.04 mmol L^?1), and K⁺ (V _M = 120.0 ± 2.4 U mg^?1, K _M = 2.74 ± 0.08 mmol L^?1) followed single saturation curves and, except for K⁺, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K _I = 12.4 ± 1.3 mol L^?1. Complementary inhibition studies suggest the presence of F₀F₁–, Na⁺-, or K⁺-ATPases, but not V(H⁺)- or Ca²⁺-ATPases, in the gill microsomal preparation. K⁺ and NH₄ ⁺ synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH₄ ⁺, and K⁺ of the gill enzyme.     

Mammalian Target of Rapamycin (mTor) Mediates Tau Protein Dyshomeostasis: IMPLICATION FOR ALZHEIMER DISEASE*

Previous evidence from post-mortem Alzheimer disease (AD) brains and drug (especially rapamycin)-oriented in vitro and in vivo models implicated an aberrant accumulation of the mammalian target of rapamycin (mTor) in tangle-bearing neurons in AD brains and its role in the formation of abnormally hyperphosphorylated tau. Compelling evidence indicated that the sequential molecular events such as the synthesis and phosphorylation of tau can be regulated through p70 S6 kinase, the well characterized immediate downstream target of mTor. In the present study, we further identified that the active form of mTor per se accumulates in tangle-bearing neurons, particularly those at early stages in AD brains. By using mass spectrometry and Western blotting, we identified three phosphoepitopes of tau directly phosphorylated by mTor. We have developed a variety of stable cell lines with genetic modification of mTor activity using SH-SY5Y neuroblastoma cells as background. In these cellular systems, we not only confirmed the tau phosphorylation sites found in vitro but also found that mTor mediates the synthesis and aggregation of tau, resulting in compromised microtubule stability. Changes of mTor activity cause fluctuation of the level of a battery of tau kinases such as protein kinase A, v-Akt murine thymoma viral oncogene homolog-1, glycogen synthase kinase 3β, cyclin-dependent kinase 5, and tau protein phosphatase 2A. These results implicate mTor in promoting an imbalance of tau homeostasis, a condition required for neurons to maintain physiological function.     

Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR

Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.     

EVOLUTIONARY INFERENCES FROM THE ANALYSIS OF EXCHANGEABILITY

Evolutionary inferences are usually based on statistical models that compare mean genotypes or phenotypes (or their frequencies) among populations. An alternative is to use the full distribution of genotypes and phenotypes to infer the “exchangeability” of individuals among populations. We illustrate this approach by using discriminant functions on principal components to classify individuals among paired lake and stream populations of threespine stickleback in each of six independent watersheds. Classification based on neutral and nonneutral microsatellite markers was highest to the population of origin and next highest to populations in the same watershed. These patterns are consistent with the influence of historical contingency (separate colonization of each watershed) and subsequent gene flow (within but not between watersheds). In comparison to this low genetic exchangeability, ecological (diet) and morphological (trophic and armor traits) exchangeability was relatively high—particularly among populations from similar habitats. These patterns reflect the role of natural selection in driving parallel adaptive changes when independent populations colonize similar habitats. Importantly, however, substantial nonparallelism was also evident. Our results show that analyses based on exchangeability can confirm inferences based on statistical analyses of means or frequencies, while also refining insights into the drivers of—and constraints on—evolutionary diversification.     

Different Head Environments in Tarantula Thick Filaments Support a?Cooperative Activation Process

Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). Structural analysis of relaxed tarantula thick filaments shows that the RLCs of the interacting free and blocked myosin heads are in different environments. This and other data suggested a phosphorylation mechanism in which Ser-35 of the free head is exposed and constitutively phosphorylated by protein kinase C, whereas the blocked head is hidden and unphosphorylated; on activation, myosin light chain kinase phosphorylates the monophosphorylated free head followed by the unphosphorylated blocked head, both at Ser-45. Our goal was to test this model of phosphorylation. Mass spectrometry of quickly frozen, intact muscles showed that only Ser-35 was phosphorylated in the relaxed state. The location of this constitutively phosphorylated Ser-35 was analyzed by immunofluorescence, using antibodies specific for unphosphorylated or phosphorylated Ser-35. In the relaxed state, myofibrils were labeled by anti-pSer-35 but not by anti-Ser-35, whereas in rigor, labeling was similar with both. This suggests that only pSer-35 is exposed in the relaxed state, while in rigor, Ser-35 is also exposed. In the interacting-head motif of relaxed filaments, only the free head RLCs are exposed, suggesting that the constitutive pSer-35 is on the free heads, consistent with the proposed mechanism.     

Reducing boll weevil populations by clipping terminal buds and removing abscised fruiting bodies

Cotton pests damaging fruiting bodies (squares and young bolls) are difficult to control and their damage results in direct yield loss. Small growers, with low technological inputs, represent a large portion of cotton growers worldwide comprising more than 76 countries; they rely mainly on cultural practices to counteract pest attack in their crops. Boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae), oviposition involves puncturing cotton squares and young bolls, causing abscission. We examined the impact on boll weevil population of collecting abscised cotton fruiting bodies and clipping plant terminals at 50% boll maturation in the field during two cotton‐growing seasons and under field cage conditions. Greatest numbers of damaged squares occurred ca. 117 days after planting and clipped plants resulted in reduction of abscised structures and adult boll weevils compared with non‐clipped plants, irrespective of cotton variety. Damaged young bolls were found ca. 128 days after planting in 2009 and 2011, but clipping had no effect. Numbers of boll weevils found in plants of the varieties BRS 201 and BRS Rubi (both in 2009) and BRS Rubi (in 2011) were, respectively, 13‐, 17‐, and 20‐fold greater when clipping plus collecting abscised fruiting bodies were not practiced. Furthermore, the average percentage of the boll weevil parasitoid Bracon vulgaris Ashmead (Hymenoptera: Braconidae) emerging from abscised and collected structures was similar between clipped and non‐clipped plant terminals in both seasons. Clipping plant terminals did not result in yield reduction and reduced adult boll weevil production. Collecting abscised reproductive structures, clipping plant terminals, and using both practices together reduced boll weevil populations by as much as 63, 57, and 79%, respectively, in cage trials. Thus, these practices cause significant impact on boll weevil populations and are feasible of adoption, especially for smallholder cotton growers.     

E. coli outbreak in a neonate intensive care unit in a general hospital in Mexico City

Nosocomial infections are a major cause of morbidity and mortality among neonates admitted to neonatal intensive care units (NICUs). The aim of this paper was to describe an outbreak of Escherichia coli among infants admitted to the NICU of the General Hospital “Dr. Manuel Gea Gonzalez” in May of 2008. The isolated E. coli strains were identified using standard biochemical methods. The susceptibilities of these strains were analysed by determining their minimal inhibitory concentrations. Following this, their molecular relationships to each other were assessed by pulsed field gel electrophoresis (PFGE) analysis and corroborated by serology. Twelve E. coli strains were isolated from blood, urine, or indwelling catheter samples from five cases of preterm infants within a 3-day period. Patients were admitted to the NICU of the general hospital and, during the outbreak, developed sepsis caused by E. coli. For four of the patients, the average age was 23 days, while one patient was a 3-month-old infant. Prior to sepsis, the infants had received assisted ventilation and hyperalimentation through a central venous catheter. Two profiles were observed by PFGE; profile A was identified as the outbreak’s cause and an outcome of cross-infection, while profile B showed genetic differences but serologically it was identified as part of the same serotype. We conclude that E. coli colonised the patients through horizontal transmission. A focal source of the microorganism in this outbreak was not identified, but cross-transmission through handling was the most probable route.     

Quinones bearing non-steroidal anti-inflammatory fragments as multitarget ligands for Alzheimer’s disease

The anti-amyloid properties shared by several quinones inspired the design of a new series of hybrids derived from the multi-target drug candidate memoquin (1). The hybrids consist of a central benzoquinone core and a fragment taken from non-steroidal anti-inflammatory drugs, connected through polyamine linkers. The new hybrids retain the potent anti-aggregating activity of the parent 1, while exhibiting micromolar AChE inhibitory activities. Remarkably, 2, 4, (R)-6 and (S)-6 were Aβ aggregation inhibitors even more potent than 1. The balanced amyloid/cholinesterase inhibitory profile is an added value that makes the present series of compounds promising leads against Alzheimer’s disease.