Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6–7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2’-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides.     

Multilocus and morphological analysis of south-eastern Iberian Wall lizards (Squamata,Podarcis)

The phylogenetic relationships among the wall lizards of the Podarcis hispanicus complex that inhabit the south-east (SE) of the Iberian Peninsula and other lineages of the complex remain unclear. In this study, four mitochondrial and two nuclear markers were used to study genetic relationships within this complex. The phylogenetic analyses based on mtDNA gene trees constructed with ML and BI, and a species tree using *BEAST support three divergent clades in this region: the Valencia, Galera and Albacete/Murcia lineages. These three lineages were also corroborated in species delimitation analyses based on mtDNA using bPTP, mPTP, GMYC, ABGD and BAPS. Bayesian inference species delimitation method (BPP) based on both nuclear data and a combined data set (mtDNA + nuclear) showed high posterior probabilities for these three SE lineages (≥0.94) and another Bayesian analysis (STACEY) based on combined data set recovered the same three groups in this region. Divergence time dating of the species tree provided an estimated divergence of the Galera lineage from the other SE group (Podarcis vaucheri, (Albacete/Murcia, Valencia)) at 12.48 Ma. During this period, the Betic–Rifian arc was isolated, which could have caused the isolation of the Galera form distributed to the south of the Betic Corridor. Although lizards from the Albacete/Murcia and Galera lineage are morphologically similar, they clearly represent distinct genetic lineages. The noteworthy separation of the Galera lineage enables us to conclude that this lineage must be considered as a new full species.     

Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria

Although diabetes has been identified as a major risk factor for atrial fibrillation, little is known about glucose metabolism in the healthy and diabetic atria. Glucose transport into the cell, the rate-limiting step of glucose utilization, is regulated by the Glucose Transporters (GLUTs). Although GLUT4 is the major isoform in the heart, GLUT8 has recently emerged as a novel cardiac isoform. We hypothesized that GLUT-4 and -8 translocation to the atrial cell surface will be regulated by insulin and impaired during insulin-dependent diabetes. GLUT protein content was measured by Western blotting in healthy cardiac myocytes and type 1 (streptozotocin-induced, T1Dx) diabetic rodents. Active cell surface GLUT content was measured using a biotinylated photolabeled assay in the perfused heart. In the healthy atria, insulin stimulation increased both GLUT-4 and -8 translocation to the cell surface (by 100% and 240%, respectively, P<0.05). Upon insulin stimulation, we reported an increase in Akt (Th308 and s473 sites) and AS160 phosphorylation, which was positively (P<0.05) correlated with GLUT4 protein content in the healthy atria. During diabetes, active cell surface GLUT-4 and -8 content was downregulated in the atria (by 70% and 90%, respectively, P<0.05). Akt and AS160 phosphorylation was not impaired in the diabetic atria, suggesting the presence of an intact insulin signaling pathway. This was confirmed by the rescued translocation of GLUT-4 and -8 to the atrial cell surface upon insulin stimulation in the atria of type 1 diabetic subjects. In conclusion, our data suggest that: 1) both GLUT-4 and -8 are insulin-sensitive in the healthy atria through an Akt/AS160 dependent pathway; 2) GLUT-4 and -8 trafficking is impaired in the diabetic atria and rescued by insulin treatment. Alterations in atrial glucose transport may induce perturbations in energy production, which may provide a metabolic substrate for atrial fibrillation during diabetes.     

Carbonic anhydrase inhibitors: Inhibition of the tumor-associated isozymes IX and XII with polyfluorinated aromatic/heterocyclic sulfonamides

The tumor-associated transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozymes IX (CA IX) and XII (CA XII) are involved in acidification of hypoxic tumors, a process correlated with poor prognosis and clinical outcome of patients harboring such tumors. This process may be reversed by inhibiting these enzymes with potent sulfonamide/sulfamate inhibitors. A series of such aromatic/heterocyclic sulfonamides incorporating 2,3,5,6-tetrafluorobenzoyl-, 2,3,5,6-tetrafluoro- phenylsulfonyl- and pentafluorophenylureido moieties has been investigated for its interaction with the catalytic domain of the human isozymes hCA IX and hCA XII. Some of these compounds showed excellent inhibitory properties against both isozymes IX and XII, with several subnanomolar inhibitors detected for the first time. These sulfonamides may constitute valuable candidates for the development of novel antitumor therapies based on the inhibition of such tumor-associated CA isozymes.     

Disentangling the link between leaf photosynthesis and turgor in fruit growth

Despite the importance of understanding plant growth, the mechanisms underlying how plant and fruit growth declines during drought remain poorly understood. Specifically, it remains unresolved whether carbon or water factors are responsible for limiting growth as drought progresses. We examine questions regarding the relative importance of water and carbon to fruit growth depending on the water deficit level and the fruit growth stage by measuring fruit diameter, leaf photosynthesis, and a proxy of cell turgor in olive (Olea europaea). Flow cytometry was also applied to determine the fruit cell division stage. We found that photosynthesis and turgor were related to fruit growth; specifically, the relative importance of photosynthesis was higher during periods of more intense cell division, while turgor had higher relative importance in periods where cell division comes close to ceasing and fruit growth is dependent mainly on cell expansion. This pattern was found regardless of the water deficit level, although turgor and growth ceased at more similar values of leaf water potential than photosynthesis. Cell division occurred even when fruit growth seemed to stop under water deficit conditions, which likely helped fruits to grow disproportionately when trees were hydrated again, compensating for periods with low turgor. As a result, the final fruit size was not severely penalized. We conclude that carbon and water processes are able to explain fruit growth, with importance placed on the combination of cell division and expansion. However, the major limitation to growth is turgor, which adds evidence to the sink limitation hypothesis.     

Colonization and extinction of land planarians (Platyhelminthes,Tricladida) in a Brazilian Atlantic Forest regrowth remnant

Long-term assessments of species assemblages are valuable tools for detecting species ecological preferences and their dispersal tracks, as well as for assessing the possible effects of alien species on native communities. Here we report a 50-year-long study on population dynamics of the four species of land flatworms (Platyhelminthes, Tricladida, Terricola) that have colonized or become extinct in a 70-year-old Atlantic Forest regrowth remnant through the period 1955–2006. On the one hand, the two initially most abundant species, which are native to the study site, Notogynaphallia ernesti and Geoplana multicolor have declined over decades and at present do not exist in the forest remnant. The extinction of these species is most likely related with their preference for open vegetation areas, which presently do not exist in the forest remnant. On the other hand, the neotropical Geoplaninae 1 and the exotic Endeavouria septemlineata were detected in the forest only very recently. The long-term study allowed us to conclude that Geoplaninae 1 was introduced into the study area, although it is only known from the study site. Endeavouria septemlineata, an active predator of the exotic giant African snail, is originally known from Hawaii. This land flatworm species was observed repeatedly in Brazilian anthropogenic areas, and this is the first report of the species in relatively well preserved native forest, which may be evidence of an ongoing adaptive process. Monitoring of its geographic spread and its ecological role would be a good practice for preventing potential damaging effects, since it also feeds on native mollusk fauna, as we observed in lab conditions. Júlio Pedroni: Granted by CNPQ–Brazil.     

Virtual brain endocast of Antifer (Mammalia: Cervidae), an extinct large cervid from South America

A diverse fossil record of Cervidae (Mammalia) has been documented in the South American Pleistocene, when these animals arrived during the Great American Biotic Interchange. Using computed tomography-scanning techniques, it is possible to access the endocranial morphology of extinct species. Here, we studied the brain endocast of the extinct late Pleistocene cervid Antifer ensenadensis from southern Brazil, one of the largest forms that lived on this continent, using comparative morphology, geometric morphometrics, and encephalization quotients. The analyzed endocasts demonstrate that A. ensenadensis had a gyrencephalic brain, showing a prominent longitudinal sinus (=sagittal superior sinus), which is also observed in the large South American cervid Blastocerus dichotomus. The encephalization quotient is within the variation of extant cervids, suggesting maintenance of the pattern of encephalization from at least the late Pleistocene. Geometric morphometric analysis suggested a clear and linear allometric trend between brain endocast size and shape, and highlights A. ensenadensis as an extreme form within the analyzed cervids regarding brain morphology.     

Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS*

The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2_SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally “opens” CB2_SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.