Reactivity of Metabolic Intermediates and Cofactor Stability under Model Early Earth Conditions

Origins of Life and Evolution of Biospheres - Understanding the emergence of metabolic pathways is key to unraveling the factors that promoted the origin of life. One popular view is that protein...     

Structural and Thermodynamic Characterization of the TYK2 and JAK3 Kinase Domains in Complex with CP-690550 and CMP-6

Janus kinases (JAKs) are critical regulators of cytokine pathways and attractive targets of therapeutic value in both inflammatory and myeloproliferative diseases. Although the crystal structures of active JAK1 and JAK2 kinase domains have been reported recently with the clinical compound CP-690550, the structures of both TYK2 and JAK3 with CP-690550 have remained outstanding. Here, we report the crystal structures of TYK2, a first in class structure, and JAK3 in complex with PAN-JAK inhibitors CP-690550 ((3R,4R)-3-[4-methyl-3-[N-methyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropionitrile) and CMP-6 (tetracyclic pyridone 2-t-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one), both of which bind in the ATP-binding cavities of both JAK isozymes in orientations similar to that observed in crystal structures of JAK1 and JAK2. Additionally, a complete thermodynamic characterization of JAK/CP-690550 complex formation was completed by isothermal titration calorimetry, indicating the critical role of the nitrile group from the CP-690550 compound. Finally, computational analysis using WaterMap further highlights the critical positioning of the CP-690550 nitrile group in the displacement of an unfavorable water molecule beneath the glycine-rich loop. Taken together, the data emphasize the outstanding properties of the kinome-selective JAK inhibitor CP-690550, as well as the challenges in obtaining JAK isozyme-selective inhibitors due to the overall structural and sequence similarities between the TYK2, JAK1, JAK2 and JAK3 isozymes. Nevertheless, subtle amino acid variations of residues lining the ligand-binding cavity of the JAK enzymes, as well as the global positioning of the glycine-rich loop, might provide the initial clues to obtaining JAK-isozyme selective inhibitors.     

New finds of charophytes in Iceland with an update on the distribution of the charophyte flora

Charophyte species new to the flora of Iceland are reported and an update on the distribution of the previously known species is given. Four species are new to Iceland; Chara aspera Willdenow, Chara contraria Braun ex Kützing, Tolypella canadensis Sawa and Tolypella glomerata (Desvaux) Leonhardi, while four species were previously known: Chara globularis Thuiller, Chara virgata Kützing, Nitella flexilis (L.) Agardh and Nitella opaca (Agardh ex Bruzelius) Agardh. The finding of the species new to Iceland add to the hitherto known worldwide distribution of those species, including a significant extension to the north.     

Indigenous Knowledge and Management of Araucaria Araucana Forest in the Chilean Andes: Implications for Native Forest Conservation

Southern Chile experienced serious deforestation during the past century and it is projected that by the year 2025 Chile will be devoid of native forests. One of the most important endemic tree species of the country and at the same time one of the most endangered ones is Araucaria araucana (Mol.) C. Koch, the monkey-puzzle tree. It grows in the Andes Mountains, homeland of the indigenous Mapuche Pewenche people who depend on this tree. This paper is based on participatory field research with a Mapuche Pewenche community in the southern Chilean Andes on their ecological knowledge, values, use and management of the Araucaria araucana forest. It attempts to reveal how indigenous people and their knowledge contribute to the sustainable management of these forests. The paper (1) illustrates the complexity of indigenous ecological knowledge of Araucaria araucana and its efficacy in native forest management, (2) explores the link between the conservation and use of biodiversity by the indigenous people, and (3) provides answers relevant to native forest management and conservation strategies ex-situ and in-situ incorporating indigenous and scientific knowledge, thus providing a contribution towards integrated natural resource management.     

Nuclear staining and relative distance for quantifying epidermal differentiation in biomarker expression profiling

Background  

The epidermal physiology results from a complex regulated homeostasis of keratinocyte proliferation, differentiation and death and is tightly regulated by a specific protein expression during cellular maturation. Cellular in silico models are considered a promising and inevitable tool for the understanding of this complex system. Hence, we need to incorporate the information of the differentiation dependent protein expression in cell based systems biological models of tissue homeostasis. Such methods require measuring tissue differentiation quantitatively while correlating it with biomarker expression intensities.     

Discovery of novel spirocyclic inhibitors of fatty acid amide hydrolase (FAAH). Part 1: identification of 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane as lead scaffolds

Herein we report the identification of two new fatty acid amide hydrolase (FAAH) inhibitor lead series with FAAH k(inact)/K(i) potency values greater than 1500M(-1)s(-1). The two novel spirocyclic cores, 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane, clearly distinguished themselves from the other spirocyclic cores on the basis of their superior potency for FAAH. Lead compounds from these two series have suitable FAAH potency and selectivity for additional medicinal chemistry optimization.     

Exploring the active site of the tungsten, iron-sulfur enzyme acetylene hydratase

The soluble tungsten, iron-sulfur enzyme acetylene hydratase (AH) from mesophilic Pelobacter acetylenicus is a member of the dimethyl sulfoxide (DMSO) reductase family. It stands out from its class as it catalyzes a nonredox reaction, the addition of H₂O to acetylene (H—C☰C—H) to form acetaldehyde (CH₃CHO). Caught in its active W(IV) state, the high-resolution three-dimensional structure of AH offers an excellent starting point to tackle its unique chemistry and to identify catalytic amino acid residues within the active site cavity: Asp13 close to W(IV) coordinated to two molybdopterin-guanosine-dinucleotide ligands, Lys48 which couples the [4Fe-4S] cluster to the W site, and Ile142 as part of a hydrophobic ring at the end of the substrate access channel designed to accommodate the substrate acetylene. A protocol was developed to express AH in Escherichia coli and to produce active-site variants which were characterized with regard to activity and occupancy of the tungsten and iron-sulfur centers. By this means, fusion of the N-terminal chaperone binding site of the E. coli nitrate reductase NarG to the AH gene improved the yield and activity of AH and its variants significantly. Results from site-directed mutagenesis of three key residues, Asp13, Lys48, and Ile142, document their important role in catalysis of this unusual tungsten enzyme.Molybdenum and tungsten are the only transition metals of the second (Mo) and third (W) row of the periodic table of elements with known biological functions (7). In their biologically active form, both metals are bound to the cofactor molybdopterin (Moco), which is present in all molybdenum and tungsten enzymes with the exception of nitrogenase, where molybdenum is coordinated to a large iron-sulfur cluster, MoFe₇S₉ (9). Virtually all organisms including plants and mammals use either molybdenum or tungsten proteins in important metabolic pathways (35). Microorganisms carry a wide variety of molybdenum enzymes, such as nitrate reductase (NAR), formate dehydrogenase (FDH), dimethyl sulfoxide reductase (DMSOR), or trimethylamine N-oxide reductase (TMAOR) (7). These enzymes are involved in either oxygen atom transfer reactions or in reductive hydroxylations. By this means, the metal shuttles between the oxidation states +IV and +VI (16). Notably, the tungsten, iron-sulfur enzyme acetylene hydratase ([AH] EC 4.2.1.112), isolated from the soluble fraction of the mesophilic anaerobe Pelobacter acetylenicus, is an exception (26). It catalyzes the hydration of acetylene to acetaldehyde via an enol intermediate as an initial step for the fermentation of acetylene by P. acetylenicus, clearly a nonredox reaction (equation 1): Except for nitrogenase, which reduces acetylene to ethylene (H₂C=CH₂), AH is the only enzyme known to accept acetylene as a substrate. However, acetylene is well known to act as an inhibitor for numerous metal-dependent enzymes (10). AH is a member of the DMSOR family and carries one [4Fe-4S] cluster and two molybdopterin-guanosine-dinucleotide (referred to as P- and Q-MGD) ligands that coordinate the tungsten atom (Fig. ​(Fig.1)1) (18). The enzyme is sensitive toward dioxygen, and its [4Fe-4S] cluster is converted to a truncated [3Fe-4S] cluster upon exposure to air, as shown by electron paramagnetic resonance (EPR) (18). In AH prepared under the exclusion of dioxygen, the EPR signal of the [3Fe-4S] cluster was absent, and reaction with sodium dithionite led to a rhombic EPR signal (g_z of 2.048, g_y of 1.939, and g_x of 1.920) originating from a [4Fe-4S]⁺ cluster. Upon oxidation with hexacyanoferrate(III), a new signal appeared (g_x of 2.007, g_y of 2.019, and g_z of 2.048; average g value [g_av] of 2.022), which was assigned to a W(V) center (18).Open in a separate windowFIG. 1.Acetylene hydratase from P. acetylenicus. (Left) Overall structure, with the [4Fe-4S] cluster and W(MGD)₂ buried inside the protein; the peptide backbone is shown in dark blue at the N-terminal end, and continues as light blue, cyan, green, and yellow to orange at the C-terminal end. (Right) [4Fe-4S] cluster and W(MGD)₂ center. C is shown in gray, N in blue, O in red, P in orange, S in yellow, Fe in brown, and W in cyan (Protein Data Bank [PDB] code 2E7Z).For catalytic activity, AH requires a strong reductant, such as sodium dithionite or titanium(III) citrate (18). Recently, the X-ray structure of AH in the reduced state could be solved at 1.26-Å resolution (28) which gave a first view of its active site: W(IV) is coordinated by four sulfur atoms delivered by the two dithiolene ligands (MGD), one cysteinyl sulfur (Cys141), and one oxygen ligand at a distance of 2.04 Å (Fig. ​(Fig.1).1). Mechanistically, the nature of this oxygen ligand is critical. The observed W-O distance of 2.04 Å is right between the values expected for a hydroxide ligand (1.9 to 2.1 Å) and a coordinated water (2.0 to 2.3 Å), thus not allowing an unequivocal assignment of the sixth ligand of the WS₅O core. Two different reaction mechanisms have been proposed: (i) nucleophilic attack of the hydroxide group and (ii) electrophilic attack of a polarized water molecule, on the C,C triple bond of acetylene (28). As a consequence of theoretical calculations, and in agreement with the observed bond distances, the active W(IV) state should favor a water ligand and therefore an electrophilic addition mechanism (28). Acetylene can access the tungsten ion through a well-defined channel close to the N-terminal domain that harbors the [4Fe-4S] cluster. One residue, Asp13, interacts with the oxygen ligand bound to the W ion, forming a short hydrogen bond of 2.4 Å. Above the W ion and the coordinated H₂O molecule, the substrate channel ends in a ring of six hydrophobic residues. These residues build a cavity with dimensions perfect for accommodating acetylene. Experiments to bind the substrate acetylene, ethylene, the inhibitor propargyl alcohol (H—C☰C—CH₂OH), and dinitrogen or carbon monoxide have failed thus far to produce a complex in the crystal. However, computer docking of one acetylene molecule at this position led to a reasonable fit, positioning the two carbon atoms of the substrate exactly above the H₂O molecule coordinated to tungsten (28).To gain further information about the reaction mechanism of AH, we initiated a study by site-directed mutagenesis and exchanged several amino acids which have been suggested to be important for catalysis at the active site cavity. To achieve this goal, we had to develop a suitable procedure for the heterologous expression of AH in Escherichia coli. Notably, E. coli uses a chaperone system for the insertion of Moco into its enzymes (6). These chaperones of the TorD superfamily act in two ways. First, they bind at the N-terminal signal sequence, similar to the sequence of the TAT export system, thereby delaying the folding of the newly synthesized molybdenum enzyme until Moco has been properly inserted (25). Second, they actively facilitate the incorporation of the molybdopterin cofactor by binding to a second, yet unknown site (13). To improve the assembly of the metal sites as well as to increase the enzymatic activity of the recombinant AH, the N-terminal chaperone binding site of the nitrate reductase, NarG, from E. coli was fused to the AH gene in the expression vector.     

A History of Abuse and Operative Delivery – Results from a European Multi-Country Cohort Study

Objective

The main aim of this study was to assess whether a history of abuse, reported during pregnancy, was associated with an operative delivery. Secondly, we assessed if the association varied according to the type of abuse and if the reported abuse had been experienced as a child or an adult.

Design

The Bidens study, a cohort study in six European countries (Belgium, Iceland, Denmark, Estonia, Norway, and Sweden) recruited 6724 pregnant women attending routine antenatal care. History of abuse was assessed through questionnaire and linked to obstetric information from hospital records. The main outcome measure was operative delivery as a dichotomous variable, and categorized as an elective caesarean section (CS), or an operative vaginal birth, or an emergency CS. Non-obstetrically indicated were CSs performed on request or for psychological reasons without another medical reason. Binary and multinomial regression analysis were used to assess the associations.

Results

Among 3308 primiparous women, sexual abuse as an adult (≥18 years) increased the risk of an elective CS, Adjusted Odds Ratio 2.12 (1.28–3.49), and the likelihood for a non-obstetrically indicated CS, OR 3.74 (1.24–11.24). Women expressing current suffering from the reported adult sexual abuse had the highest risk for an elective CS, AOR 4.07 (1.46–11.3). Neither physical abuse (in adulthood or childhood <18 years), nor sexual abuse in childhood increased the risk of any operative delivery among primiparous women. Among 3416 multiparous women, neither sexual, nor emotional abuse was significantly associated with any kind of operative delivery, while physical abuse had an increased AOR for emergency CS of 1.51 (1.05–2.19).

Conclusion

Sexual abuse as an adult increases the risk of an elective CS among women with no prior birth experience, in particular for non-obstetrical reasons. Among multiparous women, a history of physical abuse increases the risk of an emergency CS.     

Structure based design of novel irreversible FAAH inhibitors

Fatty acid amide hydrolase (FAAH) has attracted significant attention due to its promise as an analgesic target. This has resulted in the discovery of numerous chemical classes as inhibitors of this potential therapeutic target. In this paper we disclose a new series of novel FAAH irreversible azetidine urea inhibitors. In general these compounds illustrate potent activity against the rat FAAH enzyme. Our SAR studies allowed us to optimize this series resulting in the identification of compounds 13 which were potent inhibitors of both human and rat enzyme. This series of compounds illustrated good hydrolase selectivity along with good PK properties.     

Subdiaphragmatic vagal deafferentation affects body weight gain and glucose metabolism in obese male Zucker (fa/fa) rats

We investigated the effect of subdiaphragmatic vagal deafferentation (SDA) on food intake, body weight gain, and metabolism in obese (fa/fa) and lean (Fa/?) Zucker rats. Before and after recovery from surgery, food intake and body weight gain were recorded, and plasma glucose and insulin were measured in tail-prick blood samples. After implantation of a jugular vein catheter, an intravenous glucose tolerance test (IVGTT) was performed, followed by minimal modeling to estimate the insulin sensitivity index. Food intake relative to metabolic body weight (g/kg(0.75)) and daily body weight gain after surgery were lower (P < 0.05) in SDA than in sham obese but not lean rats. Before surgery, plasma glucose and insulin concentrations were lower (P < 0.05) in lean than in obese rats but did not differ between surgical groups within both genotypes. Four weeks after surgery, plasma glucose and insulin were still similar in SDA and sham lean rats but lower (P < 0.05) in SDA than in sham obese rats. IVGTT revealed a downward shift of the plasma insulin profile by SDA in obese but not lean rats, whereas the plasma glucose profile was unaffected. SDA decreased (P < 0.05) area under the curve for insulin but not glucose in obese rats. The insulin sensitivity index was higher in lean than in obese rats but was not affected by SDA in both genotypes. These results suggest that elimination of vagal afferent signals from the upper gut reduces food intake and body weight gain without affecting the insulin sensitivity index measured by minimal modeling in obese Zucker rats.