Ebola viruses (EBOVs) cause rare but highly fatal outbreaks of viral hemorrhagic fever in humans, and approved treatments for these infections are currently lacking. The Ebola VP35 protein is multifunctional, acting as a component of the viral RNA polymerase complex, a viral assembly factor, and an inhibitor of host interferon (IFN) production. Mutation of select basic residues within the C-terminal half of VP35 abrogates its dsRNA-binding activity, impairs VP35-mediated IFN antagonism, and attenuates EBOV growth in vitro and in vivo. Because VP35 contributes to viral escape from host innate immunity and is required for EBOV virulence, understanding the structural basis for VP35 dsRNA binding, which correlates with suppression of IFN activity, is of high importance. Here, we report the structure of the C-terminal VP35 IFN inhibitory domain (IID) solved to a resolution of 1.4 Å and show that VP35 IID forms a unique fold. In the structure, we identify 2 basic residue clusters, one of which is important for dsRNA binding. The dsRNA binding cluster is centered on Arg-312, a highly conserved residue required for IFN inhibition. Mutation of residues within this cluster significantly changes the surface electrostatic potential and diminishes dsRNA binding activity. The high-resolution structure and the identification of the conserved dsRNA binding residue cluster provide opportunities for antiviral therapeutic design. Our results suggest a structure-based model for dsRNA-mediated innate immune antagonism by Ebola VP35 and other similarly constructed viral antagonists.crystal structure ͉ Ebola virus ͉ RNA binding E bola viruses (EBOVs) cause severe hemorrhagic fever characterized by fever, shock, coagulation defects, and impaired immunity (1, 2). These manifestations of infection are thought to reflect subversion of the innate immune system coupled with uncontrolled viral replication, particularly in macrophages and dendritic cells (3,4). EBOV infection of these cells enhances production of proinflammatory cytokines, such as TNF-␣ and IFN (IFN)-␥, and diminishes stimulation of T cell maturation by dendritic cells (3, 4). Like other negative-strand RNA viruses that impair both innate and adaptive immunity (e.g., influenza, rabies, and measles), EBOV suppresses host IFN activities to replicate, thus resulting in serious disease (5, 6). Only individuals who survive EBOV infection show appreciable amounts of viral-specific antibodies (7), suggesting that EBOV infections lead to shutdown of early immune responses and prevent activation of adaptive immune responses.Recognition of viral particles and viral RNA, including RNA modifications such as 5Ј-triphosphate (5Ј-ppp), by cytosolic pattern recognition receptor helicases RIG-I and MDA-5 leads to activation of transcription factors, including IFN regulatory factor-3 (IRF-3), IRF-7, NF-B, and AP-1 (8-12). These transcription factors in turn induce expression of a large number of cytokines, such as IFN-␣ and IFN-␤ (13). Activated IFN genes operate in both autocrine and paracrin...

Background-Cannabis is the most widely used illicit substance worldwide, and legalization for recreational and medical purposes has substantially increased its availability and use in the United States. Objectives-Decades of research have suggested that recreational cannabis use confers risk for cognitive impairment across various domains, and structural and functional differences in the brain have been linked to early and heavy cannabis use. Methods-With substantial evidence for the role of the endocannabinoid system in neural development and understanding that brain development continues into early adulthood, the rising use of cannabis in adolescents and young adults raises major concerns. Yet some formulations of cannabinoid compounds are FDA-approved for medical uses, including applications in children. Results-Potential effects on the trajectory of brain morphology and cognition, therefore, should be considered. The goal of this review is to update and consolidate relevant findings in order to inform attitudes and public policy regarding the recreational and medical use of cannabis and cannabinoid compounds. Conclusions-The findings point to considerations for age limits and guidelines for use.

Phosphoribosylaminoimidazole-succinocarboxamide synthetase (SAICAR synthetase) converts 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) to 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide (SAICAR). The enzyme is a target of natural products that impair cell growth. Reported here are the crystal structures of the ADP and the ADP⅐CAIR complexes of SAICAR synthetase from Escherichia coli, the latter being the first instance of a CAIR-ligated SAICAR synthetase. ADP and CAIR bind to the active site in association with three Mg 2؉ , two of which coordinate the same oxygen atom of the 4-carboxyl group of CAIR; whereas, the third coordinates the ␣-and ␤-phosphoryl groups of ADP. The ADP⅐CAIR complex is the basis for a transition state model of a phosphoryl transfer reaction involving CAIR and ATP, but also supports an alternative chemical pathway in which the nucleophilic attack of L-aspartate precedes the phosphoryl transfer reaction. The polypeptide fold for residues 204 -221 of the E. coli structure differs significantly from those of the ligand-free SAICAR synthetase from Thermatoga maritima and the adenine nucleotide complexes of the synthetase from Saccharomyces cerevisiae. Conformational differences between the E. coli, T. maritima, and yeast synthetases suggest the possibility of selective inhibition of de novo purine nucleotide biosynthesis in microbial organisms.Phosphoribosylaminoimidazole-succinocarboxamide synthetase (EC 6.3.2.6, 5Ј-phosphoribosyl-4-carboxy-5-aminoimidazole:L-aspartate ligase (ADP)) (SAICAR synthetase) 2 catalyzes the eighth step in bacterial de novo purine nucleotide biosynthesis, ATP ϩ L-aspartate ϩ CAIR 3 ADP ϩ P i ϩ SAICAR. Lukens and Buchanan (1) first described the enzyme in 1959. In 1962 Miller and Buchanan (2) demonstrated its presence in a variety of life forms and reported the purification and properties of the synthetase from chicken liver. More recently, the Stubbe laboratory (3) purified SAICAR synthetase from Escherichia coli. The E. coli enzyme exhibits a rapid equilibrium random kinetic mechanism (4). SAICAR synthetase from Saccharomyces cerevisiae is a monomer (5-8) and that from Thermatoga maritima a dimer (9). Comparable enzymes from vertebrates have masses in excess of 330 kDa and possess 6 -8 identical subunits of 47 kDa (10, 11). The vertebrate systems are bifunctional, combining 5-aminoimidazole ribonucleotide carboxylase (AIR carboxylase) and SAICAR synthetase activities (10 -12).L-Alanosine can replace L-aspartate as a substrate both in vitro and in vivo for SAICAR synthetase (4, 13, 14). The product of the SAICAR synthetase reaction, L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide, is a potent inhibitor of adenylosuccinate synthetase and adenylosuccinate lyase, being the compound responsible for L-alanosine toxicity (13). Many cancers (ϳ30% of all T-cell acute lymphocytic leukemia, for instance) lack a salvage pathway for adenine nucleotides and rely entirely on de novo biosynthesis (15). L-Alanosine is toxic to cell lines of such cancers at ...

Background: The therapeutic mechanism of repetitive transcranial magnetic stimulation (rTMS) for treatment-resistant depression (TRD) may involve modulation of γ-aminobutyric acid (GABA) levels. We used proton magnetic resonance spectroscopy (MRS) to assess changes in GABA levels at the site of rTMS in the left dorsolateral prefrontal cortex (DLPFC). Methods: In 26 adults with TRD, we used Mescher-Garwood point-resolved spectroscopy (MEGA-PRESS) spectral-editing MRS to measure GABA in the left DLPFC before and after standard clinical treatment with rTMS. All participants but 1 were medicated, including 12 patients on GABA agonist agents. Results: Mean GABA in the DLPFC increased 10.0% (p = 0.017) post-rTMS in the overall sample. As well, GABA increased significantly in rTMS responders (n = 12; 23.6%, p = 0.015) but not in nonresponders (n = 14; 4.1%, p = not significant). Changes in GABA were not significantly affected by GABAergic agonists, but clinical response was less frequent (p = 0.005) and weaker (p = 0.035) in the 12 participants who were receiving GABA agonists concomitant with rTMS treatment. Limitations: This study had an open-label design in a popu lation receiving naturalistic treatment. Conclusion: Treatment using rTMS was associated with increases in GABA levels at the stimulation site in the left DLPFC, and the degree of GABA change was related to clinical improvement. Participants receiving concomitant treatment with a GABA agonist were less likely to respond to rTMS. These findings were consistent with earlier studies showing the effects of rTMS on GABA levels and support a GABAergic model of depression.

A gene encoding a glycoside hydrolase family 44 (GH44) protein from Clostridium acetobutylicum ATCC 824 was synthesized and transformed into Escherichia coli. The previously uncharacterized protein was expressed with a C-terminal His tag and purified by nickel-nitrilotriacetic acid affinity chromatography. Crystallization and X-ray diffraction to a 2.2-Å resolution revealed a triose phosphate isomerase (TIM) barrel-like structure with additional Greek key and ␤-sandwich folds, similar to other GH44 crystal structures. The enzyme hydrolyzes cellotetraose and larger cellooligosaccharides, yielding an unbalanced product distribution, including some glucose. It attacks carboxymethylcellulose and xylan at approximately the same rates. Its activity on carboxymethylcellulose is much higher than that of the isolated C. acetobutylicum cellulosome. It also extensively converts lichenan to oligosaccharides of intermediate size and attacks Avicel to a limited extent. The enzyme has an optimal temperature in a 10-min assay of 55°C and an optimal pH of 5.0.Thirteen glycoside hydrolase (GH) families, each having members related to each other by amino acid sequence, contain enzymes that hydrolyze cellulose and/or cellooligosaccharides (4; http://www.cazy.org). Among them is GH family 44 (GH44), most of whose enzymes are endoglucanases (EGs). In general, EGs are more active on longer rather than on shorter chains and are more likely to attack bonds in the interiors of carbohydrate chains than those near their termini.With one exception, GH44 enzymes are produced by bacteria, both aerobic and anaerobic. At present, 29 amino acid sequences of GH44 members have been determined (4). Often they are combined with other GHs in multienzyme proteins (Fig. 1).Not all of these GH44 enzymes have been produced in vitro, and those that have been produced have only been partially characterized. Experimental results indicate that GH44 enzymes exclusively cleave ␤-1,4 bonds between glucosyl and xylosyl residues and that they have different abilities to attack xylan, lichenan, and different cellulose forms, such as Avicel, acid-swollen cellulose, and carboxymethyl cellulose (CMC), with the presence of a carbohydrate-binding module (CBM) allowing higher activity on solid cellulose. They appear to be inactive on short oligosaccharides, like p-nitrophenyl (PNP)-␤-glucopyranoside, PNP-␤-cellobioside, and PNP-␤-xylopyranoside.Most GH families containing cellulases have at least one member with a known tertiary structure. That was not true of GH44 until Kitago et al. (15) published six different crystal structures of an EG, CelJ, from Clostridium thermocellum.