Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate

Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques(1). A catalytic mechanism, featuring a long-lived oxo-carbenium-ion intermediate, was proposed on the basis of model-building studies(2). The `Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of beta -glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining beta -glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated(3). Here we show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. We also show the three-dimensional structure of this intermediate as determined by Xray diffraction. We formulate a general catalytic mechanism for all retaining beta -glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate

Comparative population structure of <i>Plasmodium malariae</i> and <i>Plasmodium falciparum</i> under different transmission settings in Malawi

&lt;b&gt;Background:&lt;/b&gt; Described here is the first population genetic study of Plasmodium malariae, the causative agent of quartan malaria. Although not as deadly as Plasmodium falciparum, P. malariae is more common than previously thought, and is frequently in sympatry and co-infection with P. falciparum, making its study increasingly important. This study compares the population parameters of the two species in two districts of Malawi with different malaria transmission patterns - one seasonal, one perennial - to explore the effects of transmission on population structures. &lt;BR/&gt; &lt;b&gt;Methods:&lt;/b&gt; Six species-specific microsatellite markers were used to analyse 257 P. malariae samples and 257 P. falciparum samples matched for age, gender and village of residence. Allele sizes were scored to within 2 bp for each locus and haplotypes were constructed from dominant alleles in multiple infections. Analysis of multiplicity of infection (MOI), population differentiation, clustering of haplotypes and linkage disequilibrium was performed for both species. Regression analyses were used to determine association of MOI measurements with clinical malaria parameters. &lt;BR/&gt; &lt;b&gt;Results:&lt;/b&gt; Multiple-genotype infections within each species were common in both districts, accounting for 86.0% of P. falciparum and 73.2% of P. malariae infections and did not differ significantly with transmission setting. Mean MOI of P. falciparum was increased under perennial transmission compared with seasonal (3.14 vs 2.59, p = 0.008) and was greater in children compared with adults. In contrast, P. malariae mean MOI was similar between transmission settings (2.12 vs 2.11) and there was no difference between children and adults. Population differentiation showed no significant differences between villages or districts for either species. There was no evidence of geographical clustering of haplotypes. Linkage disequilibrium amongst loci was found only for P. falciparum samples from the seasonal transmission setting. &lt;BR/&gt; &lt;b&gt;Conclusions:&lt;/b&gt; The extent of similarity between P. falciparum and P. malariae population structure described by the high level of multiple infection, the lack of significant population differentiation or haplotype clustering and lack of linkage disequilibrium is surprising given the differences in the biological features of these species that suggest a reduced potential for out-crossing and transmission in P. malariae. The absence of a rise in P. malariae MOI with increased transmission or a reduction in MOI with age could be explained by differences in the duration of infection or degree of immunity compared to P. falciparum

Leptin Activates Anorexigenic POMC Neurons through a Neural Network in the Arcuate Nucleus

The administration of leptin to leptin-deficient humans, and the analogous Lepob/Lepob mice, effectively reduces hyperphagia and obesity. But common obesity is associated with elevated leptin, which suggests that obese humans are resistant to this adipocyte hormone. In addition to regulating long-term energy balance, leptin also rapidly affects neuronal activity. Proopiomelanocortin (POMC) and neuropeptide-Y types of neurons in the arcuate nucleus of the hypothalamus7 are both principal sites of leptin receptor expression and the source of potent neuropeptide modulators, melanocortins and neuropeptide Y, which exert opposing effects on feeding and metabolism. These neurons are therefore ideal for characterizing leptin action and the mechanism of leptin resistance; however, their diffuse distribution makes them difficult to study. Here we report electrophysiological recordings on POMC neurons, which we identified by targeted expression of green fluorescent protein in transgenic mice. Leptin increases the frequency of action potentials in the anorexigenic POMC neurons by two mechanisms: depolarization through a nonspecific cation channel; and reduced inhibition by local orexigenic neuropeptide-Y/GABA (g-aminobutyric acid) neurons. Furthermore, we show that melanocortin peptides have an autoinhibitory effect on this circuit. On the basis of our results, we propose an integrated model of leptin action and neuronal architecture in the arcuate nucleus of the hypothalamu

Selective and Irreversible Inhibitors of Aphid Acetylcholinesterases: Steps Toward Human-Safe Insecticides

Aphids, among the most destructive insects to world agriculture, are mainly controlled by organophosphate insecticides that disable the catalytic serine residue of acetylcholinesterase (AChE). Because these agents also affect vertebrate AChEs, they are toxic to non-target species including humans and birds. We previously reported that a cysteine residue (Cys), found at the AChE active site in aphids and other insects but not mammals, might serve as a target for insect-selective pesticides. However, aphids have two different AChEs (termed AP and AO), and only AP-AChE carries the unique Cys. The absence of the active-site Cys in AO-AChE might raise concerns about the utility of targeting that residue. Herein we report the development of a methanethiosulfonate-containing small molecule that, at 6.0 µM, irreversibly inhibits 99% of all AChE activity extracted from the greenbug aphid (Schizaphis graminum) without any measurable inhibition of the human AChE. Reactivation studies using β-mercaptoethanol confirm that the irreversible inhibition resulted from the conjugation of the inhibitor to the unique Cys. These results suggest that AO-AChE does not contribute significantly to the overall AChE activity in aphids, thus offering new insight into the relative functional importance of the two insect AChEs. More importantly, by demonstrating that the Cys-targeting inhibitor can abolish AChE activity in aphids, we can conclude that the unique Cys may be a viable target for species-selective agents to control aphids without causing human toxicity and resistance problems

Probing of Exosites Leads to Novel Inhibitor Scaffolds of HCV NS3/4A Proteinase

Hepatitis C is a treatment-resistant disease affecting millions of people worldwide. The hepatitis C virus (HCV) genome is a single-stranded RNA molecule. After infection of the host cell, viral RNA is translated into a polyprotein that is cleaved by host and viral proteinases into functional, structural and non-structural, viral proteins. Cleavage of the polyprotein involves the viral NS3/4A proteinase, a proven drug target. HCV mutates as it replicates and, as a result, multiple emerging quasispecies become rapidly resistant to anti-virals, including NS3/4A inhibitors.To circumvent drug resistance and complement the existing anti-virals, NS3/4A inhibitors, which are additional and distinct from the FDA-approved telaprevir and boceprevir α-ketoamide inhibitors, are required. To test potential new avenues for inhibitor development, we have probed several distinct exosites of NS3/4A which are either outside of or partially overlapping with the active site groove of the proteinase. For this purpose, we employed virtual ligand screening using the 275,000 compound library of the Developmental Therapeutics Program (NCI/NIH) and the X-ray crystal structure of NS3/4A as a ligand source and a target, respectively. As a result, we identified several novel, previously uncharacterized, nanomolar range inhibitory scaffolds, which suppressed of the NS3/4A activity in vitro and replication of a sub-genomic HCV RNA replicon with a luciferase reporter in human hepatocarcinoma cells. The binding sites of these novel inhibitors do not significantly overlap with those of α-ketoamides. As a result, the most common resistant mutations, including V36M, R155K, A156T, D168A and V170A, did not considerably diminish the inhibitory potency of certain novel inhibitor scaffolds we identified.Overall, the further optimization of both the in silico strategy and software platform we developed and lead compounds we identified may lead to advances in novel anti-virals

SPRING: an RCT study of probiotics in the prevention of gestational diabetes mellitus in overweight and obese women

Background: Obesity is increasing in the child-bearing population as are the rates of gestational diabetes. Gestational diabetes is associated with higher rates of Cesarean Section for the mother and increased risks of macrosomia, higher body fat mass, respiratory distress and hypoglycemia for the infant. Prevention of gestational diabetes through life style intervention has proven to be difficult. A Finnish study showed that ingestion of specific probiotics altered the composition of the gut microbiome and thereby metabolism from early gestation and decreased rates of gestational diabetes in normal weight women. In SPRING (the Study of Probiotics IN the prevention of Gestational diabetes), the effectiveness of probiotics ingestion for the prevention of gestational diabetes will be assessed in overweight and obese women

Discovery of Inhibitors of Leishmania β-1,2-Mannosyltransferases Using a Click-Chemistry-Derived Guanosine Monophosphate Library

Leishmania spp. are a medically important group of protozoan parasites that synthesize a novel intracellular carbohydrate reserve polymer termed mannogen. Mannogen is a soluble homopolymer of β-1,2-linked mannose residues that accumulates in the major pathogenic stages in the sandfly vector and mammalian host. While several steps in mannogen biosynthesis have been defined, none of the enzymes have been isolated or characterized. We report the development of a simple assay for the GDP-mannose–dependent β-1,2-mannosyltransferases involved in mannogen synthesis. This assay utilizes octyl α-d-mannopyranoside to prime the formation of short mannogen oligomers up to 5 mannose residues. This assay was used to screen a focussed library of 44 GMP-triazole adducts for inhibitors. Several compounds provided effective inhibition of mannogen β-1,2-mannosyltransferases in a cell-free membrane preparation. This assay and inhibitor compounds will be useful for dissecting the role of different mannosyltransferases in regulating de novo biosynthesis and elongation reactions in mannogen metabolism

Practice change in chronic conditions care: an appraisal of theories

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Background Management of chronic conditions can be complex and burdensome for patients and complex and costly for health systems. Outcomes could be improved and costs reduced if proven clinical interventions were better implemented, but the complexity of chronic care services appears to make clinical change particularly challenging. Explicit use of theories may improve the success of clinical change in this area of care provision. Whilst theories to support implementation of practice change are apparent in the broad healthcare arena, the most applicable theories for the complexities of practice change in chronic care have not yet been identified. Methods We developed criteria to review the usefulness of change implementation theories for informing chronic care management and applied them to an existing list of theories used more widely in healthcare. Results Criteria related to the following characteristics of chronic care: breadth of the field; multi-disciplinarity; micro, meso and macro program levels; need for field-specific research on implementation requirements; and need for measurement. Six theories met the criteria to the greatest extent: the Consolidate Framework for Implementation Research; Normalization Process Theory and its extension General Theory of Implementation; two versions of the Promoting Action on Research Implementation in Health Services framework and Sticky Knowledge. None fully met all criteria. Involvement of several care provision organizations and groups, involvement of patients and carers, and policy level change are not well covered by most theories. However, adaptation may be possible to include multiple groups including patients and carers, and separate theories may be needed on policy change. Ways of qualitatively assessing theory constructs are available but quantitative measures are currently partial and under development for all theories. Conclusions Theoretical bases are available to structure clinical change research in chronic condition care. Theories will however need to be adapted and supplemented to account for the particular features of care in this field, particularly in relation to involvement of multiple organizations and groups, including patients, and in relation to policy influence. Quantitative measurement of theory constructs may present difficulties