Model for calcium binding to gamma-carboxyglutamic acid residues of proteins: crystal structure of calcium alpha-ethylmalonate

The crystal structure of a Ca2+ salt of alpha-ethylmalonic acid was determined from three-dimensional X-ray diffraction data. The dicarboxylate anion represents the functional side chain of gamma-carboxyglutamic acid (Gla) residues, which are implicated as essential calcium-binding ligands in a variety of proteins. The alpha-ethylmalonate ion chelates the Ca2+ ion in a bidentate manner that involves an O atom from each of the two malonate carboxylate groups. This type of binding arises from the constrained arrangement of carboxylate ligands in the malonate group and may be of significance to the calcium-binding properties of Gla-containing sites in proteins. The Ca2+-malonate chelation forms a six-membered ring, which is stabilized by interactions that are consistent with the preferred stereochemistries of both calcium-carboxylate and metal-malonate complexes. No other interactions are observed between Ca2+ ions and alpha-ethylmalonate ions that depend upon the malonate juxtaposition of two carboxylate groups. The potential for this type of binding distinguishes Gla residues from the monocarboxylate residues, aspartate and glutamate, and confers a novel calcium-chelation ability upon Gla-containing sites in proteins.     

Prevalence of methotrexate intolerance in rheumatoid arthritis and psoriatic arthritis

Introduction

The aim of this study was to determine the prevalence of gastrointestinal and behavioural symptoms occurring before (anticipatory/associative) and after methotrexate (MTX) administration, termed MTX intolerance, in rheumatoid (RA) and psoriatic arthritis (PsA).

Methods

Methotrexate Intolerance Severity Score (MISS), previously validated in juvenile idiopathic arthritis patients, was used to determine MTX intolerance prevalence in 291 RA/PsA patients. The MISS consisted of four domains: abdominal pain, nausea, vomiting and behavioural symptoms, occurring upon, prior to (anticipatory) and when thinking of MTX (associative). MTX intolerance was defined as ≥6 on the MISS with ≥1 point on anticipatory and/or associative and/or behavioural items.

Results

A total of 123 patients (42.3%) experienced at least one gastrointestinal adverse effect. The prevalence of MTX intolerance was 11%. MTX intolerance prevalence was higher in patients on parenteral (20.6%) than on oral MTX (6.2%) (p < 0.001).

Conclusion

Besides well-known gastrointestinal symptoms after MTX, RA and PsA patients experienced these symptoms also before MTX intake. RA and PsA patients on MTX should be closely monitored with the MISS for early detection of MTX intolerance, in order to intervene timely and avoid discontinuation of an effective treatment.     

Dermatological conditions during TNF-α-blocking therapy in patients with rheumatoid arthritis: a prospective study

Various dermatological conditions have been reported during tumor necrosis factor (TNF)-α-blocking therapy, but until now no prospective studies have been focused on this aspect. The present study was set up to investigate the number and nature of clinically important dermatological conditions during TNF-α-blocking therapy in patients with rheumatoid arthritis (RA). RA patients starting on TNF-α-blocking therapy were prospectively followed up. The numbers and natures of dermatological events giving rise to a dermatological consultation were recorded. The patients with a dermatological event were compared with a group of prospectively followed up RA control patients, naive to TNF-α-blocking therapy and matched for follow-up period. 289 RA patients started TNF-α-blocking therapy. 128 dermatological events were recorded in 72 patients (25%) during 911 patient-years of follow-up. TNF-α-blocking therapy was stopped in 19 (26%) of these 72 patients because of the dermatological event. More of the RA patients given TNF-α-blocking therapy (25%) than of the anti-TNF-α-naive patients (13%) visited a dermatologist during follow-up (P < 0.0005). Events were recorded more often during active treatment (0.16 events per patient-year) than during the period of withdrawal of TNF-α-blocking therapy (0.09 events per patient-year, P < 0.0005). The events recorded most frequently were skin infections (n = 33), eczema (n = 20), and drug-related eruptions (n = 15). Other events with a possible relation to TNF-α-blocking therapy included vasculitis, psoriasis, drug-induced systemic lupus erythematosus, dermatomyositis, and a lymphomatoid-papulosis-like eruption. This study is the first large prospective study focusing on dermatological conditions during TNF-α-blocking therapy. It shows that dermatological conditions are a significant and clinically important problem in RA patients receiving TNF-α-blocking therapy.     

Phospho-N-acetyl-muramyl-pentapeptide translocase from Escherichia coli: catalytic role of conserved aspartic acid residues

Phospho-N-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-N-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of Escherichia coli translocase 1 as a C-terminal hexahistidine (C-His6) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His6 fusions. Enzymatic analysis established that all three mutations eliminated translocase 1 activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-N-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile.     

Catalytic mechanism of C-C hydrolase MhpC from Escherichia coli: kinetic analysis of His263 and Ser110 site-directed mutants

C-C hydrolase MhpC (2-hydroxy-6-keto-nona-1,9-dioic acid 5,6-hydrolase) from Escherichia coli catalyses the hydrolytic C-C cleavage of the meta-ring fission product on the phenylpropionic acid catabolic pathway. The crystal structure of E. coli MhpC has revealed a number of active-site amino acid residues that may participate in catalysis. Site-directed mutants of His263, Ser110, His114, and Ser40 have been analysed using steady-state and stopped-flow kinetics. Mutants H263A, S110A and S110G show 10(4)-fold reduced catalytic efficiency, but still retain catalytic activity for C-C cleavage. Two distinct steps are observed by stopped-flow UV/Vis spectrophotometry, corresponding to ketonisation and C-C cleavage: H263A exhibits very slow ketonisation and C-C cleavage, whereas S110A and S110G exhibit fast ketonisation, an intermediate phase, and slow C-C cleavage. H114A shows only twofold-reduced catalytic efficiency, ruling out a catalytic role, but shows a fivefold-reduced K(M) for the natural substrate, and an ability to process an aryl-containing substrate, implying a role for His114 in positioning of the substrate. S40A shows only twofold-reduced catalytic efficiency, but shows a very fast (500 s(-1)) interconversion of dienol (317 nm) to dienolate (394 nm) forms of the substrate, indicating that the enzyme accepts the dienol form of the substrate. These data imply that His263 is responsible for both ketonisation of the substrate and for deprotonation of water for C-C cleavage, a novel catalytic role in a serine hydrolase. Ser110 has an important but non-essential role in catalysis, which appears not to be to act as a nucleophile. A catalytic mechanism is proposed involving stabilisation of reactive intermediates and activation of a nucleophilic water molecule by Ser110.     

Polymerase chain reaction amplification of single-stranded DNA containing a base analog, 2-chloradenine.

By utilization of polymerase chain reaction techniques, single-stranded DNA of defined length and sequence containing a purine analog, 2-chloroadenine, in place of adenine was synthesized. This was accomplished by a combination of standard polymerase chain amplification reactions with Thermus aquaticus DNA polymerase in the presence of four normal deoxynucleoside triphosphates, M13 duplex DNA as template, and two primers to generate double-stranded DNA 118 bases in length. An asymmetric polymerase chain reaction, which produced an excess of single-stranded 98-base DNA, was then conducted with 2-chloro-2'-deoxy-adenosine 5'-triphosphate in place of dATP and with only one primer that annealed internal to the original two primers. Standard polymerase chain reaction techniques alone conducted in the presence of the analog as the fourth nucleotide did not produce duplex DNA that was modified within both strands. This asymmetric technique allows the incorporation of an altered nucleotide at specific sites into large quantities of single-stranded DNA without using chemical phosphoramidite synthesis procedures and circumvents the apparent inability of DNA polymerase to synthesize fully substituted double-stranded DNA during standard amplification reactions. The described method will permit the study of the effects of modified bases in template DNA on a variety of protein-DNA interactions and enzymes.     

Aggregation patterns of bile salts: crystal structure of calcium cholate chloride heptahydrate

Crystals of calcium cholate chloride heptahydrate, CaC24H39O7Cl . 7H2O, are monoclinic, space group P2(1), with a = 11.918(2), b = 8.636(1), c = 15.302(3) A, beta = 97.93(3) degrees, V = 1559.9(8) A3, and Z = 2. A trial structure was obtained by Patterson and Fourier techniques and was refined by full-matrix least-squares calculations using absorption corrected CuK-alpha diffractometer data. The final R index is 0.047. The crystal structure contains bilayer-type arrangements, with hydrophobic portions of cholate rings sandwiched between layers of polar groups that are interacting with calcium ions and water molecules. The calcium ion is coordinated to five water molecules and to the two carboxylate oxygen atoms of the cholate residue. Two additional water molecules are involved only in crystal packing through the formation of hydrogen bonds. Cholate-cholate hydrophobic interactions involve contacts between the hydrocarbon portions of the carboxylate sidechains and the A and B rings. This results in a staggered packing pattern that is nearly identical to that found in crystals of sodium cholate and rubidium deoxycholate. Similar bilayer aggregation patterns may also be involved in the formation of bile salt micelles in aqueous media. The characteristic bilayer packing arrangement can accommodate a variety of cation-binding patterns, as evidenced by the finding that calcium, sodium, and rubidium ions interact with the polar faces of the bilayers in different ways. The carboxylate sidechain displays two different conformations in the crystal structure of calcium cholate chloride heptahydrate. Variation in sidechain conformation may be of importance in the adjustment required to accommodate different cation coordination schemes.     

Feasibility of a liver transcriptomics approach to assess bovine treatment with the prohormone dehydroepiandrosterone (DHEA)

Background  

Within the European Union the use of growth promoting agents in animal production is prohibited. Illegal use of natural prohormones like dehydroepiandrosterone (DHEA) is hard to prove since prohormones are strongly metabolized in vivo. In the present study, we investigated the feasibility of a novel effect-based approach for monitoring abuse of DHEA. Changes in gene expression profiles were studied in livers of bull calves treated orally (PO) or intramuscularly (IM) with 1000 mg DHEA versus two control groups, using bovine 44K DNA microarrays. In contrast to controlled genomics studies, this work involved bovines purchased at the local market on three different occasions with ages ranging from 6 to 14 months, thereby reflecting the real life inter-animal variability due to differences in age, individual physiology, season and diet.     

Relative shortening and functional tethering of spinal cord in adolescent scoliosis – Result of asynchronous neuro-osseous growth, summary of an electronic focus group debate of the IBSE

There is no generally accepted scientific theory for the causes of adolescent idiopathic scoliosis (AIS). As part of its mission to widen understanding of scoliosis etiology, the International Federated Body on Scoliosis Etiology (IBSE) introduced the electronic focus group (EFG) as a means of increasing debate on knowledge of important topics. This has been designated as an on-line Delphi discussion. The Statement for this debate was written by Dr WCW Chu and colleagues who examine the spinal cord to vertebral growth interaction during adolescence in scoliosis. Using the multi-planar reconstruction technique of magnetic resonance imaging they investigated the relative length of spinal cord to vertebral column including ratios in 28 girls with AIS (mainly thoracic or double major curves) and 14 age-matched normal girls. Also evaluated were cerebellar tonsillar position, somatosensory evoked potentials (SSEPs), and clinical neurological examination. In severe AIS compared with normal controls, the vertebral column is significantly longer without detectable spinal cord lengthening. They speculate that anterior spinal column overgrowth relative to a normal length spinal cord exerts a stretching tethering force between the two ends, cranially and caudally leading to the initiation and progression of thoracic AIS. They support and develop the Roth-Porter concept of uncoupled neuro-osseous growth in the pathogenesis of AIS which now they prefer to term ' asynchronous neuro-osseous growth'. Morphological evidence about the curve apex suggests that the spinal cord is also affected, and a 'double pathology' is suggested. AIS is viewed as a disorder with a wide spectrum and a common neuroanatomical abnormality namely, a spinal cord of normal length but short relative to an abnormally lengthened anterior vertebral column. Neuroanatomical changes and/or abnormal neural function may be expressed only in severe cases. This asynchronous neuro-osseous growth concept is regarded as one component of a larger concept. The other component relates to the brain and cranium of AIS subjects because abnormalities have been found in brain (infratentorial and supratentorial) and skull (vault and base). The possible relevance of systemic melatonin-signaling pathway dysfunction, platelet calmodulin levels and putative vertebral vascular biology to the asynchronous neuro-osseous growth concept is discussed. A biomechanical model to test the spinal component of the concept is in hand. There is no published research on the biomechanical properties of the spinal cord for scoliosis specimens. Such research on normal spinal cords includes movements (kinematics), stress-strain responses to uniaxial loading, and anterior forces created by the stretched cord in forward flexion that may alter sagittal spinal shape during adolescent growth. The asynchronous neuro-osseous growth concept for the spine evokes controversy. Dr Chu and colleagues respond to five other concepts of pathogenesis for AIS and suggest that relative anterior spinal overgrowth and biomechanical growth modulation may also contribute to AIS pathogenesis.     

The effects of population and thermal acclimation on the growth,condition and cold responsive mRNA expression of age-0 lake sturgeon (Acipenser fulvescens)

In Manitoba, Canada, wild lake sturgeon (Acipenser fulvescens) populations exist along a latitudinal gradient and are reared in hatcheries to bolster threatened populations. We reared two populations of lake sturgeon, one from each of the northern and southern ends of Manitoba and examined the effects of typical hatchery temperatures (16°C) as well as 60-day acclimation to elevated rearing temperatures (20°C) on mortality, growth and condition throughout early development. Additionally, we examined the cold shock response, which may be induced during stocking, through the hepatic mRNA expression of genes involved in the response to cold stress and homeoviscous adaptation (HSP70, HSP90a, HSP90b, CIRP and SCD). Sturgeon were sampled after 1 day and 1 week following stocking into temperatures of 8, 6 and 4°C in a controlled laboratory environment. The southern population showed lower condition and higher mortality during early life than the northern population while increased rearing temperature impacted the growth and condition of developing northern sturgeon. During the cold shock, HSP70 and HSP90a mRNA expression increased in all sturgeon treatments as stocking temperature decreased, with higher expression observed in the southern population. Expression of HSP90b, CIRP and SCD increased as stocking temperature decreased in northern sturgeon with early acclimation to 20°C. Correlation analyses indicated the strongest molecular relationships were in the expression of HSP90b, CIRP and SCD, across all treatments, with a correlation between HSP90b and body condition in northern sturgeon with early acclimation to 20°C. Together, these observations highlight the importance of population and rearing environment throughout early development and on later cellular responses induced by cold stocking temperatures.