Triazolopyrimidine herbicides are potent inhibitors of Aspergillus fumigatus acetohydroxyacid synthase and potential antifungal drug leads.

Aspergillus fumigatus is a fungal pathogen whose effects can be debilitating and potentially fatal in immunocompromised patients. Current drug treatment options for this infectious disease are limited to just a few choices (e.g. voriconazole and amphotericin B) and these themselves have limitations due to potentially adverse side effects. Furthermore, the likelihood of the development of resistance to these current drugs is ever present. Thus, new treatment options are needed for this infection. A new potential antifungal drug target is acetohydroxyacid synthase (AHAS; EC 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway, and a target for many commercial herbicides. In this study, we have expressed, purified and characterised the catalytic subunit of AHAS from A. fumigatus and determined the inhibition constants for several known herbicides. The most potent of these, penoxsulam and metosulam, have Ki values of 1.8 ± 0.9 nM and 1.4 ± 0.2 nM, respectively. Molecular modelling shows that these compounds are likely to bind into the herbicide binding pocket in a mode similar to Candida albicans AHAS. We have also shown that these two compounds inhibit A. fumigatus growth at a concentration of 25 µg/mL. Thus, AHAS inhibitors are promising leads for the development of new anti-aspergillosis therapeutics.

Lipid biochemical changes detected in normal appearing white matter of chronic multiple sclerosis by spectral coherent Raman imaging.

Multiple sclerosis (MS) exhibits demyelination, inflammatory infiltration, axonal degeneration, and gliosis, affecting widespread regions of the central nervous system (CNS). While white matter MS lesions have been well characterized pathologically, evidence indicates that the MS brain may be globally altered, with subtle abnormalities found in grossly normal appearing white matter (NAWM). These subtle changes are difficult to investigate by common methods such as histochemical stains and conventional magnetic resonance imaging. Thus, the prototypical inflammatory lesion likely represents the most obvious manifestation of a more widespread involvement of the CNS. We describe the application of spectral coherent anti-Stokes Raman Scattering (sCARS) microscopy to study such changes in chronic MS tissue particularly in NAWM. Subtle changes in myelin lipid biochemical signatures and intra-molecular disorder of fatty acid acyl chains of otherwise normal-appearing myelin were detected, supporting the notion that the biochemical involvement of the MS brain is far more extensive than conventional methods would suggest.

Product release is rate-limiting for catalytic processing by the Dengue virus protease.

Dengue Virus (DENV) is the most prevalent global arbovirus, yet despite an increasing burden to health care there are currently no therapeutics available to treat infection. A potential target for antiviral drugs is the two-component viral protease NS2B-NS3pro, which is essential for viral replication. Interactions between the two components have been investigated here by probing the effect on the rate of enzyme catalysis of key mutations in a mobile loop within NS2B that is located at the interface of the two components. Steady-state kinetic assays indicated that the mutations greatly affect catalytic turnover. However, single turnover and fluorescence experiments have revealed that the mutations predominantly affect product release rather than substrate binding. Fluorescence analysis also indicated that the addition of substrate triggers a near-irreversible change in the enzyme conformation that activates the catalytic centre. Based on this mechanistic insight, we propose that residues within the mobile loop of NS2B control product release and present a new target for design of potent Dengue NS2B-NS3 protease inhibitors.

What drives MRI-measured cortical atrophy in multiple sclerosis?

BACKGROUND: Cortical atrophy, assessed with magnetic resonance imaging (MRI), is an important outcome measure in multiple sclerosis (MS) studies. However, the underlying histopathology of cortical volume measures is unknown. OBJECTIVE: We investigated the histopathological substrate of MRI-measured cortical volume in MS using combined post-mortem imaging and histopathology. METHODS: MS brain donors underwent post-mortem whole-brain in-situ MRI imaging. After MRI, tissue blocks were systematically sampled from the superior and inferior frontal gyrus, anterior cingulate gyrus, inferior parietal lobule, and superior temporal gyrus. Histopathological markers included neuronal, axonal, synapse, astrocyte, dendrite, myelin, and oligodendrocyte densities. Matched cortical volumes from the aforementioned anatomical regions were measured on the MRI, and used as outcomes in a nested prediction model. RESULTS: Forty-five tissue blocks were sampled from 11 MS brain donors. Mean age at death was 68±12 years, post-mortem interval 4±1 hours, and disease duration 35±15 years. MRI-measured regional cortical volumes varied depending on anatomical region. Neuronal density, neuronal size, and axonal density were significant predictors of GM volume. CONCLUSIONS: In patients with long-standing disease, neuronal and axonal pathology are the predominant pathological substrates of MRI-measured cortical volume in chronic MS.

Transmigration of beta amyloid specific heavy chain antibody fragments across the in vitro blood-brain barrier.

Previously selected amyloid beta recognizing heavy chain antibody fragments (VHH) affinity binders derived from the Camelid heavy chain antibody repertoire were tested for their propensity to cross the blood-brain barrier (BBB) using an established in vitro BBB co-culture system. Of all tested VHH, ni3A showed highest transmigration efficiency which is, in part, facilitated by a three amino acid substitutions in its N-terminal domain. Additional studies indicated that the mechanism of transcellular passage of ni3A is by active transport. As VHH ni3A combines the ability to recognize amyloid beta and to cross the BBB, it has potential as a tool for non-invasive in vivo imaging and as efficient local drug targeting moiety in patients suffering from cerebral amyloidosis such as Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA).

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase.

To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier-either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies "open" and "closed" CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.

Differential receptor-mediated drug targeting to the diseased brain.

The brain is not directly accessible for intravenously administered macro- and most small molecular drugs because of the presence of the blood-brain barrier (BBB). In this respect the BBB functions as a physical and metabolic barrier which is presented by the endothelial cells in brain capillaries. In order to overcome the BBB, therapeutic compounds have been targeted to internalizing receptors at the BBB. In this review we summarize the different approaches that have been described in current literature, including the possible difficulties for clinical application. Particularly, we focus on the possible impact of brain diseases on receptor-mediated transport to the BBB/brain and how this may affect various targeting strategies. Moreover, it is our opinion that a differential drug targeting/delivery approach should be applied to treat central nervous system (CNS) diseases that are related to the BBB alone, and for CNS diseases that are related to both the brain and the BBB.

Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase.

Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of approximately 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the approximately 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans.

[Investigation of the entrance dose of standardized chest radiographs]. / Untersuchungen über die Einfalldosis bei standardisierten Aufnahmen der Lunge.

In 50 chest radiographs obtained in PA projection with the patient erect and the radiographic techniques kept constant, entrance dose, entrance surface dose and dose area product were measured and compared with the measurements obtained with Plexiglas and Alderson phantoms. Following the recommendations of the International Commission on Radiological Protection, the "absorbed dose in air" at the point of intersection of the X-ray beam with the surface of the patient was used as reference. For patients with the same diameter, the measured dose deviated up to +/- 50 % from the "Reference Man" according to the ICRP 23 as well as from the "Representative Patient" according to the ICRP 73. The exposure data produced by the equipment corresponded to the measured values with minor deviations, but the dose values expressed as entrance surface dose showed major deviation in comparison with the entrance dose due to differences in the back-scatter factor. The values of entrance dose calculated from the dose area product measurements showed additional uncertainties due to different field sizes. Moreover, a sex-specific dose area product value was identified. These results reveal that the values of the "ICRP Reference Man" differ in many situations from the values produced by the examined patient due to differences in radiation transmission. This has consequences for determining diagnostic reference values and investigating recorded radiation exposure. This will be explained and substantiated.

A purple acid phosphatase from sweet potato contains an antiferromagnetically coupled binuclear Fe-Mn center.

A purple acid phosphatase from sweet potato is the first reported example of a protein containing an enzymatically active binuclear Fe-Mn center. Multifield saturation magnetization data over a temperature range of 2 to 200 K indicates that this center is strongly antiferromagnetically coupled. Metal ion analysis shows an excess of iron over manganese. Low temperature EPR spectra reveal only resonances characteristic of high spin Fe(III) centers (Fe(III)-apo and Fe(III)-Zn(II)) and adventitious Cu(II) centers. There were no resonances from either Mn(II) or binuclear Fe-Mn centers. Together with a comparison of spectral properties and sequence homologies between known purple acid phosphatases, the enzymatic and spectroscopic data strongly indicate the presence of catalytic Fe(III)-Mn(II) centers in the active site of the sweet potato enzyme. Because of the strong antiferromagnetism it is likely that the metal ions in the sweet potato enzyme are linked via a mu-oxo bridge, in contrast to other known purple acid phosphatases in which a mu-hydroxo bridge is present. Differences in metal ion composition and bridging may affect substrate specificities leading to the biological function of different purple acid phosphatases.

Gene therapy: future therapy for erectile dysfunction.

Advances in molecular biological techniques, completion of the Human Genome Project, and the ensuing age of molecular medicine, in conjuction with the sum of a decades-long accumulation of knowledge of the physiology of erection and the pathophysiology of erectile dysfunction have converged to make gene therapy for erectile dysfunction a distinct possibility. In short, both the intrinsic complexities of mechanisms responsible for ensuring normal erection and the multifactorial nature of erectile dysfunction ensure that there is a relatively vast number of physiologically relevant molecular targets for gene therapy. As such, perhaps it is not surprising that virtually every preclinical gene therapy strategy/target examined thus far has been largely successful in ameliorating conditions associated with compromised erectile function in vivo and/or in vitro. This report highlights the goals and strategies of gene therapy for erectile dysfunction and reviews the strategies that initially have been employed. In short, the preclinical data, while still quite preliminary in many regards, are nonetheless quite impressive and encouraging. If similar success is obtained in clinical trials, gene therapy for erectile dysfunction may provide the first concrete "proof of concept" for using gene therapy in the treatment of human smooth muscle disorders.

Purple acid phosphatases from bacteria: similarities to mammalian and plant enzymes.

Mammalian and plant purple acid phosphatases have similar active site structures despite low sequence identity (<20%). Although no bacterial enzyme has been purified, a sequence database search revealed that genes that could encode potential purple acid phosphatases may be restricted to a small number of organisms (i.e. myco- and cyanobacteria). Analysis of their deduced amino acid sequences and predicted secondary structures indicates that the cyanobacterial enzyme is similar to both the mammalian and the recently discovered low-molecular-weight plant purple acid phosphatases, while the mycobacterial enzyme is homologous to the fungal and high-molecular-weight plant purple acid phosphatases. Homology models indicate that both bacterial proteins appear to be similar to mammalian purple acid phosphatases in the immediate vicinity of the active site. It is likely that these enzymes act as Fenton-type catalysts in order to prevent damage caused by reactive oxygen species generated by invaded host cells (M. tuberculosis) or by the light-harvesting complex (Synechocystis sp.).

Identification of mammalian-like purple acid phosphatases in a wide range of plants.

Purple acid phosphatases (PAPs) comprise a family of binuclear metal-containing hydrolases, members of which have been isolated from plants, mammals and fungi. Polypeptide chains differ in size (animal approximately 35kDa, plant approximately 55kDa) and exhibit low sequence homology between kingdoms but all residues involved in co-ordination of the metal ions are invariant. A search of genomic databases was undertaken using a sequence pattern which includes the conserved residues. Several novel potential PAP sequences were detected, including the first known examples from bacterial sources. Ten plant ESTs were also identified which, although possessing the conserved sequence pattern, were not homologous throughout their sequences to previously known plant PAPs. Based on these EST sequences, novel cDNAs from sweet potato, soybean, red kidney bean and Arabidopsis thaliana were cloned and sequenced. These sequences are more closely related to mammalian PAP than to previously characterized plant enzymes. Their predicted secondary structure is similar to that of the mammalian enzyme. A model of the sweet potato enzyme was generated based on the coordinates of pig PAP. These observations strongly suggest that the cloned cDNA sequences represent a second group of plant PAPs with properties more similar to the mammalian enzymes than to the high molecular weight plant enzymes.

Lack of antimicrobial effect on periodontopathic bacteria by ultrasonic and sonic scalers in vitro.

BACKGROUND: The purpose of this study was to assess the antimicrobial effects of a sonic and ultrasonic scaler generally used for subgingival scaling on gram-negative and gram-positive periodontopathic bacteria. METHOD: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Campylobacter rectus, or Peptostreptococcus micros were suspended in Schaedler's broth medium and treated by a sonic or a magnetostrictive ultrasonic scaler for 30 s and 150 s in vitro. Bacterial suspensions treated by an ultrasonic cell disruptor served as a positive control and untreated bacterial suspensions served as a negative control. Following sonication, samples were serially diluted, streaked on blood agar plates and incubated for 2-5 days at 37 degrees C. RESULTS: Treatment by the sonic or ultrasonic scaler for up to 150 s did not reduce the viability of any of the tested periodontal pathogens. Compared to untreated controls, the viability of A. actinomycetemcomitans and P. gingivalis was significantly (p<0.05) reduced only following ultrasonication with the cell disruptor after 30 s (0.72 and 0.54 log CFU/ml, respectively) and of A. actinomycetemcomitans, P. gingivalis, C. rectus, and P. micros after 150 s (1.98, 1.34, 1.95 and 1.98 log CFU/ml, respectively). CONCLUSION: The data of the study may indicate that the assessed sonic and ultrasonic scaler used for subgingival debridement do not result in killing of the tested periodontal pathogens.

Binuclear metal centers in plant purple acid phosphatases: Fe-Mn in sweet potato and Fe-Zn in soybean.

Purple acid phosphatases comprise a family of binuclear metal-containing acid hydrolases, representatives of which have been found in animals, plants, and fungi. The goal of this study was to characterize purple acid phosphatases from sweet potato tubers and soybean seeds and to establish their relationship with the only well-characterized plant purple acid phosphatase, the FeIII-ZnII-containing red kidney bean enzyme. Metal analysis indicated the presence in the purified sweet potato enzyme of 1.0 g-atom of iron, 0.6-0.7 g-atom of manganese, and small amounts of zinc and copper. The soybean enzyme contained 0.8-0.9 g-atom of iron, 0.7-0.8 g-atom of zinc per subunit, and small amounts of manganese, copper, and magnesium. Both enzymes exhibited visible absorption maxima at 550-560 nm, with molar absorption coefficients of 3200 and 3300 M(-1) cm(-1), respectively, very similar to the red kidney bean enzyme. Substrate specificities were markedly different from those of the red kidney bean enzyme. A cloning strategy was developed based on N-terminal sequences of the sweet potato and soybean enzymes and short sequences around the conserved metal ligands of the mammalian and red kidney bean enzymes. Three sequences were obtained, one from soybean and two from sweet potato. All three showed extensive sequence identity (>66%) with red kidney bean purple acid phosphatase, and all of the metal ligands were conserved. The combined results establish that these enzymes are binuclear metalloenzymes: Fe-Mn in the sweet potato enzyme and Fe-Zn in soybean. The sweet potato enzyme is the first well-defined example of an Fe-Mn binuclear center in a protein.

Crystallization and preliminary X-ray diffraction data for a purple acid phosphatase from sweet potato.

Purple acid phosphatase from sweet potato is a homodimer of 110 kDa. Two forms of the enzyme have been characterized. One contains an Fe-Zn centre similar to that previously reported for red kidney bean purple acid phosphatase. Another isoform, the subject of this work, is the first confirmed example of an Fe-Mn-containing enzyme. Crystals of this protein have been grown from PEG 6000. They have unit-cell parameters a = b = 118.4, c = 287.4 A and have the symmetry of space group P6(5)22, with one dimer per asymmetric unit. Diffraction data collected using a conventional X--ray source from a cryocooled crystal extend to 2.90 A resolution. The three-dimensional structure of the enzyme will provide insight into the coordination of this novel binuclear metal centre.

Heterologous expression of human transketolase.

Transketolase belongs to the family of thiamin diphosphate dependent enzymes. The aim of this study was to establish a bacterial expression system for human transketolase in order to investigate the functional characteristics of mammalian transketolases. The level of recombinant human enzyme expressed in Escherichia coli was modest. Purification of recombinant transketolase and separation from the E. coli enzyme has been greatly simplified by means of a non-cleavable hexa-histidine tag. The highest specific activity was 13.5 U/mg and the K(m) values were 0.27 +/- 0.02 and 0.51 +/- 0.05 mM for the substrates D-xylulose 5-phosphate and D-ribose 5-phosphate, respectively. Binding of cofactors to the apoenzyme showed the expected hysteresis. Without preincubation, the K(m) values for thiamin diphosphate and for Mg2+ were, respectively, 4.1 +/- 0.8 and 2.5 +/- 0.4 microM, but after 1 h of preincubation these values were 85 +/- 16 nM and 0.74 +/- 0.23 microM. The kinetic constants are similar to those of the native enzyme purified from human erythrocytes. Despite the modest expression level the reported system is well suited to a variety of functional studies.

Properties and functions of the thiamin diphosphate dependent enzyme transketolase.

This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a divalent metal ion for its activity. The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall flux of labelled substrates remains controversial. Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined. Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions. In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound. Transketolase is ubiquitous and more than 30 full-length sequences are known. The encoded protein sequences contain two motifs of high homology; one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif. All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives. Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications. Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny. However, a modification of transketolase is a marker for Alzheimer's disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition. The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals.

The role of His113 and His114 in pyruvate decarboxylase from Zymomonas mobilis.

Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP) and a divalent cation as essential cofactors. Recently, the three-dimensional structures of the enzyme from two yeasts have been determined. While these structures shed light on the binding of the cofactors and the reaction mechanism, the interactions between the substrate pyruvate and the enzyme remain unclear. We have used PDC from Zymomonas mobilis as a model for these enzymes in order to study substrate binding. The recombinant enzyme was expressed in Escherichia coli. High yield, simplicity of purification, high stability and simple kinetics make this model well suited for these studies. Activity measurements in the pH range between 5.8 and 8.5 indicated that a His residue may be involved in substrate binding. Analysis of an alignment of all known PDC protein sequences showed two invariant His residues (His113 and His114) which, according to the crystal structure of yeast PDC, are in the vicinity of the active site. Here we demonstrate that replacement of His114 by Gln does not have a great effect on cofactor and substrate binding. However, the k(cat) is decreased indicating that His114 may assist in catalysis. In contrast, substitution of His113 by Gln renders the enzyme completely inactive. This mutant has decreased affinity for both cofactors, as revealed by measurements of tryptophan fluorescence quenching. However, this decreased affinity is insufficient to account for the complete loss of activity. Despite its inability to support overall catalysis, this [Gln113]PDC mutant is capable of releasing acetaldehyde from 2-(1-hydroxyethyl)thiamin diphosphate supplied exogenously. It is proposed that upon substrate binding, His113 is placed close to C2 of the thiazole ring. Subsequent deprotonation of this atom leads to a conformational change that allows a flexible loop (residues 105-112) that precedes His113 to close over the active site. Hence, replacement of His113 by another residue interferes with this closure of the active site and thus disrupts the catalytic process.