COVALENT LIPOPROTEIN(A) ASSEMBLY: Characterization of Oxidase Activity Responsible for Catalyzing Covalent Lipoprotein(a) Assembly

Lipoprotein(a) (Lp(a)) has been identified as an emerging risk factor for the development of vascular diseases. The Lp(a) particle is assembled in a 2-step process upon secretion of the LDL and apo(a) components from hepatocytes. Work done by the Koschinsky group has identified an oxidase-like activity present in the conditioned medium (CM) harvested from human hepatoma (HepG2), as well as HEK 293 (human endothelian kidney) cells that catalyzes the rate of covalent Lp(a) formation. We have taken a candidate enzyme approach to identifying this oxidase activity. Specifically, we have proposed that the QSOX (Quiescin/sulfhydryl oxidase) is responsible for catalysis of covalent Lp(a) assembly. An oxidase activity assay developed by Dr. Thorpe (University of Delaware) was used to detect QSOX1 in CM harvested from cultured cell lines that catalyze covalent Lp(a) assembly. In addition, the QSOX1 transcript was identified in each cell line and quantified with the use of Real-Time RT-PCR. Quantitative assays of covalent Lp(a) assembly were performed to study some characteristics of the unkwown oxidase activity. First, conditioned medium was dialyzed through a 5 kDa cutoff, as this has previously been shown to reduce the aforementioned oxidase activity. Purified QSOX was then added back to the reaction and the rate of catalysis was observed. The addition of QSOX appeared to enhance the rate of covalent Lp(a) assembly in a dose-dependent manner. Additional covalent Lp(a) assembly assays were performed where various chemicals were added to determine whether Lp(a) assembly was affected. The addition of EDTA did not affect covalent assembly, suggesting that the oxidase activity may not be metallo-dependent. Moreover, dose-dependent addition of Calcium, DTT, Copper and glutathione to dialyzed medium also did not affect the rate of Lp(a) assembly. Taken together, these studies will aid in identifying the nature of the oxidase activity that catalyzes covalent Lp(a) assembly. This will provide us with valuable information on how Lp(a) particles are assembled, and may lead to the development of drugs inhibiting Lp(a) formation.

The Effect of Illuminant Position on Perceived Curvature

In shaded scenes surface features can appear either concave or convex, depending upon the viewers judment about the direction of the prevailing illuminant. If other curvature cues are added to the image this ambiguity can be removed. However, it is not clear to what extent, if any, illuminant positin exerts an influence on the perceived magnitude of surface curvature. Subjects were presented with pairs of spherical surface patches in a curavture matching task. The patches were defined by shading and texture cues. The percevied curvature of a standard patch was measured as a function of light source position. We found a clear effect of light source position on apparent curvature. Perceived curvature decreased as light source tilt increased and as light source slant decreased. We also found that the strength of this effect is determined partly by a surface's reflectance function and partly by the relative weight of the texture cue. When a specular component was added to the stimuli, the effect of light source orientation was weakened. The weight of the texture cue was manipulated by disrupting the regular distribution of texture elements. We found an inverse relationship between the strength of the effecct and the weight of the texture cue: lowering the texture cue weight resulted in an enhancement of the illuminant position effect.

A possible model of benzimidazole binding to beta-tubulin disclosed by invoking an inter-domain movement.

Although it is well established that benzimidazole (BZMs) compounds exert their therapeutic effects through binding to helminth beta-tubulin and thus disrupting microtubule-based processes in the parasites, the precise location of the benzimidazole-binding site on the beta-tubulin molecule has yet to be determined. In the present study, we have used previous experimental data as cues to help identify this site. Firstly, benzimidazole resistance has been correlated with a phenylalanine-to-tyrosine substitution at position 200 of Haemonchus contortus beta-tubulin isotype-I. Secondly, site-directed mutagenesis studies, using fungi, have shown that other residues in this region of the protein can influence the interaction of benzimidazoles with beta-tubulin. However, the atomic structure of the alphabeta-tubulin dimer shows that residue 200 and the other implicated residues are buried within the protein. This poses the question: how might benzimidazoles interact with these apparently inaccessible residues? In the present study, we present a mechanism by which those residues generally believed to interact with benzimidazoles may become accessible to the drugs. Furthermore, by docking albendazole-sulphoxide into a modelled H. contortus beta-tubulin molecule we offer a structural explanation for how the mutation conferring benzimidazole resistance in nematodes may act, as well as a possible explanation for the species-specificity of benzimidazole anthelmintics.

A complex zinc finger controls the enzymatic activities of nidovirus helicases.

Nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) encode a nonstructural protein, called nsp10 in arteriviruses and nsp13 in coronaviruses, that is comprised of a C-terminal superfamily 1 helicase domain and an N-terminal, putative zinc-binding domain (ZBD). Previously, mutations in the equine arteritis virus (EAV) nsp10 ZBD were shown to block arterivirus reproduction by disrupting RNA synthesis and possibly virion biogenesis. Here, we characterized the ATPase and helicase activities of bacterially expressed mutant forms of nsp10 and its human coronavirus 229E ortholog, nsp13, and correlated these in vitro activities with specific virus phenotypes. Replacement of conserved Cys or His residues with Ala proved to be more deleterious than Cys-for-His or His-for-Cys replacements. Furthermore, denaturation-renaturation experiments revealed that, during protein refolding, Zn2+ is essential for the rescue of the enzymatic activities of nidovirus helicases. Taken together, the data strongly support the zinc-binding function of the N-terminal domain of nidovirus helicases. nsp10 ATPase/helicase deficiency resulting from single-residue substitutions in the ZBD or deletion of the entire domain could not be complemented in trans by wild-type ZBD, suggesting a critical function of the ZBD in cis. Consistently, no viral RNA synthesis was detected after transfection of EAV full-length RNAs encoding ATPase/helicase-deficient nsp10 into susceptible cells. In contrast, diverse phenotypes were observed for mutants with enzymatically active nsp10, which in a number of cases correlated with the activities measured in vitro. Collectively, our data suggest that the ZBD is critically involved in nidovirus replication and transcription by modulating the enzymatic activities of the helicase domain and other, yet unknown, mechanisms.

Herpes simplex virus type 1 and Alzheimer's disease: the autophagy connection.

The causes of Alzheimer's disease (AD) and of the characteristic pathological features—amyloid plaques and neurofibrillary tangles—of AD brain are unknown, despite the enormous resources provided over the years for their investigation. Indeed, the only generally accepted risk factors are age, Down syndrome, carriage of the type 4 allele of the apolipoprotein E gene (APOE-e 4), and possibly brain injury. Following the authors' previous studies implicating herpes simplex virus type 1 (HSV1) in brain of APOE-e 4 carriers as a major cause of AD, the authors propose here, on the basis of their and others' recent studies, that not only does HSV1 generate the main components of amyloid plaques and neurofibrillary tangles (NFTs)—ß -amyloid (Aß) and abnormally phosphorylated tau but also, by disrupting autophagy, it prevents degradation of these aberrant proteins, leading to their accumulation and deposition, and eventually to AD.

Selective synthesis of chlorophosphoramidites using ionic liquids

A range of chlorophosphoramidites have been prepared in ionic liquids and compared with material synthesised in molecular solvents. Through the use of ionic liquids as reaction media the moisture sensitivity and impurity issues hampering existing traditional synthetic routes have been eased. Not only can stock chemicals be used without purification, but the reactions may be conducted at room temperature and at high concentrations. Furthermore, reaction times are reduced and rapid addition of reagents is possible whilst retaining tight control over product selectivity. Beyond their role as reaction media, ionic liquids also present a unique storage medium for these highly moisture sensitive chlorophosphoramidites.

A framework for assessing the vulnerability of food systems to future shocks

Modem society depends on complex agro-ecological and trading systems to provide food for urban residents, yet there are few tools available to assess whether these systems are vulnerable to future disturbances. We propose a preliminary framework to assess the vulnerability of food systems to future shocks based on landscape ecology's 'Panarchy Framework'. According to Panarchy, ecosystem vulnerability is determined by three generic characteristics: (1) the wealth available in the system, (2) how connected the system is, and (3) how much diversity exists in the system. In this framework, wealthy, non-diverse, tightly connected systems are highly vulnerable. The wealth of food systems can be measured using the approach pioneered by development economists to assess how poverty affects food security. Diversity can be measured using the tools investors use to measure the diversity of investment portfolios to assess financial risk. The connectivity of a system can be evaluated with the tools chemists use to assess the pathways chemicals use to flow through the environment. This approach can lead to better tools for creating policy designed to reduce vulnerability, and can help urban or regional planners identify where food systems are vulnerable to shocks and disturbances that may occur in the future. (c) 2005 Elsevier Ltd. All rights reserved.

A simple and robust method for connecting small-molecule drugs using gene-expression signatures

Background

Interaction of a drug or chemical with a biological system can result in a gene-expression profile or signature characteristic of the event. Using a suitably robust algorithm these signatures can potentially be used to connect molecules with similar pharmacological or toxicological properties by gene expression profile. Lamb et al first proposed the Connectivity Map [Lamb et al (2006), Science 313, 1929–1935] to make successful connections among small molecules, genes, and diseases using genomic signatures.

Results

Here we have built on the principles of the Connectivity Map to present a simpler and more robust method for the construction of reference gene-expression profiles and for the connection scoring scheme, which importantly allows the valuation of statistical significance of all the connections observed. We tested the new method with two randomly generated gene signatures and three experimentally derived gene signatures (for HDAC inhibitors, estrogens, and immunosuppressive drugs, respectively). Our testing with this method indicates that it achieves a higher level of specificity and sensitivity and so advances the original method.

Conclusion

The method presented here not only offers more principled statistical procedures for testing connections, but more importantly it provides effective safeguard against false connections at the same time achieving increased sensitivity. With its robust performance, the method has potential use in the drug development pipeline for the early recognition of pharmacological and toxicological properties in chemicals and new drug candidates, and also more broadly in other 'omics sciences.

Gelation of ionic liquids using a cross-linked poly(ethylene glycol) gel matrix

Conductive ionic liquid -poly(ethylene glycol) (IL-PEG) gels have been prepared by gelation of the hydrophobic ionic liquid 1-hexyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [(C(6)mim] [NTf2]) by the cross-linking reaction of disuccinimidylpropyl PEG monomers with four-arm tetraamine PEG cross-linkers. This is the first time that a crosslinked PEG matrix, such as this, has been used to gel nonaqueous solvents. Initial studies screening other ionic liquids as solvents indicate that the gelation of the ionic liquid is both cation and anion dependent with smaller, coordinating cations disrupting or preventing gel formation.

Activation of Alkanes by Gold-Modified Lanthanum Oxide

Functionalization of alkanes is much sought after for the production of fine and bulk chemicals. In particular, the oxidative activation of alkanes and their conversion to ethene and propene has been studied extensively, owing to the use of these alkenes in polymerization reactions. The greater reactivity of the products in comparison with the reactants has proven a major issue in this reaction as this can result in overoxidation, producing CO and CO2 and, therefore, reducing the alkene yield. Herein, the first application of supported gold catalysts for the direct activation of C2+ aliphatic alkanes with oxygen to form alkenes is demonstrated. This catalyst is particularly notable as it is highly active, selective to propene and ethene, and stable on stream over a 48 h period. Maintaining cationic gold is thought to be critical for the stability and this catalyst design provides the possibility of applying gold-based catalysts over a much wider temperature range than has been reported.

Human Dental Pulp Fibroblasts Express the “Cold-sensing” Transient Receptor Potential Channels TRPA1 and TRPM8

Introduction: Transient receptor potential (TRP) channels comprise a group of nonselective calcium-permeable cationic channels, which are polymodal sensors of environmental stimuli such as thermal changes and chemicals. TRPM8 and TRPA1 are cold-sensing TRP channels activated by moderate cooling and noxious cold temperatures, respectively. Both receptors have been identified in trigeminal ganglion neurones, and their expression in nonneuronal cells is now the focus of much interest. The aim of this study was to investigate the molecular and functional expression of TRPA1 and TRPM8 in dental pulp fibroblasts.
Methods: Human dental pulp fibroblasts were derived from healthy molar teeth. Gene and protein expression was determined by polymerase chain reaction and Western blotting. Cellular localization was investigated by immunohistochemistry, and TRP functionality was determined by Ca2+ microfluorimetry.
Results: Polymerase chain reaction and Western blotting showed gene and protein expression of both TRPA1 and TRPM8 in fibroblast cells in culture. Immunohistochemistry studies showed that TRPA1 and TRPM8 immunoreactivity co-localized with the human fibroblast surface protein. In Ca2+ microfluorimetry studies designed to determine the functionality of TRPA1 and TRPM8 in pulp fibroblasts, we showed increased intracellular calcium ([Ca2+]i) in response to the TRPM8 agonist menthol, the TRPA1 agonist cinnamaldehyde, and to cool and noxious cold stimuli, respectively. The responses to agonists and thermal stimuli were blocked in the presence of specific TRPA1 and TRPM8 antagonists.
Conclusions: Human dental pulp fibroblasts express TRPA1 and TRPM8 at the molecular, protein, and functional levels, indicating a possible role for fibroblasts in mediating cold responses in human teeth.

Combining bio- and chemo-catalysis: from enzymes to cells, from petroleum to biomass

In the future, biomass will continue to emerge as a viable source of chemicals. The development of new industries that utilize bio-renewables provides opportunities for innovation. For example, bio- and chemo-catalysts can be combined in 'one pot' to prepare chemicals of commercial value. This has been demonstrated using isolated enzymes and whole cells for a variety of chemical transformations. The one-pot approach has been successfully adopted to convert chemicals derived from biomass, and, in our opinion, it has an important role to play in the design of a more sustainable chemical industry. To implement new one-pot bio- and chemo-catalytic processes, issues of incompatibility must be overcome; the strategies for which are discussed in this opinion article.

Relative quantification of the proteomeic changes associated with the mycotoxin zearalenone in the H295R steroidogenesis model

Zearalenone (ZEN) is a mycotoxin with endocrine disrupting effects having vast economic implications in e.g. pig farming. Structurally, ZEN resembles 17b-estradiol, and thus is able to bind to estrogen receptors (ER) in target cells. Because of this, it is also classified as a non-steroidal estrogen, a phytoestrogen, a mycoestrogen, and a growth promoter. Quantitative proteomic analysis was undertaken using stable-isotope labeling by amino acids in cell culture (SILAC) upon exposure of the steroidogenesis cell model H295R with ZEN to elucidate its effect on protein regulation. ZEN significantly regulated 21 proteins, including proteins with known endocrine disrupting effects and several oncogenes. In addition, network analysis using Ingenuity Pathway Analysis showed that ZEN affected the oxidative phosphorylation pathway and the mitochondrial dysfunction pathway, both previously reported to be involved in endocrine dysfunction.

Hydrophobic substances induce water stress in microbial cells

Ubiquitous noxious hydrophobic substances, such as hydrocarbons, pesticides and diverse industrial chemicals, stress biological systems and thereby affect their ability to mediate biosphere functions like element and energy cycling vital to biosphere health. Such chemically diverse compounds may have distinct toxic activities for cellular systems; they may also share a common mechanism of stress induction mediated by their hydrophobicity. We hypothesized that the stressful effects of, and cellular adaptations to, hydrophobic stressors operate at the level of water : macromolecule interactions. Here, we present evidence that: (i) hydrocarbons reduce structural interactions within and between cellular macromolecules, (ii) organic compatible solutes-metabolites that protect against osmotic and chaotrope-induced stresses-ameliorate this effect, (iii) toxic hydrophobic substances induce a potent form of water stress in macromolecular and cellular systems, and (iv) the stress mechanism of, and cellular responses to, hydrophobic substances are remarkably similar to those associated with chaotrope-induced water stress. These findings suggest that it may be possible to devise new interventions for microbial processes in both natural environments and industrial reactors to expand microbial tolerance of hydrophobic substances, and hence the biotic windows for such processes.

Cdk1 participates in BRCA1-dependent S phase checkpoint control in response to DNA damage.

Cdk2 and cdk1 are individually dispensable for cell-cycle progression in cancer cell lines because they are able to compensate for one another. However, shRNA-mediated depletion of cdk1 alone or small molecule cdk1 inhibition abrogated S phase cell-cycle arrest and the phosphorylation of a subset of ATR/ATM targets after DNA damage. Loss of DNA damage-induced checkpoint control was caused by a reduction in formation of BRCA1-containing foci. Mutation of BRCA1 at S1497 and S1189/S1191 resulted in loss of cdk1-mediated phosphorylation and also compromised formation of BRCA1-containing foci. Abrogation of checkpoint control after cdk1 depletion or inhibition in non-small-cell lung cancer cells sensitized them to DNA-damaging agents. Conversely, reduced cdk1 activity caused more potent G2/M arrest in nontransformed cells and antagonized the response to subsequent DNA damage. Cdk1 inhibition may therefore selectively sensitize BRCA1-proficient cancer cells to DNA-damaging treatments by disrupting BRCA1 function.