Computational analyses of the catalytic and heparin-binding sites and their interactions with glycosaminoglycans in glycoside hydrolase family 79 endo-d-glucuronidase (heparanase)

Mammalian heparanase is an endo-β-glucuronidase associated with cell invasion in cancer metastasis, angiogenesis and inflammation. Heparanase cleaves heparan sulfate proteoglycans in the extracellular matrix and basement membrane, releasing heparin/heparan sulfate oligosaccharides of appreciable size. This in turn causes the release of growth factors, which accelerate tumor growth and metastasis. Heparanase has two glycosaminoglycan-binding domains; however, no three-dimensional structure information is available for human heparanase that can provide insights into how the two domains interact to degrade heparin fragments. We have constructed a new homology model of heparanase that takes into account the most recent structural and bioinformatics data available. Heparin analogs and glycosaminoglycan mimetics were computationally docked into the active site with energetically stable ring conformations and their interaction energies were compared. The resulting docked structures were used to propose a model for substrates and conformer selectivity based on the dimensions of the active site. The docking of substrates and inhibitors indicates the existence of a large binding site extending at least two saccharide units beyond the cleavage site (toward the nonreducing end) and at least three saccharides toward the reducing end (toward heparin-binding site 2). The docking of substrates suggests that heparanase recognizes the N-sulfated and O-sulfated glucosamines at subsite +1 and glucuronic acid at the cleavage site, whereas in the absence of 6-O-sulfation in glucosamine, glucuronic acid is docked at subsite +2. These findings will help us to focus on the rational design of heparanase-inhibiting molecules for anticancer drug development by targeting the two heparin/heparan sulfate recognition domains.

A comparative structural bioinformatics analysis of the insulin receptor family ectodomain based on phylogenetic information

The insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor-related receptor (IRR) are covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible mechanical trigger of the activation of the IR that involves a slight ‘twist’ rotation of the last two fibronectin type III domains in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from amphibians to mammals.

Polymorphic low penetrance genes and breast cancer : The role of genes involved in metabolism of xenobiotics, estrogens and reactive oxygen species

Various endogenous and exogenous factors have been reported to increase the risk of breast cancer. Many of those are related to prolonged lifetime exposure to estrogens. Furthermore, a positive family history of breast cancer and certain benign breast diseases are known to increase the risk of breast cancer. The role of lifestyle factors, such as use of alcohol and smoking has been an area of intensive study. Alcohol has been found to increase the risk of breast cancer, whereas the role of smoking has remained obscure. A multitude of enzymes are involved in the metabolism of estrogens and xenobiotics including the carcinogens found in tobacco smoke. Many of the metabolic enzymes exhibit genetic polymorphisms that can lead to inter-individual differences in their abilities to modify hazardous substrates. Therefore, in presence of a given chemical exposure, one subgroup of women may be more susceptible to breast carcinogenesis, since they carry unfavourable forms of the polymorphic genes involved in the metabolism of the chemical. In this work, polymorphic genes encoding for cytochrome P450 (CYP) 1A1 and 1B1, N-acetyl transferase 2 (NAT2), sulfotransferase 1A1 (SULT1A1), manganese superoxide dismutase (MnSOD) and vitamin D receptor (VDR) were investigated in relation to breast cancer susceptibility in a Finnish population. CYP1A1, CYP1B1 and SULT1A1 are involved in the metabolism of both estrogens and xenobiotics, whereas NAT2 is involved only in the latter. MnSOD is an antioxidant enzyme protecting cells from oxidative damage. VDR, in turn, mediates the effects of the active form of vitamin D (1,25(OH)2D3, calcitriol) on maintenance of calcium homeostasis and it has anti-proliferative effects in many cancer cells. A 1.3-fold (95% CIs 1.01-1.73) increased risk of breast cancer was seen among women who carried the NAT2 slow acetylator genotype and a 1.5-fold (95% CI 1.1-2.0) risk was found in women with a MnSOD variant A allele containing genotypes compared to women with the NAT2 rapid acetylator genotype or to those with the MnSOD VV genotype, respectively. Instead, women with the VDR a allele containing genotypes were found to be at a decreased risk for breast cancer (OR 0.73; 95% CI 0.54-0.98) compared to women with the AA genotype. No significant overall associations were found between SULT1A1 or CYP genotypes and breast cancer risk, whereas a combination of the CYP1B1 432Val allele containing genotypes with the NAT2 slow acetylator genotypes posed a 1.5-fold (95% CI 1.03-2.24) increased risk. Moreover, NAT2 slow acetylator genotype was found to be confined to women with an advanced stage of breast cancer (stages III and IV). Further evidence for the association of xenobiotic metabolising genes with breast cancer risk was found when active smoking was taken into account. Women who smoked less than 10 cigarettes/day and carried at least one CYP1B1 432Val variant allele, were at 3.1-fold (95% CI 1.32-7.12) risk of breast cancer compared to women who smoked the same amount but did not carry the variant allele. Furthermore, the risk was significantly increased with increasing number of the CYP1B1 432Val alleles (p for trend 0.005). In addition, women who smoked less than 5 pack-years and carried the NAT2 slow acetylator genotype were at a 2.6-fold (95% CI 1.01-6.48) increased risk of breast cancer compared to women who smoked the same amount but carried the NAT2 rapid acetylator genotype. Furthermore, the combination of the CYP1B1 432Val allele and the NAT2 slow acetylator genotype increased the risk of breast cancer by 2.5-fold (95% CI 1.11-5.45) among ever smokers. Instead, the MnSOD A allele was found to be a risk factor among postmenopausal long-term smokers (>15 years of smoking) (OR 5.1; 95% CI 1.4-18.4) or among postmenopausal women who had smoked more than 10 cigarettes/day (OR 5.5; 95% CI 1.3-23.4) compared to women who had similar smoking habits but carried the MnSOD V/V genotype. Similarly, within subgroups of postmenopausal women who were using oral contraceptives, hormone replacement therapy or alcohol, women carrying the MnSOD A allele genotypes seemed to be at increased risk of breast cancer compared to women with the MnSOD V/V genotype. A positive family history of breast cancer and high parity were shown to be inversely associated with breast cancer risk among women carrying the VDR ApaI a allele or among premenopausal women carrying the SULT1A1*2 allele, respectively.

Transcriptional Silencing of a tRNA1Gly Copy from within a Multigene Family Is Modulated by Distal cis Elements

Individual copies of tRNA1Gly from within the multigene family in Bombyx mori could be classified based on in vitro transcription in homologous nuclear extracts into three categories of highly, moderately, or weakly transcribed genes. Segregation of the poorly transcribed gene copies 6 and 7, which are clustered in tandem within 425 base pairs, resulted in enhancement of their individual transcription levels, but the linkage itself had little influence on the transcriptional status. For these gene copies, when fused together generating a single coding region, transcription was barely detectable, which suggested the presence of negatively regulating elements located in the far flanking sequences. They exerted the silencing effect on transcription overriding the activity of positive regulatory elements. Systematic analysis of deletion, chimeric, and mutant constructs revealed the presence of a sequence element TATATAA located beyond 800 nucleotides upstream to the coding region acting as negative modulator, which when mutated resulted in high level transcription. Conversely, a TATATAA motif reintroduced at either far upstream or far downstream flanking regions exerted a negative effect on transcription. The location of cis-regulatory sequences at such farther distances from the coding region and the behavior of TATATAA element as negative regulator reported here are novel. These element(s) could play significant roles in activation or silencing of genes from within a multigene family, by recruitment or sequestration of transcription factors.

Novel CDNF/MANF protein family : Molecular structure, expression and neurotrophic activity

Neurotrophic factors (NTFs) are secreted proteins which promote the survival of neurons, formation and maintenance of neuronal contacts and regulate synaptic plasticity. NTFs are also potential drug candidates for the treatment of neurodegenerative diseases. Parkinson’s disease (PD) is mainly caused by the degeneration of midbrain dopaminergic neurons. Current therapies for PD do not stop the neurodegeneration or repair the affected neurons. Thus, search of novel neurotrophic factors for midbrain dopaminergic neurons, which could also be used as therapeutic proteins, is highly warranted. In the present study, we identified and characterized a novel protein named conserved dopamine neurotrophic factor (CDNF), a homologous protein to mesencephalic astrocyte-derived neurotrophic factor (MANF). Others have shown that MANF supports the survival of embryonic midbrain dopaminergic neurons in vitro, and protects cultured cells against endoplasmic reticulum (ER) stress. CDNF and MANF form a novel evolutionary conserved protein family with characteristic eight conserved cysteine residues in their primary structure. The vertebrates have CDNF and MANF encoding genes, whereas the invertebrates, including Drosophila and Caenorhabditis have a single homologous CDNF/MANF gene. In this study we show that CDNF and MANF are secreted proteins. They are widely expressed in the mammalian brain, including the midbrain and striatum, and in several non-neuronal tissues. We expressed and purified recombinant human CDNF and MANF proteins, and tested the neurotrophic activity of CDNF on midbrain dopaminergic neurons using a 6-hydroxydopamine (6-OHDA) rat model of PD. In this model, a single intrastriatal injection of CDNF protected midbrain dopaminergic neurons and striatal dopaminergic fibers from the 6-OHDA toxicity. Importantly, an intrastriatal injection of CDNF also restored the functional activity of the nigrostriatal dopaminergic system when given after the striatal 6-OHDA lesion. Thus, our study shows that CDNF is a potential novel therapeutic protein for the treatment of PD. In order to elucidate the molecular mechanisms of CDNF and MANF activity, we resolved their crystal structure. CDNF and MANF proteins have two domains; an amino (N)-terminal saposin-like domain and a presumably unfolded carboxy (C)-terminal domain. The saposin-like domain, which is formed by five α-helices and stabilized by three intradomain disulphide bridges, may bind to lipids or membranes. The C-terminal domain contains an internal cysteine bridge in a CXXC motif similar to that of thiol/disulphide oxidoreductases and isomerases, and may thus facilitate protein folding in the ER. Our studies suggest that CDNF and MANF are novel potential therapeutic proteins for the treatment of neurodegenerative diseases. Future studies will reveal the neurotrophic and cytoprotective mechanisms of CDNF and MANF in more detail.

The BglG group of antiterminators: a growing family of bacterial regulators

The product of the bglG gene of Escherichia coli was among the first bacterial antiterminators to be identified and characterized. Since the elucidation ten years ago of its role in the regulation of the bgl operon of E. coli,a large number of homologies have been discovered in both Gram-positive and Gram-negative bacteria. Often the homologues of BglG in other organisms are also involved in regulating β-glucoside utilization. Surprisingly, in many cases, they mediate antitermination to regulate a variety of other catabolic functions. Because of the high degree of conservation of the cis-acting regulatory elements, antiterminators from one organism can function in another. Generally the antiterminator protein itself is negatively regulated by phosphorylation by a component of the phosphotransferase system. This family of proteins thus represents a highly evolved regulatory system that is conserved across evolutionarily distant genuses.

Revision of the family Alycidae (Acariformes, Acari), with special reference to European species

The thesis provides a proposal to divide Alycidae G. Canestrini & Fanzago into two subfamilies and four tribes. This new hierarchy is based on a reassessment and reranking of new and previously known synapomorphies of the clusters concerned by cladistic analysis, using 60 morphological characters for 48 ingroup species. The basic characters of the taxa are illustrated either by SEM micrographs (Scanning Electron Microscopy) or by outline drawings. The presented classification includes the definitions of Alycini G. Canestrini & Fanzago new rank; Bimichaeliini Womersley new rank; Petralycini new rank; and the (re)descriptions of Alycus C.L. Koch, Pachygnathus Dugès, Amphialycus Zachvatkin, Bimichaelia Thor and Laminamichaelia gen. nov. The species described or redescribed are: Pachygnathus wasastjernae sp. nov. from Kvarken (Merenkurkku), Finland; Pachygnathus villosus Dugès (in Oken); Alycus roseus C.L. Koch; Alycus denasutus (Grandjean) comb. and stat. nov.; Alycus trichotus (Grandjean) comb. nov.; Alycus marinus (Schuster) comb. nov.; Amphialycus (Amphialycus) pentophthalmus Zachvatkin; Amphialycus (Amphialycus) leucogaster (Grandjean); and Amphialycus (Orthacarus) oblongus (Halbert) comb. nov.; Bimichaelia augustana (Berlese); Bimichaelia sarekensis Trägårdh; Laminamichaelia setigera (Berlese) comb. nov.; Laminamichelia arbusculosa (Grandjean) comb. nov.; Laminamichelia subnuda (Berlese) comb. nov. and Petralycus unicornis Grandjean. Fourteen nominal species were found to be junior synonymies. The importance of sensory organs in taxonomy is well recognized, but inclusion of the elaborate skin pattern seemed to improve essentially the usefulness of the prodorsal sensory area. The detailed pictures of the prodorsa of the European alycids could be used like passport photographs for the species. A database like this of prodorsa of other mite taxa as well might be an answer to future needs of species identification in soil zoology, ecology and conservation.

Time-driven life cycle cost estimation system for product family design

This research develops a design support system, which is able to estimate the life cycle cost of different product families at the early stage of product development. By implementing the system, a designer is able to develop various cost effective product families in a shorter lead-time and minimise the destructive impact of the product family on the environment.

Ion exchange of sodium chloride and sodium bicarbonate solutions using strong acid cation resins in relation to coal seam water treatment

Coal seam gas production has resulted in the production of large volumes of associated water which contains dissolved salts dominated by sodium chloride and sodium bicarbonate. Ion exchange using synthetic resins has been proposed as a method for desalination of coal seam water to make it suitable for various beneficial reuse options. This study investigated the behaviour of solutions of sodium chloride and sodium bicarbonate with respect to exchange with Lanxess S108H strong acid cation (SAC) resin. Equilibrium isotherms were created for solutions of NaCl and NaHCO3 and an actual sample of coal seam water from the Surat Basin in southern Queensland. The exchange of sodium ions arising from sodium bicarbonate was found to be considerably more favourable than exchange of sodium ions from sodium chloride solutions. This latter behaviour was attributed to the secondary decomposition of bicarbonate species under acidic conditions which resulted in the evolution of carbon dioxide and formation of water. The isotherm profiles could not be satisfactorily fitted by a single isotherm model such as the Langmuir expression. Instead, two Langmuir equations had to be simultaneously applied in order to fit the sections of the isotherm attributable to sodium ion exchange from sodium bicarbonate and sodium chloride. The shape of the isotherm profile was dependent upon the ratio of sodium chloride to sodium bicarbonate in solution and there was a high degree of correlation between simulated and actual coal seam water solutions.