Modelling family 2 cystatins and their interaction with papain

Cystatins are extensively studied cysteine protease inhibitors, found in wide range of organisms with highly conserved structural folds. S-type of cystatins is well known for their abundance in saliva, high selectivity and poorer activity towards host cysteine proteases in comparison to their immediate ancestor cystatin C. Despite more than 90% sequence similarity, the members of this group show highly dissimilar binding affinity towards papain. Cystatin M/E is a potent inhibitor of legumain and papain like cysteine proteases and recognized for its involvement in skin barrier formation and potential role as a tumor suppressor gene. However, the structures of these proteins and their complexes with papain or legumain are still unknown. In the present study, we have employed computational methods to get insight into the interactions between papain and cystatins. Three-dimensional structures of the cystatins are generated by homology modelling, refined with molecular dynamics simulation, validated through numerous web servers and finally complexed with papain using ZDOCK algorithm in Discovery Studio. A high degree of shape complementarity is observed within the complexes, stabilized by numerous hydrogen bonds (HB) and hydrophobic interactions. Using interaction energy, HB and solvent accessible surface area analyses, we have identified a series of key residues that may be involved in papain–cystatin interaction. Differential approaches of cystatins towards papain are also noticed which are possibly responsible for diverse inhibitory activity within the group. These findings will improve our understanding of fundamental inhibitory mechanisms of cystatin and provide clues for further research.     

Wild plants eaten in childhood: a retrospective of Estonia in the 1970s–1990s

In this ethnobotanical study, the authors provide the first quantitative analysis of the use of wild edible plants in Estonia, describing the domains and assessing the food importance of different species. The information was collected using free‐listing written questionnaires and concerned plants used by the respondents in their childhood. As part of a major study, this article covers the responses of professionals with some botanical education at vocational or university level, to ensure the greatest possible reliability without using voucher specimens. Fifty‐eight respondents provided information on the use of 137 plant taxa, corresponding to approximately 6% of the native and naturalized vascular plants of Estonia. According to use frequency, the most typical wild food plant of Estonia is a fruit, eaten raw as a snack. The results clearly signal that the majority of famine and food shortage plants had already been forgotten by the end of the 20^th century, but new plants have been introduced as green vegetables for making salads. Despite changes in the nomenclature of the plants, the use of wild food plants in Estonia was still thriving at the turn of the 20^th century, covering many domains already forgotten in urbanized modern Europe. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 239–253.     

Activity of tocopherol oxidase in Phaseolus coccineus seedlings

In the present study, we have demonstrated that membrane-free extracts of etiolated shoots of Phaseolus coccineus seedlings show tocopherol oxidase activity. For this reaction, presence of membrane lipids, such as lecithin and mixture of plant lipids was required. The rate of the reaction was the highest for α-tocopherol and decreased in the order α ? β > γ > δ tocopherols. In the case of α-tocopherol, the main oxidation product was α-tocopherolquinone, while for the other tocopherol homologues the dominant products were other derivatives. When the enzyme activity was measured in leaves, hypocotyls and roots of etiolated seedlings of P. coccineus, the oxidase activity was the highest in extracts of leaves and decreased towards the roots where no activity was detected. The effect of hydrogen peroxide and of different inhibitors on the reaction suggest that tocopherol oxidase does not belong to peroxidases or flavin oxidases but rather to multi-copper oxidases, such as polyphenol oxidases or laccases. On the other hand, catechol, the well-known substrate of polyphenol oxidases and laccases, was not oxidized by the enzyme, indicating a high substrate specificity of the tocopherol oxidase.     

Comparative study of key phosphorus and nitrogen metabolizing enzymes in mycorrhizal and non-mycorrhizal plants of Dendrobium chrysanthum Wall. ex Lindl.

The role of mycorrhizal fungi in overcoming nutrient limitation to plant growth by enhancing nutrient acquisition, especially phosphorus (P) and nitrogen (N), is well documented. However, in orchids, the importance of mycorrhizal fungi in nutrient acquisition is not as extensively studied as in other plants. Therefore, an in vitro culture system to study the effects of mycorrhizal association on P and N metabolizing enzymes, viz., acid phosphatase, alkaline phosphatase, nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) in Dendrobium chrysanthum was developed. After 90 days of mycorrhizal treatment, activities of acid phosphatase, alkaline phosphatase, NR, NiR and GS were higher in mycorrhizal plantlets than in control plantlets. The hardened plantlets that were initially treated with mycorrhiza under in vitro conditions also showed higher activities of the enzymes investigated. These mycorrhizal plantlets showed higher survival (96.33 %), shoot length (3.7 cm) and shoot fresh weight (0.359 g) as compared to control after 120 days of hardening. The results presented in this study suggest that mycorrhizal association might have increased the assimilation of P and N in D. chrysanthum plantlets, indicating the importance of mycorrhiza in orchids.     

On race: 34 conversations in a time of crisis

The purpose of this Research Note is to offer an analysis of the goals and achievements of former President Clinton's Initiative on Race from its announcement in June 1997 through spring 2000. This Initiative was the first attempt in nearly thirty years by a United States' President to seriously and systematically address the issue of "race". Three conclusions emerge: first, the Clinton Race Initiative accomplished more than most social scientists and the public are aware of; second, the Initiative is incomplete because the President has not released his own proposed book-length analysis on race; and, third, a number of structural and political factors significantly limited the "success" of the Initiative.     

Metabolic products of soluble epoxide hydrolase are essential for monocyte chemotaxis to MCP-1 in vitro and in vivo

Monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis is a major event in inflammatory disease. Our prior studies have demonstrated that MCP-1-dependent chemotaxis requires release of arachidonic acid (AA) by activated cytosolic phospholipase A₂ (cPLA₂). Here we investigated the involvement of AA metabolites in chemotaxis. Neither cyclooxygenase nor lipoxygenase pathways were required, whereas pharmacologic inhibitors of both the cytochrome-P450 (CYP) and the soluble epoxide hydrolase (sEH) pathways blocked monocyte chemotaxis to MCP-1. To verify specificity, we demonstrated that the CYP and sEH products epoxyeiscosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs), respectively, restored chemotaxis in the presence of the inhibitors, indicating that sEH-derived products are essential for MCP-1-driven chemotaxis. Importantly, DHETs also rescued chemotaxis in cPLA₂-deficient monocytes and monocytes with blocked Erk1/2 activity, because Erk controls cPLA₂ activation. The in vitro findings regarding the involvement of CYP/sEH pathways were further validated in vivo using two complementary approaches measuring MCP-1-dependent chemotaxis in mice. These observations reveal the importance of sEH in MCP-1-regulated monocyte chemotaxis and may explain the observed therapeutic value of sEH inhibitors in treatment of inflammatory diseases, cardiovascular diseases, pain, and even carcinogenesis. Their effectiveness, often attributed to increasing EET levels, is probably influenced by the impairment of DHET formation and inhibition of chemotaxis.     

The Amyloid Precursor Protein (APP) Triplicated Gene Impairs Neuronal Precursor Differentiation and Neurite Development through Two Different Domains in the Ts65Dn Mouse Model for Down Syndrome

Intellectual disability in Down syndrome (DS) appears to be related to severe proliferation impairment during brain development. Recent evidence shows that it is not only cellular proliferation that is heavily compromised in DS, but also cell fate specification and dendritic maturation. The amyloid precursor protein (APP), a gene that is triplicated in DS, plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification, and neuronal maturation. APP influences these processes via two separate domains, the APP intracellular domain (AICD) and the soluble secreted APP. We recently found that the proliferation impairment of neuronal precursors (NPCs) from the Ts65Dn mouse model for DS was caused by derangement of the Shh pathway due to overexpression of patched1(Ptch1), its inhibitory regulator. Ptch1 overexpression was related to increased levels within the APP/AICD system. The overall goal of this study was to determine whether APP contributes to neurogenesis impairment in DS by influencing in addition to proliferation, cell fate specification, and neurite development. We found that normalization of APP expression restored the reduced neuronogenesis, the increased astrogliogenesis, and the reduced neurite length of trisomic NPCs, indicating that APP overexpression underpins all aspects of neurogenesis impairment. Moreover, we found that two different domains of APP impair neuronal differentiation and maturation in trisomic NPCs. The APP/AICD system regulates neuronogenesis and neurite length through the Shh pathway, whereas the APP/secreted AP system promotes astrogliogenesis through an IL-6-associated signaling cascade. These results provide novel insight into the mechanisms underlying brain development alterations in DS.     

Generic Escherichia coli Contamination of Spinach at the Preharvest Stage: Effects of Farm Management and Environmental Factors

The objective of this study was to determine the effects of farm management and environmental factors on preharvest spinach contamination with generic Escherichia coli as an indicator of fecal contamination. A repeated cross-sectional study was conducted by visiting spinach farms up to four times per growing season over a period of 2 years (2010 to 2011). Spinach samples (n = 955) were collected from 12 spinach farms in Colorado and Texas as representative states of the Western and Southwestern United States, respectively. During each farm visit, farmers were surveyed about farm-related management and environmental factors using a questionnaire. Associations between the prevalence of generic E. coli in spinach and farm-related factors were assessed by using a multivariable logistic regression model including random effects for farm and farm visit. Overall, 6.6% of spinach samples were positive for generic E. coli. Significant risk factors for spinach contamination with generic E. coli were the proximity (within 10 miles) of a poultry farm, the use of pond water for irrigation, a >66-day period since the planting of spinach, farming on fields previously used for grazing, the production of hay before spinach planting, and the farm location in the Southwestern United States. Contamination with generic E. coli was significantly reduced with an irrigation lapse time of >5 days as well as by several factors related to field workers, including the use of portable toilets, training to use portable toilets, and the use of hand-washing stations. To our knowledge, this is the first report of an association between field workers'' personal hygiene and produce contamination with generic E. coli at the preharvest level. Collectively, our findings support that practice of good personal hygiene and other good farm management practices may reduce produce contamination with generic E. coli at the preharvest level.