Biocatalysts for multicomponent Biginelli reaction: bovine serum albumin triggered waste-free synthesis of 3,4-dihydropyrimidin-2-(1H)-ones

Bovine serum albumin (BSA) promoted simple and efficient one-pot procedure was developed for the direct synthesis of 3,4-dihydropyrimidin-2(1H)-ones including potent mitotic kinesin Eg5 inhibitor monastrol under mild reaction conditions. The catalyst recyclability and gram scale synthesis have also been demonstrated to enhance the practical utility of process.     

Force Generation in Lamellipodia Is a Probabilistic Process with Fast Growth and Retraction Events

Polymerization of actin filaments is the primary source of motility in lamellipodia and it is controlled by a variety of regulatory proteins. The underlying molecular mechanisms are only partially understood and a precise determination of dynamical properties of force generation is necessary. Using optical tweezers, we have measured with millisecond (ms) temporal resolution and picoNewton (pN) sensitivity the force-velocity (Fv) relationship and the power dissipated by lamellipodia of dorsal root ganglia neurons. When force and velocity are averaged over 3-5 s, the Fv relationships can be flat. On a finer timescale, random occurrence of fast growth and subsecond retractions become predominant. The maximal power dissipated by lamellipodia over a silica bead with a diameter of 1 μm is 10⁻¹⁶ W. Our results clarify the dynamical properties of force generation: i), force generation is a probabilistic process; ii), underlying biological events have a bandwidth up to at least 10 Hz; and iii), fast growth of lamellipodia leading edge alternates with local retractions.     

Focus Issue on Plant Cell Walls: A Comprehensive Toolkit of Plant Cell Wall Glycan-Directed Monoclonal Antibodies

A collection of 130 new plant cell wall glycan-directed monoclonal antibodies (mAbs) was generated with the aim of facilitating in-depth analysis of cell wall glycans. An enzyme-linked immunosorbent assay-based screen against a diverse panel of 54 plant polysaccharides was used to characterize the binding patterns of these new mAbs, together with 50 other previously generated mAbs, against plant cell wall glycans. Hierarchical clustering analysis was used to group these mAbs based on the polysaccharide recognition patterns observed. The mAb groupings in the resulting cladogram were further verified by immunolocalization studies in Arabidopsis (Arabidopsis thaliana) stems. The mAbs could be resolved into 19 clades of antibodies that recognize distinct epitopes present on all major classes of plant cell wall glycans, including arabinogalactans (both protein- and polysaccharide-linked), pectins (homogalacturonan, rhamnogalacturonan I), xyloglucans, xylans, mannans, and glucans. In most cases, multiple subclades of antibodies were observed to bind to each glycan class, suggesting that the mAbs in these subgroups recognize distinct epitopes present on the cell wall glycans. The epitopes recognized by many of the mAbs in the toolkit, particularly those recognizing arabinose- and/or galactose-containing structures, are present on more than one glycan class, consistent with the known structural diversity and complexity of plant cell wall glycans. Thus, these cell wall glycan-directed mAbs should be viewed and utilized as epitope-specific, rather than polymer-specific, probes. The current world-wide toolkit of approximately 180 glycan-directed antibodies from various laboratories provides a large and diverse set of probes for studies of plant cell wall structure, function, dynamics, and biosynthesis.Cell walls play important roles in the structure, physiology, growth, and development of plants (Carpita and Gibeaut, 1993). Plant cell wall materials are also important sources of human and animal nutrition, natural textile fibers, paper and wood products, and raw materials for biofuel production (Somerville, 2007). Many genes thought to be responsible for plant wall biosynthesis and modification have been identified (Burton et al., 2005; Lerouxel et al., 2006; Mohnen et al., 2008), and 15% of the Arabidopsis (Arabidopsis thaliana) genome is likely devoted to these functions (Carpita et al., 2001). However, phenotypic analysis in plants carrying cell wall-related mutations has proven particularly difficult. First, cell wall-related genes are often expressed differentially and at low levels between cells of different tissues (Sarria et al., 2001). Also, plants have compensatory mechanisms to maintain wall function in the absence of a particular gene (Somerville et al., 2004). Thus, novel tools and approaches are needed to characterize wall structures and the genes responsible for their synthesis and modification.Monoclonal antibodies (mAbs) developed against cell wall polymers have emerged as an important tool for the study of plant cell wall structure and function (Knox, 2008). Previous studies have utilized mAbs that bind epitopes present on rhamnogalacturonan I (RG-I; Freshour et al., 1996; Jones et al., 1997; Willats et al., 1998; McCartney et al., 2000; Clausen et al., 2004; Altaner et al., 2007), homogalacturonan (Willats et al., 2001; Clausen et al., 2003), xylogalacturonan (Willats et al., 2004), xylans and arabinoxylans (McCartney et al., 2005), xyloglucan (Freshour et al., 1996, 2003; Marcus et al., 2008), arabinogalactan(protein) (Pennell et al., 1991; Puhlmann et al., 1994; Dolan et al., 1995; Smallwood et al., 1996), and extensins (Smallwood et al., 1995) to localize these epitopes in plant cells and tissues. In addition, mAbs have been used to characterize plants carrying mutations in genes thought to be associated with cell wall biosynthesis and metabolism (Orfila et al., 2001; Seifert, 2004; Persson et al., 2007; Cavalier et al., 2008; Zabotina et al., 2008). Despite their utility, the available set of mAbs against carbohydrate structures is relatively small given the structural complexity of wall polymers (Ridley et al., 2001; O''Neill and York, 2003), and knowledge of their epitope specificity is limited. Thus, additional mAbs specific to diverse epitope structures and methods for rapid epitope characterization are needed (Somerville et al., 2004).Here, we report the generation of 130 new mAbs that bind to diverse epitopes present on a broad spectrum of plant cell wall glycans. In addition, approximately 50 previously reported or generated mAbs were included in the ELISA-based screens used to group the antibodies according to their binding patterns against a diverse panel of 54 polysaccharides. The resulting ELISA data were analyzed by hierarchical clustering to illustrate the relationships between the available mAbs. Nineteen groups of mAbs were identified from the clustering analysis. Some initial information regarding possible epitopes recognized by some of these antibodies could be inferred from the clustering analysis.     

Cannabinoid-2 receptor limits inflammation,oxidative/nitrosative stress,and cell death in nephropathy

Cisplatin is an important chemotherapeutic agent; however, its nephrotoxicity limits its clinical use. Enhanced inflammatory response and oxidative/nitrosative stress seem to play a key role in the development of cisplatin-induced nephropathy. Activation of cannabinoid-2 (CB₂) receptors with selective agonists exerts anti-inflammatory and tissue-protective effects in various disease models. We have investigated the role of CB₂ receptors in cisplatin-induced nephrotoxicity using the selective CB₂ receptor agonist HU-308 and CB₂ knockout mice. Cisplatin significantly increased inflammation (leukocyte infiltration, CXCL1/2, MCP-1, TNFα, and IL-1β levels) and expression of adhesion molecule ICAM-1 and superoxide-generating enzymes NOX2, NOX4, and NOX1 and enhanced ROS generation, iNOS expression, nitrotyrosine formation, and apoptotic and poly(ADP-ribose) polymerase-dependent cell death in the kidneys of mice, associated with marked histopathological damage and impaired renal function (elevated serum BUN and creatinine levels) 3 days after the administration of the drug. CB₂ agonist attenuated the cisplatin-induced inflammatory response, oxidative/nitrosative stress, and cell death in the kidney and improved renal function, whereas CB₂ knockouts developed enhanced inflammation and tissue injury. Thus, the endocannabinoid system, through CB₂ receptors, protects against cisplatin-induced kidney damage by attenuating inflammation and oxidative/nitrosative stress, and selective CB₂ agonists may represent a promising novel approach to preventing this devastating complication of chemotherapy.     

Identification of SD-0006, a potent diaryl pyrazole inhibitor of p38 MAP kinase

Starting from an initial HTS screening lead, a novel series of C(5)-substituted diaryl pyrazoles were developed that showed potent inhibition of p38α kinase. Key to this outcome was the switch from a pyridyl to pyrimidine at the C(4)-position leading to analogs that were potent in human whole blood based cell assay as well as in a number of animal efficacy models for rheumatoid arthritis. Ultimately, we identified a clinical candidate from this substrate; SD-0006.     

Photoprotective compounds from marine organisms

The substantial loss in the stratospheric ozone layer and consequent increase in solar ultraviolet radiation on the earth’s surface have augmented the interest in searching for natural photoprotective compounds in organisms of marine as well as freshwater ecosystems. A number of photoprotective compounds such as mycosporine-like amino acids (MAAs), scytonemin, carotenoids and several other UV-absorbing substances of unknown chemical structure have been identified from different organisms. MAAs form the most common class of UV-absorbing compounds known to occur widely in various marine organisms; however, several compounds having UV-screening properties still need to be identified. The synthesis of scytonemin, a predominant UV-A-photoprotective pigment, is exclusively reported in cyanobacteria. Carotenoids are important components of the photosynthetic apparatus that serve both light-harvesting and photoprotective functions, either by direct quenching of the singlet oxygen or other toxic reactive oxygen species or by dissipating the excess energy in the photosynthetic apparatus. The production of photoprotective compounds is affected by several environmental factors such as different wavelengths of UVR, desiccation, nutrients, salt concentration, light as well as dark period, and still there is controversy about the biosynthesis of various photoprotective compounds. Recent studies have focused on marine organisms as a source of natural bioactive molecules having a photoprotective role, their biosynthesis and commercial application. However, there is a need for extensive work to explore the photoprotective role of various UV-absorbing compounds from marine habitats so that a range of biotechnological and pharmaceutical applications can be found.