Diastereoselective synthesis of glycosylated prolines as alpha-glucosidase inhibitors and organocatalyst in asymmetric aldol reaction

1,3-Dipolar cycloaddition of azomethine ylides and glycosyl E-olefins in presence of LDA led to diastereoselective formation of C-glycosylated proline esters. The selected esters on regioselective hydrolysis with LiOH gave C-glycosyl prolines. Few of the proline esters exhibited very good alpha-glucosidase inhibitory activity. The organocatalytic activity of the proline derivatives in a prototype Aldol reaction has also been investigated.     

Evaluation of the presence of reduced nicotinamide adenine dinucleotide phosphate in bacterial metabolites used as immunostimulators and its role in nitric oxide induction

Bacterial metabolites that act as immunostimulators have aroused interest because of their therapeutic potential in several immune disorders. These metabolites are complex, heterogeneous, and comprise numerous immune‐boosting biomolecules. To better understand their immune stimulatory properties, characterization of their components is essential. An ether extract of metabolites from nine bacterial species was analyzed for the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) or other fluorophores. This metabolite in combination with bile lipids is a licensed immune stimulatory drug. Excitation of the extract at 340 nm resulted in fluorescence with an emission maximum of around 410 nm, which is fairly specific for NADH and NADPH. Reverse‐phase‐HPLC and electro‐spray ionization‐mass analysis confirmed the presence of NADPH in the bacterial metabolites. Quantification by glutathione reductase assay indicated 11.90 ± 0.01 µM of NADPH in the metabolites. Further characterization of the individual bacterial extracts of the metabolite confirmed the presence of NADPH. Subsequently, studies were performed to evaluate the role/s of NADPH in immune‐stimulatory drugs. NADPH is known to be involved in production of nitric oxide (NO), which has versatile roles in the immune system. The biological function of NADPH in NO induction by RAW 264.7 (mouse macrophage) cells was evaluated and it was found that bacterial NADPH has a significant role in inducing NO and that NADPH from individual bacterial extracts is capable of inducing NO. Investigation on the stability and biological potency of NADPH in bacterial metabolites is important because of NADPH's wide therapeutic applications, most of which are associated with its role in NO induction.     

Pattern of Resource Allocation of Six Plantago Species with Different Breeding Systems

Plantago exhibit great deal of differences in the breeding system. The reproductive effort calculated on the basis of, (i) dry biomass of foliar and floral parts and (ii) seed output-weight (mg) per unit leaf area (cm²), exhibits relation with breeding system. The predominantly inbreeding taxa invest higher reproductive effort compared to their outbreeding allies. In terms of sex allocation strategies, the outbreeding species like P. lanceolata, P. lagopus invest more to the development of floral features and to male functions. On the contrary, inbreeding species such as P. patagonica, P. drummondii, and P. ovata invest greater resources to the female function. Received 13 April 1998/ Accepted in revised form 6 November 1998     

A genetic model for in vivo proximity labelling of the mammalian secretome

Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalysed proximity labelling enables the identification of proteins within a specific cellular compartment. Here, we report a BirA*G3 mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mouse, widespread labelling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) proteomics revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, highlighting both conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte-specific activation of BirA*G3 highlighted liver-specific biotinylated secretome profiles. The BirA*G3 mouse model demonstrates enhanced labelling efficiency and tissue specificity over viral transduction approaches and will facilitate a deeper understanding of secretory protein interplay in development, and in healthy and diseased adult states.     

Advancing Edge Speeds of Epithelial Monolayers Depend on Their Initial Confining Geometry

Collective cell migrations are essential in several physiological processes and are driven by both chemical and mechanical cues. The roles of substrate stiffness and confinement on collective migrations have been investigated in recent years, however few studies have addressed how geometric shapes influence collective cell migrations. Here, we address the hypothesis that the relative position of a cell within the confinement influences its motility. Monolayers of two types of epithelial cells—MCF7, a breast epithelial cancer cell line, and MDCK, a control epithelial cell line—were confined within circular, square, and cross-shaped stencils and their migration velocities were quantified upon release of the constraint using particle image velocimetry. The choice of stencil geometry allowed us to investigate individual cell motility within convex, straight and concave boundaries. Cells located in sharp, convex boundaries migrated at slower rates than those in concave or straight edges in both cell types. The overall cluster migration occurred in three phases: an initial linear increase with time, followed by a plateau region and a subsequent decrease in cluster speeds. An acto-myosin contractile ring, present in the MDCK but absent in MCF7 monolayer, was a prominent feature in the emergence of leader cells from the MDCK clusters which occurred every ~125 μm from the vertex of the cross. Further, coordinated cell movements displayed vorticity patterns in MDCK which were absent in MCF7 clusters. We also used cytoskeletal inhibitors to show the importance of acto-myosin bounding cables in collective migrations through translation of local movements to create long range coordinated movements and the creation of leader cells within ensembles. To our knowledge, this is the first demonstration of how bounding shapes influence long-term migratory behaviours of epithelial cell monolayers. These results are important for tissue engineering and may also enhance our understanding of cell movements during developmental patterning and cancer metastasis.     

Variability in Foliar Essential Oils among Different Morphotypes of Lantana Species Complexes,and Its Taxonomic and Ecological Significance

The genus Lantana has many species complexes, and L. camara is one of the aggressive alien weedy species complexes; species delimitation in these complexes is a nightmare for taxonomists. We examined the diversity in the chemical composition of foliar essential oils among morphotypes of Lantana species complexes inhabiting the same ecological gradient, and its taxonomic and ecological significance. The yields of essential oils varied from 0.1 to 0.79% in foliar hydrodistillates of eleven morphotypes, and a total of 39 chemical constituents were detected by GC/MS. The quantitative and qualitative variability in the composition of essential oils among morphotypes was very high, and hence they represent chemotypes. The diversity observed in the composition of essential oils appears to be of genetic origin and thus of taxonomic value. The formation of distinct clusters and sub‐clusters at high distance cluster combine values also substantiates that the patterns of distribution of chemical constituents among morphotypes can be used in delimiting species and infraspecific taxa within the species complexes. The presence of β‐caryophyllene and other such compounds, which are known to prevent herbivory, in morphotypes of Lantana species complexes suggest that these compounds may provide selective advantage to Lantana over native species in the invasion of new and disturbed habitats.     

Gamma-glutamyl transpeptidase from two plant growth promoting rhizosphere fluorescent pseudomonads

Glutathione is the most abundant non-protein thiol compound present in many cells. Because this molecule is involved in many physiological processes, each cell maintains a critical level of glutathione. Gamma-glutamyl transpeptidase (GGT, E.C.2.3.2.2) is the key enzyme involved in the glutathione cycle. In the present study, GGT was isolated from two plant growth promoting rhizosphere isolates, Pseudomonas protegens strain Pf-5 and Pseudomonas fluorescens strain PfT-1. GGT in these strains is located in the periplasm and possessed good hydrolytic activity at pH 8.0. Strains Pf-5 and PfT-1 showed maximum enzyme activity when grown at 30–35 °C. The ggt gene from both the strains was cloned in pGEM-T cloning vector and sequenced. Subsequently, GGT expressed in Escherichia coli BL21(DE3) using the pET-28a(+) expression vector was purified and characterized. The enzymes are active in a wide range of pH and some divalent cations significantly enhanced the hydrolytic activity. These enzymes showed higher thermal stability as compared to those of other mesophilic strains, as they retained ~50 % of activity at 50 °C even after 12 h of incubation. The enzymes could also tolerate up to 3.0 M NaCl.     

Identification and Origin of N-Linked β-d-N-Acetylglucosamine Monosaccharide Modifications on Arabidopsis Proteins

Many plant proteins are modified with N-linked oligosaccharides at asparagine-X-serine/threonine sites during transit through the endoplasmic reticulum and the Golgi. We have identified a number of Arabidopsis (Arabidopsis thaliana) proteins with modifications consisting of an N-linked N-acetyl-d-glucosamine monosaccharide (N-GlcNAc). Electron transfer dissociation mass spectrometry analysis of peptides bearing this modification mapped the modification to asparagine-X-serine/threonine sites on proteins that are predicted to transit through the endoplasmic reticulum and Golgi. A mass labeling method was developed and used to study N-GlcNAc modification of two thioglucoside glucohydrolases (myrosinases), TGG1 and TGG2 (for thioglucoside glucohydrolase). These myrosinases are also modified with high-mannose (Man)-type glycans. We found that N-GlcNAc and high-Man-type glycans can occur at the same site. It has been hypothesized that N-GlcNAc modifications are generated when endo-β-N-acetylglucosaminidase (ENGase) cleaves N-linked glycans. We examined the effects of mutations affecting the two known Arabidopsis ENGases on N-GlcNAc modification of myrosinase and found that modification of TGG2 was greatly reduced in one of the single mutants and absent in the double mutant. Surprisingly, N-GlcNAc modification of TGG1 was not affected in any of the mutants. These data support the hypothesis that ENGases hydrolyze high-Man glycans to produce some of the N-GlcNAc modifications but also suggest that some N-GlcNAc modifications are generated by another mechanism. Since N-GlcNAc modification was detected at only one site on each myrosinase, the production of the N-GlcNAc modification may be regulated.Many plant proteins are modified with N-linked (Glc)₃(Man)₉(GlcNAc)₂ glycan(s) at the Asn of the Asn-X-Ser/Thr sequon, where X is any amino acid except Pro, as they enter the endoplasmic reticulum (ER; Pattison and Amtmann, 2009). These N-glycans are then modified by the removal and addition of sugars as the protein transits through the secretory system. Through this process, a diverse array of glycan structures can be generated. These glycan structures have been divided into four categories (Lerouge et al., 1998). High-Man-type glycans are produced in the ER. Complex-, paucimannosidic-, and hybrid-type glycans, which are produced in the Golgi, have Xyl and Fuc attached to the protein-proximal Man and GlcNAc of their (Man)₃(GlcNAc)₂ common core (Fig. 1).Open in a separate windowFigure 1.PNGase and ENGase reactions. PNGase and ENGase cleave the common core of N-linked glycans, which is composed of GlcNAc (closed squares) and Man (closed circles), at the sites indicated. In plants, the common core can be modified with Fuc (open triangle), which inhibits PNGase F, and Xyl (inverted triangle). When a glycan is removed by PNGase F, the modified Asn (N) is converted to an Asp (D).N-Glycans influence protein conformation, stability, and activity. They play a major role in the quality-control mechanisms that (1) retain misfolded proteins in the ER until they become properly folded and (2) target terminally misfolded proteins for destruction by the endoplasmic reticulum-associated protein degradation (ERAD) pathway (Vembar and Brodsky, 2008). During ERAD, misfolded proteins are exported to the cytosol, the glycans are removed and recycled, and the proteins are degraded by the 26S proteasome. There is also precedence for glycosylation status regulating the activity of plant proteins. Concanavalin A (Con A), a lectin from jackbean (Canavalia ensiformis), is synthesized as an inactive high-Man precursor protein that is deglycosylated as part of the processing that converts it to an active lectin (Min et al., 1992; Sheldon and Bowles, 1992).Modification of the Asn of the Asn-X-Ser/Thr sequon with an N-linked N-acetyl-d-glucosamine monosaccharide (N-GlcNAc) occurs in fungi (Hase et al., 1982) and has recently been discovered in animals (Chalkley et al., 2009; Wang et al., 2010). N-GlcNAc modification also occurs in plants. The Asn-X-Ser/Thr sequon of ribosome-inactivating proteins from sponge gourd (Luffa cylindrica) and pokeweed (Phytolacca americana) is modified with N-GlcNAc (Islam et al., 1991; Zeng et al., 2003). Matrix-assisted laser-desorption ionization time-of-flight analysis of trypsin fragments derived from antibodies expressed in transgenic maize (Zea mays) and tobacco (Nicotiana tabacum) detects fragments with masses consistent with N-GlcNAc modification (Bakker et al., 2006; Rademacher et al., 2008).Several mechanisms for the origin of this modification have been proposed. It has been proposed that cytosolic endo-β-N-actetylglucosaminidase (ENGase) produces the N-GlcNAc modification when it removes larger N-linked glycans (Fig. 1). However, ENGase is more active toward free oligosaccharides than glycoproteins (Chantret and Moore, 2008), and it is unclear how cytosol-localized ENGase gains access to these glycoproteins, which are not generally localized in the cytosol. One possibility is that ENGase is acting on misfolded proteins that have been exported to the cytosol for degradation by the ERAD pathway. Alternatively, it is possible that N-GlcNAc modifications are an artifact that is generated during sample preparation. Thus, it remains an open question how N-GlcNAc modifications are produced and if they have a role in the cell.Here, we report the discovery of a number of Arabidopsis (Arabidopsis thaliana) proteins bearing N-GlcNAc modifications that are attached to the Asn of the Asn-X-Ser/Thr sites. We present evidence that these modifications occur in planta. An analysis of mutants indicates that, in at least one case, the modification is produced by Arabidopsis ENGases, but it also suggests that some N-GlcNAc modifications are generated by another mechanism.