A lupane derivative and the 13C NMR chemical shifts of some lupanols from Pleurostylia opposita

A new lupane derivative isolated from Pleurostylia opposita has been assigned the structure 6,β-hydroxy-lup-20(29)-en-3-one, using spectral evidence and chemical interconversions. The ¹³C NMR spectral assignments of 20-hydroxy-lupan-3-one, 6β, 20-dihydroxy-lupan-3-one, 6β,28-dihydroxy-lup-20(29)-en-3-one and 20-hydroxy-lupane-3, 6-dione previously isolated from the same plant are also reported.     

Diffusion of low density lipoprotein-receptor complex on human fibroblasts

Diffusion of the complex consisting of low density lipoprotein (LDL) bound to its receptor on the surface of human fibroblasts has been measured with the help of an intensely fluorescent, biologically active LDL derivative, dioctadecylindocarbocyanine LDL (dil(3)-LDL). Fluorescence photobleaching recovering and direct video observations of the Brownian motion of individual LDL-receptor complexes yielded diffusion coefficients for the slow diffusion on cell surfaces and fast diffusion on membrane blebs, respectively. At 10 degrees C, less that 20 percent of the LDL-receptor complex was measurably diffusible either on normal human fibroblasts GM-3348 or on LDL-receptor- internalization-defective J.D. cells GM-2408A. At 21 degrees and 28 degrees C, the diffusion fractions of approximately 75 and 60 percent, respectively, on both cell lines. The lipid analog nitrobenzoxadiazolephosphatidylcholine (NBD-PC) diffused in the GM-2408A cell membrane at 1.5x10(-8) cm(2)/sec at 22 degrees C. On blebs induced in GM-2408A cell membranes, the dil(3)-LDL receptor complex diffusion coefficient increased to approximately 10(-9) cm(2)/s, thus approaching the maximum theoretical predictions for a large protein in the viscous lipid bilayer. Cytoskeletal staining of blebs with NBD- phallacidin, a fluorescent probe specific for F-actin, indicated that loss of the bulk of the F-actin cytoskeleton accompanied the release of the natural constraints on later diffusion observed on blebs. This work shows that the internalization defect of J.D. is not due to immobilization of the LDL-receptor complex since its diffusibility is sufficient to sustain even the internalization rates observed in the native fibroblasts. Nevertheless, as with many other cell membrane receptors, the diffusion coefficient of the LDL-receptor complex is at least two orders of magnitude slower on native membrane than the viscous limit approached on cell membrane blebs where it is released from lateral constraints. However, LDL-receptor diffusion may not limit LDL internalization in normal human fibroblasts.     

Bacteriostatic heterocycles from Euodia lunu-ankenda

A new constituent characterized as 8-acetyl-3,4-dihydroxy-5,7-dimethoxy-2,2-dimethylchroman has been isolated together with alloevodionol-7-methyl ether, 4-methoxy-1-methyl-2(1H)quinolinone, evolitrine, isoevodionol and its methyl ether from the aerial parts of Euodia lunu-ankenda. Its structure was confirmed by its transformation to alloevodionol-7-methyl ether. 4-Methoxy-1-methyl-2(1H)quinolinone and its isomer were synthesized by a modified procedure.     

Pre-Microporation Improves Outcome of Pancreatic Islet Labelling for Optical and <Superscript>19</Superscript>F MR Imaging

Background

In vitro labelling of cells and small cell structures is a necessary step before in vivo monitoring of grafts. We modified and optimised a procedure for pancreatic islet labelling using bimodal positively charged poly(lactic-co-glycolic acid) nanoparticles with encapsulated perfluoro crown ethers and indocyanine green dye via microporation and compared the method with passive endocytosis.

Results

Pancreatic islets were microporated using two pulses at various voltages. We tested a standard procedure (poration in the presence of nanoparticles) and a modified protocol (pre-microporation in a buffer only, and subsequent islet incubation with nanoparticles on ice for 10 min).We compared islet labelling by microporation with labelling by endocytosis, i.e. pancreatic islets were incubated for 24 h in a medium with suspended nanoparticles.In order to verify the efficiency of the labelling procedures, we used ¹⁹F magnetic resonance imaging, optical fluorescence imaging and confocal microscopy.The experiment confirmed that microporation, albeit fast and effective, is invasive and may cause substantial harm to islets. To achieve sufficient poration and to minimise the reduction of viability, the electric field should be set at 20 kV/m (two pulses, 20 ms each).Poration in the presence of nanoparticles was found to be unsuitable for the nanoparticles used. The water suspension of nanoparticles (which served as a surfactant) was slightly foamy and microbubbles in the suspension were responsible for sparks causing the destruction of islets during poration. However, pre-microporation (poration of islets in a buffer only) followed by 10-min incubation with nanoparticles was safer.

Conclusions

For labelling of pancreatic islets using poly(lactic-co-glycolic acid) nanoparticles, the modified microporation procedure with low voltage was found to be safer than the standard microporation procedure. The modified procedure was fast, however, efficiency was lower compared to endocytosis.

     

Fusion of family VI cellulose binding domains to Bacillus halodurans xylanase increases its catalytic activity and substrate-binding capacity to insoluble xylan

A tandem repeat of the family VI cellulose binding domain (CBD) from Clostridium stercorarium xylanase (XylA) was fused at the carboxyl-terminus of Bacillus halodurans xylanase (XylA). B. halodurans XylA is an enzyme which is active in the alkaline region of pH and lacks a CBD. The constructed chimera was expressed in Escherichia coli, purified to homogeneity, and then subjected to detailed characterization. The chimeric enzyme displayed pH activity and stability profiles similar to those of the parental enzyme. The optimal temperature of the chimera was observed at 60 °C and the enzyme was stable up to 50 °C. Binding studies with insoluble polysaccharides indicated that the chimera had acquired an increased affinity for oat spelt xylan and acid-swollen cellulose. The bound chimeric enzyme was desorbed from insoluble substrates with sugars and soluble polysaccharides, indicating that the CBDs also possess an affinity for soluble sugars. Overall, the chimera displayed a higher level of hydrolytic activity toward insoluble oat spelt xylan than its parental enzyme and a similar level of activity toward soluble xylan.     

Neutralization of Clostridium difficile toxin A using antibody combinations

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.Key words: Clostridium difficile, toxin neutralization, therapeutic antibody, cell wall binding domains, repeat proteins, CROPs, mAb combinationThe most common cause of nosocomial antibiotic-associated diarrhea is the gram-positive, spore-forming anaerobic bacillus Clostridium difficile (C. difficile). Infection can be asymptomatic or lead to acute diarrhea, colitis, and in severe instances, pseudomembranous colitis and toxic megacolon.^1,2The pathological effects of C. difficile have long been linked to two secreted toxins, A and B.^3,4 Some strains, particularly the virulent and antibiotic-resistant strain 027 with toxinotype III, also produce a binary toxin whose significance in the pathogenicity and severity of disease is still unclear.⁵ Early studies including in vitro cell-killing assays and ex vivo models indicated that toxin A is more toxigenic than toxin B; however, recent gene manipulation studies and the emergence of virulent C. difficile strains that do not express significant levels of toxin A (termed “A⁻ B⁺”) suggest a critical role for toxin B in pathogenicity.^6,7Toxins A and B are large multidomain proteins with high homology to one another. The N-terminal region of both toxins enzymatically glucosylates small GTP binding proteins including Rho, Rac and CDC42,^8,9 leading to altered actin expression and the disruption of cytoskeletal integrity.^9,10 The C-terminal region of both toxins is composed of 20 to 30 residue repeats known as the clostridial repetitive oligopeptides (CROPs) or cell wall binding (CWB) domains due to their homology to the repeats of Streptococcus pneumoniae LytA,^11–14 and is responsible for cell surface recognition and endocytosis.^12,15–17C. difficile-associated diarrhea is often, but not always, induced by antibiotic clearance of the normal intestinal flora followed by mucosal C. difficile colonization resulting from preexisting antibiotic resistant C. difficile or concomitant exposure to C. difficile spores, particularly in hospitals. Treatments for C. difficile include administration of metronidazole or vancomycin.^2,18 These agents are effective; however, approximately 20% of patients relapse. Resistance of C. difficile to these antibiotics is also an emerging issue^19,20 and various non-antibiotic treatments are under investigation.^20–25Because hospital patients who contract C. difficile and remain asymptomatic have generally mounted strong antibody responses to the toxins,^26,27 active or passive immunization approaches are considered hopeful avenues of treatment for the disease. Toxins A and B have been the primary targets for immunization approaches.^20,28–33 Polyclonal antibodies against toxins A and B, particularly those that recognize the CWB domains, have been shown to effectively neutralize the toxins and inhibit morbidity in rodent infection models.³¹ Monoclonal antibodies (mAbs) against the CWB domains of the toxins have also demonstrated neutralizing capabilities; however, their activity in cell-based assays is significantly weaker than that observed for polyclonal antibody mixtures.^33–36We investigated the possibility of creating a cocktail of two or more neutralizing mAbs that target the CWB domain of toxin A with the goal of synthetically re-creating the superior neutralization properties of polyclonal antibody mixtures. Using the entire CWB domain of toxin A, antibodies were raised in rodents and screened for their ability to neutralize toxin A in a cell-based assay. Two mAbs, 3358 and 3359, that (1) both independently demonstrated marginal neutralization behavior and (2) did not cross-block one another from binding toxin A were identified. We report here that 3358 and 3359 use differing mechanisms to modify CWB-domain association with CHO cell surfaces and combine favorably to reduce toxin A-mediated cell lysis.     

Triterpenes from the leaves of Pleurostylia opposita

Friedelin, friedelan-3β-ol, sitosterol, α-amyrin, 6β,20-dihydroxylupan-3-one, lup-20(29)-en-3β,6β-diol, 6β,28-dihydroxylup-20(29)-en-3-one and dulcitol were isolated from the leaves of Pleurostylia opposita. The distribution of lupanes in the Celastraceae and their chemotaxonomic significance is discussed.     

Docking studies of Vitamin C,Vitamin E,Damnacanthal and Scopoletin with human lens gamma D-crystalline

Vitamin C, Vitamin E, scopoletin and damnacanthal are the major constituents of Noni (Morinda citrifolia). These compounds are known to have good medicinal properties and they are known to act as antioxidants. Loss of vision in elderly is due to opaqueness of the lens proteins such as gamma-D-crystallin during oxidative stress conditions. Therefore, it is of importance to find the potential interaction of Vitamin C, Vitamin E, Scopoletin and Damnacanthal with the lens protein gamma-D-crystallin. Hence, their physical binding to gamma-D crystallin (PDB ID: 2G98) was evaluated using molecular and structural docking procedures. Results show the potential binding of all the above anti-oxidants to gamma-D-crystalline with equal affinity. Thus, the role of cumulative anti-oxidant effect in Noni fruit juice through their potential yet predicted interaction with the lens protein gamma-D-crystallin is implied for cataract treatment.     

New alkaloids from Aegle marmelos

Four new alkaloids, O-(3,3-dimethylallyl)-halfordinol, N-2-ethoxy-2-(4-methoxyphenyl)ethylcinnamamide, N-2-methoxy-2-[4-(3′,3′-dimethyl     

Preparation and characterization of a thermostable and biodegradable biopolymers using natural cross-linker

The present study describes preparation and characterization of a thermally stable and biodegradable biopolymer using collagen and a natural polymer, alginic acid (AA). Required concentration of alginic acid and collagen was optimized and the resulting biopolymer was characterized for, degree of cross-linking, mechanical strength, thermal stability, biocompatibility (toxicity) and biodegradability. Results reveal, the degree of cross-linking of alginic acid (at 1.5% concentration) with collagen was calculated as 75%, whereas it was 83% with standard cross-linking agent, glutaraldehyde (at 1.5% concentration). The AA cross-linked biopolymer was stable up to 245°C and Exhibits 5-6-fold increase in mechanical (tensile) strength compared to plain collagen (native) materials. However, glutaraldehyde cross-linked material exhibits comparatively less thermal stability and brittle in nature (low tensile strength). With regard to cell toxicity, no cytotoxicity was observed for AA cross-linked material when tested with mesenchymal cells and found degradable when treated with collagenase enzyme. The nature of bonding pattern and the reason for thermal stability of AA cross-linked collagen biopolymer was discussed in detail with the help of bioinformatics. A supplementary file on efficacy of AACC as a wound dressing material is demonstrated in detail with animal model studies.