Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13

Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594–1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)_n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.     

Identification of the peptides of the crystals of Bacillus thuringiensis var israelensis involved in the mosquito larvicidal activity

Tryptic digestion of the proteins from the purified crystals of B.thuringiensis var israelensis resulted in the decline of high molecular weight peptides without the loss of mosquito larvicidal activity, measured after immobilization of the digests with DEAE- Sephadex A 50 beads. Amongst the peptides generated (less than 44 kDa), a 21 kDa peptide was immunoreactive to the crystal antiserum. Analysis of the peptides released from spores of the toxic (Cry+) and non-toxic (Cry-) strains has revealed a pattern in which only the 26kDa peptide was missing in the Cry-strain. Sporulation and crystal formation were dissociated by the addition of the antibiotic netropsin, which could also inhibit the crystal assembly, without considerable decrease of the larvicidal activity and retention of the 26kDa peptide. These results implicate the 26kDa peptide in the larvicidal action.     

Treatment of encephalomyocarditis virus-induced central nervous system demyelination with monoclonal anti-T-cell antibodies.

Infection of BALB/c mice with the M variant of encephalomyocarditis virus resulted in the development of a paralytic syndrome in 7 to 10 days. The paralysis was maximal during the period of viral clearance; most of the animals recovered from the initial deficit and showed no delayed recurrences. Pathologically, the white matter of brain and spinal cord showed well-demarcated areas of perivascular cuffing, demyelination, and, during recovery, remyelination by oligodendrocytes--all suggestive of postinfectious encephalomyelitis. Depletion of either the CD4 or CD8 subset of T cells in vivo with the appropriate monoclonal antibody, GK1.5 or 2.43, respectively, administered one day (24 h) prior to infection was sufficient to limit the development of the paralytic syndrome by 79% (GK1.5) and 82% (2.43).     

Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation

The developmental fate of cells in the epiblast of early-primitive-streak-stage mouse embryos was assessed by studying the pattern of tissue colonisation displayed by lac Z-expressing cells grafted orthotopically to nontransgenic embryos. Results of these fate-mapping experiments revealed that the lateral and posterior epiblast contain cells that will give rise predominantly to mesodermal derivatives. The various mesodermal populations are distributed in overlapping domains in the lateral and posterior epiblast, with the embryonic mesoderm such as heart, lateral, and paraxial mesoderm occupying a more distal position than the extraembryonic mesoderm. Heterotopic grafting of presumptive mesodermal cells results in the grafted cells adopting the fate appropriate to the new site, reflecting a plasticity of cell fate determination before ingression. The first wave of epiblast cells that ingress through the primitive streak are those giving rise to extraembryonic mesoderm. Cells that will form the mesoderm of the yolk sac and the amnion make up a major part of the mesodermal layer of the midprimitive-streak-stage embryo. Cells that are destined for embryonic mesoderm are still found within the epiblast, but some have been recruited to the distal portion of the mesoderm. By the late-primitive-streak-stage, the mesodermal layer contains only the precursors of embryonic mesoderm. This suggests that there has been a progressive displacement of the midstreak mesoderm to extraembryonic sites, which is reminiscent of that occurring in the overlying endodermal tissue. The regionalisation of cell fate in the late-primitive-streak mesoderm bears the same spatial relationship as their ancestors in the epiblast prior to cell ingression. This implies that both the position of the cells in the proximal-distal axis and their proximity to the primitive streak are major determinants for the patterning of the embryonic mesoderm. © 1995 Wiley-Liss, Inc.     

Molecular structure of antitumor drug steffimycin and modelling of its binding to DNA.

The molecular and crystal structure of steffimycin have been determined by single crystal X-ray diffraction to 0.9 angstrom resolution. The triclinic crystals are in the space group P1, with the unit cell dimensions of a = 8.606(3) angstrom, b = 22.168(7) angstrom, c = 8.448(2) angstrom, alpha = 97.56(3) degrees, beta = 95.97(2) degrees, gamma = 87.94(3) degrees, Z = 2. The structure was solved by direct methods and refined by the full-matrix least-squares method to a final R value of 0.065 with 3405 (Inet greater than 2.0 sigma (Inet] observed reflections using the NRCVAX software package. The crystal lattice includes 2 independent steffimycin, 3 water and one 2-methyl-2,4-pentanediol molecules. The conformation of steffimycin is grossly similar to other anthracycline antibiotics including daunorubicin. The crystal packing interactions of steffimycin suggest a preferred stacking of the aglycone chromophore of the antibiotic which resembles the intercalative interactions seen in the daunorubicin-d(CGTACG) (Wang et al., Biochemistry 26, 1152 (1987] and nogalamycin-d(CGT(pS)ACG) (Liaw et al., Biochemistry 28, 9913 (1989] complexes. The atomic coordinates data from these complexes were used to model the intercalative binding of steffimycin to DNA. The models were then stereochemically idealized by the constraint refinement program NUCLSQ. Subsequently XPLOR software package was used for energy minimization of these models in vacuo. The model building studies suggest that steffimycin has a higher CpG base sequence specificity over the TpA step, similar to that of daunorubicin and nogalamycin.     

Influenza viruses: transmission between species

The only direct evidence for transmission of influenza viruses between species comes from studies on swine influenza viruses. Antigenically and genetically identical Hsw1N1 influenza viruses were isolated from pigs and man on the same farm in Wisconsin, U.S.A. The isolation of H3N2 influenza viruses from a wide range of lower animals and birds suggests that influenza viruses of man can spread to the lower orders. Under some conditions the H3N2 viruses can persist for a number of years in some species. The isolation, from aquatic birds, of a large number of influenza A viruses that possess surface proteins antigenically similar to the viruses isolated from man, pigs and horses provides indirect evidence for inter-species transmission. There is now a considerable body of evidence which suggests that influenza viruses of lower animals and birds may play a role in the origin of some of the pandemic strains of influenza A viruses. There is no direct evidence that the influenza viruses in aquatic birds are transmitted to man, but they may serve as a genetic pool from which some genes may be introduced into humans by recombination. Preliminary evidence suggests that the molecular basis of host range and virulence may be related to the RNA segments coding for one of the polymerase proteins (P3) and for the nucleoprotein (NP).     

Treatment planning and dosimetric comparison study on two different volumetric modulated arc therapy delivery techniques

Aim

To compare and evaluate the performance of two different volumetric modulated arc therapy delivery techniques.

Background

Volumetric modulated arc therapy is a novel technique that has recently been made available for clinical use. Planning and dosimetric comparison study was done for Elekta VMAT and Varian RapidArc for different treatment sites.

Materials and methods

Ten patients were selected for the planning comparison study. This includes 2 head and neck, 2 oesophagus, 1 bladder, 3 cervix and 2 rectum cases. Total dose of 50 Gy was given for all the plans. All plans were done for RapidArc using Eclipse and for Elekta VMAT with Monaco treatment planning system. All plans were generated with 6 MV X-rays for both RapidArc and Elekta VMAT. Plans were evaluated based on the ability to meet the dose volume histogram, dose homogeneity index, radiation conformity index, estimated radiation delivery time, integral dose and monitor units needed to deliver the prescribed dose.

Results

RapidArc plans achieved the best conformity (CI_95% = 1.08 ± 0.07) while Elekta VMAT plans were slightly inferior (CI_95% = 1.10 ± 0.05). The in-homogeneity in the PTV was highest with Elekta VMAT with HI equal to 0.12 ± 0.02 Gy when compared to RapidArc with 0.08 ± 0.03. Significant changes were observed between the RapidArc and Elekta VMAT plans in terms of the healthy tissue mean dose and integral dose. Elekta VMAT plans show a reduction in the healthy tissue mean dose (6.92 ± 2.90) Gy when compared to RapidArc (7.83 ± 3.31) Gy. The integral dose is found to be inferior with Elekta VMAT (11.50 ± 6.49) × 10⁴ Gy cm³ when compared to RapidArc (13.11 ± 7.52) × 10⁴ Gy cm³. Both Varian RapidArc and Elekta VMAT respected the planning objective for all organs at risk. Gamma analysis result for the pre-treatment quality assurance shows good agreement between the planned and delivered fluence for 3 mm DTA, 3% DD for all the evaluated points inside the PTV, for both VMAT and RapidArc techniques.

Conclusion

The study concludes that a variable gantry speed with variable dose rate is important for efficient arc therapy delivery. RapidArc presents a slight improvement in the OAR sparing with better target coverage when compared to Elekta VMAT. Trivial differences were noted in all the plans for organ at risk but the two techniques provided satisfactory conformal avoidance and conformation.     

Distinct glycosyltransferases synthesize E-selectin ligands in human vs. mouse leukocytes

The binding of selectins to carbohydrate epitopes expressed on leukocytes is the first step in a multi-step cell adhesion cascade that controls the rate of leukocyte recruitment at sites of inflammation. The glycans that function as selectin-ligands are post-translationally synthesized by the serial action of Golgi resident enzymes called glycosyltransferases (glycoTs). Whereas much of our current knowledge regarding the role of glycoTs in constructing selectin-ligands comes from reconstituted biochemical investigations or murine models, tools to assess the impact of these enzymes on the human ligands are relatively underdeveloped. This is significant since the selectin-ligands, particularly those that bind E-selectin, vary between different leukocyte cell populations and they are also different in humans compared with mice. To address this shortcoming, a recent study by Buffone et al. (2013) outlines a systematic strategy to knockdown upto three glycoTs simultaneously in human leukocytes. The results suggest that the fucosyltransferases (FUTs) regulating selectin-ligand synthesis may be species-specific. In particular, they demonstrate that FUT9 plays a significant role during human, but not mouse, leukocyte-endothelial interactions. Overall, this article discusses the relative roles of the FUTs during human L-, E-, and P-selectin-ligand biosynthesis, and the potential that the knockdown strategy outlined here may assess the role of other glycoTs in human leukocytes also.     

Partial Purification and Characterization of a Bacteriocin DT24 Produced by Probiotic Vaginal Lactobacillus brevis DT24 and Determination of its Anti-Uropathogenic Escherichia coli Potential

The emergence of antibiotic resistance has increased the interest for finding new antimicrobials in the past decade. Probiotic Lactic acid bacteria producing antimicrobial proteins like bacteriocin can be excellent agents for development as novel therapeutic agents and complement to conventional antibiotic therapy. Uropathogenic Escherichia coli, most causative agent of Urinary tract infection, has developed resistance to various antibiotics. In the present investigation, antibacterial substance like bacteriocin (Bacteriocin DT24) produced by probiotic Lactobacillus brevis DT24 from vaginal sample of healthy Indian woman was partially purified and characterized. It was efficiently working against various pathogens, that is, Uropathogenic E. coli, Enterococcus faecium, Enterococcus faecalis and Staphylococcus aureus. The antimicrobial peptide was relatively heat resistant and also active over a broad range of pH 2–10. It has been partially purified by ammonium sulfate precipitation and gel filtration chromatography and checked on reverse-phase high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bacteriocin DT24 was approximately 7-kDa protein. The peptide is inactivated by proteolytic enzymes, trypsin and lipase but not when treated with catalase, α-amylase and pepsin. It showed bacteriostatic mode of action against uropathogenic E. coli. Such characteristics indicate that this bacteriocin-producing probiotic may be a potential candidate for alternative agents to control urinary tract infections and other pathogens.