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.     

Determinants of expression variability

The amount of tissue-specific expression variability (EV) across individuals is an essential characteristic of a gene and believed to have evolved, in part, under functional constraints. However, the determinants and functional implications of EV are only beginning to be investigated. Our analyses based on multiple expression profiles in 41 primary human tissues show that a gene’s EV is significantly correlated with a number of features pertaining to the genomic, epigenomic, regulatory, polymorphic, functional, structural and network characteristics of the gene. We found that (i) EV of a gene is encoded, in part, by its genomic context and is further influenced by the epigenome; (ii) strong promoters induce less variable expression; (iii) less variable gene loci evolve under purifying selection against copy number polymorphisms; (iv) genes that encode inherently disordered or highly interacting proteins exhibit lower variability; and (v) genes with less variable expression are enriched for house-keeping functions, while genes with highly variable expression tend to function in development and extra-cellular response and are associated with human diseases. Thus, our analysis reveals a number of potential mediators as well as functional and evolutionary correlates of EV, and provides new insights into the inherent variability in eukaryotic gene expression.     

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).     

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).     

Influence of bacterial streak disease development on changes in phenolics,soluble amino acids and carbohydrates of rice leaves

The healthy leaves of rice cultivar ‘BJ 1’ resistant to bacterial leaf streak pathogen (Xanthomonas translucens f. sp.oryzicola) contained higher quantities of total phenolic compounds, reducing and nonreducing sugars than the susceptible cultivar ’IR 8’, while the leaves of cultivar ’IR 8’ possessed larger concentration of total soluble amino acids than the resistant cultivar ’BJ 1’. In the leaves of cultivar ‘BJ 1’, the disease development caused an initial decrease in the concentration of phenols followed by an increase at later stages. As a result of inoculation, soluble carbohydrates and amino acids generally decreased in the leaves of resistant cultivar ‘BJ 1’, in contrast to an increase in their concentration in the leaves of cultivar ‘IR 8’.     

Oxidative dealkylation of a hindered phenol catalyzed by copper (II) bis benzimidazole diamide complex

The oxidative dealkylation of 2,4,6-tri-tert-butylphenol (TTBP) has been investigated using molecular oxygen and [Cu(NO₃(GBHA)](NO₃) as catalyst, where GBHA is N,N′-bis((benzimidazol-2-yl)methyl)hexanediamide [(a) M. Gupta, P. Mathur, R.J. Butcher, Inorg. Chem. 40 (2001) 878; (b) M. Gupta, S.K. Das, P. Mathur, A.W. Cordes, Inorg. Chim. Acta 353 (2003) 197; (c) S. Tehlan, M.S. Hundal, P. Mathur, Inorg. Chem. 43 (2004) 6589; (d) F. Afreen, P. Mathur, A. Rheingold, Inorg. Chim. Acta 358 (2005) 1125.]. X-ray structural characterization of complex [Cu(NO₃)(GBHA)](NO₃) · CH₃OH confirms that the Cu (II) ion is in a distorted square pyramidal geometry (τ = 0.168). The TTBP oxidation reaction proceeds via tri-tert-butylphenoxyl radical producing two products 2,6-di-tert-butyl-1,4-benzoquinone (A) and 4,6-di-tert-butyl-1,2-benzoquinone (B). Both A and B have been well characterized by ¹H NMR, ¹³C NMR, UV-Vis and mass data.