Singlet oxygen inactivates protein tyrosine phosphatase-1B by oxidation of the active site cysteine

Singlet oxygen ((1)O(2)), an electronically excited form of molecular oxygen, is a mediator of biological effects of ultraviolet A radiation, stimulating signaling cascades in human cells. We demonstrate here that (1)O(2) generated by photosensitization or by thermodecomposition of 3,3'-(1,4-naphthylidene)dipropionate-1,4-endoperoxide inactivates isolated protein tyrosine phosphatases (PTPases). PTPase activities of PTP1B or CD45 were abolished by low concentrations of (1)O(2), but were largely restored by post-treatment with dithiothreitol. Electrospray ionization mass spectrometry analysis of tryptic digests of PTP1B exposed to (1)O(2) revealed oxidation of active-site Cys215 as the only cysteine residue oxidized. In summary, (1)O(2) may activate signaling cascades by interfering with phosphotyrosine dephosphorylation.     

The influence of earthworm-processed pig manure on the growth and productivity of marigolds.

The effects of additions of earthworm-processed pig manure (vermicompost) on the growth and productivity of French marigold (Tagetes patula) plants were evaluated under glasshouse conditions. Marigolds were germinated and grown in a standard commercial greenhouse container medium (Metro-Mix 360), substituted with 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% (by volume) pig manure vermicompost. The control consisted of Metro-Mix 360 alone without vermicompost. Plants were supplied regularly with a complete mineral nutrient solution for 121 days. The greatest vegetative growth resulted from substitution of Metro-Mix 360 with 30% and 40% pig manure vermicompost, and the lowest growth was in the potting mixtures containing 90% and 100% vermicompost. Most flower buds occurred in the potting mixtures containing 40% pig manure vermicompost (19.4 buds), and fewest in the potting mixtures containing 100% vermicompost. Marigolds grown in Metro-Mix 360 substituted with 90% and 100% pig manure vermicompost had the fewest and smallest flowers. After substitution of Metro-Mix 360 with 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% vermicompost, the marigold roots were larger than those of plants grown in the Metro-Mix 360 controls. Substitution of Metro-Mix 360 with any concentration of pig manure vermicompost, with all needed nutrients provided, increased the overall nitrate-nitrogen concentrations of the marigold leaf tissues at flowering stage. Some of the marigold growth and productivity enhancement, resulting from substitution of Metro-Mix 360 with pig manure vermicompost, may be explained by nutritional factors; However, other, factors, such as plant-growth regulators and humates, might have also been involved since all plants were supplied regularly with all required nutrients.     

A denaturing gradient gel electrophoresis assay for sensitive detection of p53 mutations

p53 is a tumor suppressor gene located on 17p, a region of the human genome frequently deleted in tumors. Mutation of the p53 gene is an important step leading to development of many forms of human cancer. To simplify the analysis of tumors for p53 point mutations, we describe a GC-clamped denaturing gradient gel assay for detecting single-base substitutions within highly conserved regions of the p53 gene. This assay alows for efficient screening of tumors for single-base substitutions within the p53 gene and can be used to facilitate sequence analysis of p53 point mutations.     

Probing ADAMTS13 Substrate Specificity using Phage Display

Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2’ and P11’, for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13–VWF exosite interactions outside of VWF73.     

In vivo activation of quiescent B cells by anti-immunoglobulin. I. Induction of latent VH allotype production in adult rabbits by treatment with heterologous antibodies or antibody fragments

We demonstrated previously that injection of adult rabbits with homologous anti-VHa1 allotype antibody induces the appearance in serum of genetically unexpected (latent) a1 immunoglobulin (Ig) and a1-like internal images. We have now investigated the mechanism for this induction effect and have found that treatment of animals with polyclonal goat or monoclonal mouse anti-a1 antibody similarly results in production of unexpected a1 determinants. The N-terminal amino acid sequences of deblocked heavy chains showed that latent a1 Ig induced by monoclonal antibody treatment was identical to nominal a1 Ig at 18 of 19 residues, with the one amino acid difference at position 13 probably due to a single nucleotide change. Like nominal a1, these molecules expressed multiple VHa1 framework epitopes, as demonstrated by direct immunoelectron microscopic visualization of immune complexes. Whereas F(ab)'2 fragments of rabbit anti-a1 antibody retained inducing activity, F(ab) fragments were effective only when administered in an aggregated form; this result suggests that cross-linking of surface Ig receptors plays a critical role in the induction process. We conclude that all rabbits contain dormant B cells expressing germ-line-encoded latent allotypes, and that in vivo administration of anti-Ig antibody causes activation of these cells directly rather than through perturbation of an allotype regulatory network.     

Overview of the Oldest Existing Set of Substrate-optimized Anaerobic Processes: Digestive Tracts

Over millions of years, living organisms have explored and optimized the digestion of a wide variety of substrates. Engineers who develop anaerobic digestion processes for waste treatment and energy production can learn much from this accumulated ‘experience’. The aim of this work is a survey based on the comparison of 190 digestive tracts (vertebrate and insect) considered as ‘reactors’ and their anaerobic processes. Within a digestive tract, each organ is modeled as a type of reactor (continuous stirred-tank, such reactors in series, plug-flow or batch) associated with chemical aspects such as pH or enzymes. Based on this analysis, each complete digestion process has been rebuilt and classified in accordance with basic structures which take into account the relative size of the different reactors. The results show that all animal digestive structures can be grouped within four basic types. Size and/or position in the structure of the different reactors (pre/post treatment and anaerobic microbial digestion) are closely correlated to the degradability of the feed (substrate). Major common features are: (i) grinding, (ii) an extreme pH compartment, and (iii) correlation between the size of the microbial compartment and the degradability of the feed. Thus, shared answers found by animals during their evolution can be a source of inspiration for engineers in designing optimal anaerobic processes.