A simple vertebrate collagenase assay using soluble radioactive collagen substrate

A highly sensitive assay for vertebrate collagenase has been developed using [14C]proline- or [3H]proline-labeled collagen as soluble substrate. The substrate was easy to prepare, gave high specific activity (1.4 X 10(6) cpm/mg collagen), and was stable at -20 degrees C for a long period. The digestion reaction for the assay was done at 21 degrees C to minimize the cleavage of collagen by proteases other than collagenase and to protect the 3/4 and 1/4 cleavage fragments of collagen from being further attacked by proteases. The cleaved products were denatured and then separated from undigested native collagen by precipitation with 1 M NaCl at pH 3.5. The conditions selected for denaturation and separation gave better discrimination between the cleaved products and uncleaved substrate than did conditions used in some other assays. The digestion products can be examined further by gel electrophoresis at the end of the assay to confirm the activity of vertebrate collagenase. This assay can also be adapted to assess telopeptidase activity independently of collagenase activity.     

Maxi-circles and mini-circles in kinetoplast DNA from trypanosoma cruzi

Glyceryl trinitrate specifically required cysteine, whereas NaNO2 at concentrations less than 10 mM required one of several thiols or ascorbate, to activate soluble guanylate cyclase from bovine coronary artery. However, guanylate cyclase activation by nitroprusside or nitric oxide did not require the addition of thiols or ascorbate. Whereas various thiols enhanced activation by nitroprusside, none of the thiols tested enhanced activation by nitric oxide. S-Nitrosocysteine, which is formed when cysteine reacts with either NO-2 or nitric oxide, was a potent activator of guanylate cyclase. Similarly, micromolar concentrations of the S-nitroso derivatives of penicillamine, GSH and dithiothreitol, prepared by reacting the thiol with nitric oxide, activated guanylate cyclase. Guanylate cyclase activation by S-nitrosothiols resembled that by nitric oxide and nitroprusside in that activation was inhibited by methemoglobin, ferricyanide and methylene blue. Similarly, guanylate cyclase activation by glyceryl trinitrae plus cysteine, and by NaNO2 plus either a thiol or ascorbate, was inhibited by methemoglobin, ferricyanide and methylene blue. These data suggest that the activation of guanylate cyclase by each of the compounds tested may occur through a common mechanism, perhaps involving nitric oxide. Moreover, these findings suggest that S-nitrosothiols could act as intermediates in the activation of guanylate cyclase by glyceryl trinitrate, NaNO2 and possibly nitroprusside.     

Species variation in BrdUrd-induced sister-chromatid exchanges.

Peripheral blood lymphocytes from cattle, pigs, sheep and humans were cultured in the presence of 0.5, 1, 2, 5, 10 or 20 micrograms/ml of BrdUrd. Sister-chromatid exchanges were scored in 25 second-division metaphases from each donor at each level of the chemical. Dose--response curves for all 4 species increased steeply to 2 micrograms/ml; above this level, SCE numbers increased less rapidly but maintained a linear relationship to increasing BrdUrd concentration. Comparisons of the straight-line portions of the dose--response curves showed human cells to be significantly more sensitive to increasing BrdUrd level than cow or pig cells and different from sheep at the 10% level of confidence.     

Plasma Membrane Profiling Defines an Expanded Class of Cell Surface Proteins Selectively Targeted for Degradation by HCMV US2 in Cooperation with UL141

Human cytomegalovirus (HCMV) US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I). Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin β1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2’s impact on HCMV pathogenesis.     

Identification of the sites in opsin modified by photoactivated azido[125I]iodobenzene.

Opsin labelled with photoactivated 1-azido-4-[125I]iodobenzene was proteolysed in situ with Staphylococcus aureus V8 proteinase to yield two radioactive membrane-bound fragments. These were separated, cleaved with CNBr and the resultant peptides sequenced in order to locate the radiolabelled residues. In the whole molecule, there was clear evidence for modification of at least 20 sites, identified as derivatives of cysteine, tryptophan, tyrosine, histidine and lysine residues. The probe primary reacted, therefore, with nucleophilic substituents. The positions of the modified sites relative to the confines of the phospholipid bilayer were consistent with all other studies on the disposition of the polypeptide chain. The location of these sites substantiated an earlier suggestion that not all the transmembrane segments should be regarded as continuous regular alpha-helices.