Cysteine protease activity is up-regulated in inflamed ankle joints of rats with adjuvant-induced arthritis and decreases with in vivo administration of a vinyl sulfone cysteine protease inhibitor.

OBJECTIVE: Cysteine proteases are postulated to play a role in tissue destruction in the joints of animals with arthritis. The purpose of the present study was to confirm the concept that cysteine proteases are enzymes involved in the pathology of rheumatoid arthritis (RA). METHODS: Arthritis was induced in Lewis rats by adjuvant injection (adjuvant-induced arthritis [AIA] model) and scored for inflammation. At necropsy, the rear paws were either fixed in formalin and assigned a histologic score (based on synovial cell proliferation, cartilage erosion, bone erosion, and fibroproliferative pannus) or frozen, cryosectioned, and assayed for enzyme activity either by in situ cytochemical staining with a post-azo-coupling method using a chromogenic substrate (Z-arg-arg-MNA) or by a novel assay placing the tissue section directly in a cuvette using the fluorogenic substrate Z-arg-arg-AMC. RESULTS: Enzymatic activity, measured either in frozen sections in situ or in the cuvette assay, was positively correlated with joint destruction (r = 0.7) and inflammation (r = 0.8). Activity was not inhibited significantly by Pefabloc (a serine protease inhibitor), EDTA (a metalloprotease inhibitor), or pepstatin A (an aspartyl protease inhibitor) but was inhibited by E-64 and vinyl sulfone irreversible inhibitors of cysteine proteases. The effect of one of the vinyl sulfone cysteine protease inhibitors, Mu-Leu-HomoPhe-vinylsulfone, was tested in vivo by dietary administration at 2.2 mg/kg/day in the AIA model; this resulted in a significant decrease in inflammation and in the amount of cysteine protease activity measured in the joint tissue. CONCLUSION: Cysteine protease activity levels increase in the diseased state and may be an important target for designing small molecule inhibitors to reduce the inflammation and tissue destruction associated with RA.

Isoforms of agrin are widely expressed in the developing rat and may function as protease inhibitors.

The agrin family of extracellular matrix proteins may be important in the formation of the neuromuscular junction. Using in situ hybridization with a probe recognizing all agrin isoforms, we demonstrate that it is widely expressed during mammalian embryogenesis. In the developing rat, particularly high levels of expression are found in the dorsal root and cranial ganglia, gut, whisker rudiments, penis, snout, teeth, retina, hippocampus, cerebral cortex and the lining of brain ventricles. Functional analysis of the recombinant rat protein shows that it is a potent inhibitor of the proteases trypsin, chymotrypsin and plasmin but not thrombin or the plasminogen activators. We conclude that agrin and its isoforms may play multiple roles in mammalian development including the regulation of proteolysis in the extracellular matrix.

The use of 33P-labeled oligonucleotides for in situ hybridization of vertebrate embryo frozen sections.

In situ hybridization was performed on frozen sections of whole E16 rat embryos or whole E7 chick embryos using oligonucleotide probes tagged with a 33P-poly A tail. We used two antisense oligonucleotide probes: a 57-mer rat agrin oligo and a 60-mer chicken B-cadherin oligo. After exposure to Hyperfilm beta max for three days, mRNA was detected in distinct organs and tissues of the embryo. After dipping the slides in liquid emulsion and exposing them for two weeks, the grains could be localized to a specific cellular layer, for example, the ganglion layer of the retina or the luminal epithelium of the gizzard. It is concluded that 33P autoradiography is excellent for defining gross areas or cellular layers of mRNA in a vertebrate embryo with a resolution of about 20 microns.

The effect of periodate oxidation and alpha-mannosidase treatment on Dolichos biflorus lectin.

The effects of periodate oxidation and alpha-mannosidase treatment of the Dolichos biflorus lectin were determined. Destruction by periodate of 16% of the mannose residues of the lectin had no effect on its ability to agglutinate type A erythrocytes, precipitate blood group A + H substance or to be precipitated by concanavalin A. Removal of up to 40% of the mannose by either periodate or alpha-mannosidase rendered the lectin nonprecipitable by concanavalin A. The lectin treated by alpha-mannosidase retained its ability to agglutinate erythrocytes and precipitate blood group A + H substance, but the lectin treated with periodate lost most of its activity. The results suggest that the complete integrity of the carbohydrate unit of the lectin is not necessary for its activity and that the periodate may be affecting the protein portion of the molecule as well as its carbohydrate residues. No conversion of form A to form B of the lectin was observed with either periodate oxidation or alpha-mannosidase treatment.

Iodination of Xenopus laevis histone F2a1 in chromatin.

The reaction of calf thymus and Xenopus laevis histones with radioactive iodine has been studied under various conditions that affect chromatin structure. All histones from both species contain at least one tyrosine residue and, in a denaturing solvent, all the the histones react with iodine. Histone F2a1 has been studied in detail. Calf thymus F2a1 is known to contain four tyrosyls and all four react with iodine. In high voltage paper electrophoresis, the tyrosine-containing peptides from calf co-migrate with those from Xenopus F2a1, suggesting that the amino acid sequence is strongly conserved between these two species. Therefore, the published calf thymus F2a1 sequence has been used to order the Xenopus F2a1 peptides within the molecules. When gently isolated native chromatin is iodinated in a low ionic strength medium 60% of the radioactivity in F2a1 is in tyrosyl 88, 30% in tyrosyl 51, and tyrosyl 72 and 98 have almost no radioactivity. Reagents which remove the protein from the DNA (2 M NaCl) or partially disrupt protein tertiary structure (5 M urea) increase the reactivity of each of the four tyrosyls five- to tenfold, suggesting that all four are protected about equally by the overall folding of the chromatin. Isolated F2a1 iodinated in the presence of 10 M urea shows uniform labeling in each of the four peptides, suggesting that tyrosyl 72 and 98 are afforded some protection solely by protein-protein interactions. The stepwise removal of histones in increasing NaCl concentrations differentially increases the availability of each F2a1 tyrosyl. The preferential exposure of tyrosyl 88 coincides with the removal of the majority of F1 histones at 0.5 M NaCl while the gradual and stepwise increase in reactivity of tyrosyl 51, 72, and 98 correlates with the gradual removal of histones other than F1. Radioactive iodination of chromatin has been shown to be a sensitive probe for detecting changes in the association state (or conformation) of histone F2a1.