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Type II collagen is one of the predominant extracellular matrix macromolecules in cartilage responsible for maintenance of integrity of this specialized tissue. We showed previously that interleukin-1 (IL-1) and interferon-gamma (IFN-gamma) are capable of decreasing the levels of alpha 1(II) procollagen mRNA and suppressing the synthesis of type II collagen in cultured human chondrocytes. Data reported here show that these effects of IL-1 and IFN-gamma on the expression of the human type II collagen gene (COL2A1) are mediated primarily at the transcriptional level. This conclusion is based on three types of experimental evidence: (1) in nuclear run-off assays, preincubation of chondrocytes with either IL-1 or IFN-gamma decreased COL2A1 transcription; (2) experiments with the protein synthesis inhibitor cycloheximide and the transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) indicated that the suppression of alpha 1(II) procollagen mRNA by IL-1 could not be ascribed to decreased mRNA stability; and (3) a plasmid (pCAT-B/4.0) containing 4.0 kb of 5'-flanking sequences of COL2A1 (-577/+3428), encompassing the promoter, exon 1 and the putative enhancer sequence in the first intron, linked to the chloramphenicol acetyltransferase (CAT) reporter gene, was transfected in human chondrocytes. A high level of expression of pCAT-B/4.0 was observed in human chondrocytes incubated with an insulin-containing serum substitute that is permissive for expression of the COL2A1 gene. Expression of pCAT-B/4.0 in these cells was inhibited by either IL-1 or IFN-gamma. Furthermore, expression of pCAT-B/4.0 was not detected in human dermal fibroblasts. When the putative enhancer fragment in the first intron was removed, the expression in chondrocytes was greatly reduced. These studies demonstrate that expression of COL2A1 is tissue specific and that suppression by either IL-1 or IFN-gamma is mediated primarily at the transcriptional level.

We studied the mechanism of resistance to imipenem in three clinical isolates of Pseudomonas aeruginosa. Two of these isolates arose from imipenem-susceptible strains isolated during therapy with imipenem and were associated with treatment failure. One of these two strains had previously been broadly resistant to beta-lactams; the second acquired resistance to imipenem alone. One isolate of the third strain was resistant to imipenem but susceptible to other antipseudomonal beta-lactams. No isolate contained beta-lactamase activity capable of hydrolyzing imipenem at a detectable rate. Studies of the penicillin-binding proteins of all isolates revealed no differences in the number of proteins, molecular weight of, affinity for penicillin, or affinity for imipenem in any isolate. In each case the resistant isolate lacked one or more outer membrane proteins that were present in a susceptible isolate of the same strain. The observed alterations in outer membrane proteins may be associated with diminished permeability of the bacterial outer membrane to imipenem and may be the major factor responsible for resistance in these isolates.