Anergic T cells are defective in both jun NH2-terminal kinase and mitogen-activated protein kinase signaling pathways.

T helper type 1 cells (Th1) become anergic when stimulated through the antigen receptor in the absence of costimulation. They do not produce IL-2 or proliferate in response to subsequent stimulation. Previous studies have indicated that anergic T cells are defective in the trnsactivational activity of the transcription factor, AP-1, which is required for optimal IL-2 transcription. Using two murine Th1 cell clones, we demonstrate that anergic Th1 cells have defects in both jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activities. These kinases have been shown to be important for the upregulation of AP-1 activity. Furthermore, our data show that ERK and JNK activities are restored when anergy is induced in the presence of the protein synthesis inhibitor cycloheximide, or when anergic T cells are allowed to proliferate in response to exogenous IL-2. These treatments have previously been shown to prevent or reverse the anergic state. Our results suggest that defects in both JNK and ERK may result in the decreased AP-1 activity and the reduced IL-2 transcription observed in anergic T cells.

Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations.

Complementation analyses using minimal recombinant clones showed that all known pdx point mutations, which cause pyridoxine (vitamin B6) or pyridoxal auxotrophy, are located in the pdxA, pdxB, serC, pdxJ, and pdxH genes. Antibiotic enrichments for chromosomal transposon mutants that require pyridoxine (vitamin B6) or pyridoxal led to the isolation of insertions in pdxA, pdxB, and pdxH but not in pdxJ. This observation suggested that pdxJ, like pdxA, pdxB, and serC, might be in a complex operon. To test this hypothesis, we constructed stable insertion mutations in and around pdxJ in plasmids and forced them into the bacterial chromosome. Physiological properties of the resulting insertion mutants were characterized, and the DNA sequence of pdxJ and adjacent regions was determined. These combined approaches led to the following conclusions: (i) pdxJ is the first gene in a two-gene operon that contains a gene, temporarily designated dpj, essential for Escherichia coli growth; (ii) expression of the rnc-era-recO and pdxJ-dpj operons can occur independently, although the pdxJ-dpj promoter may lie within recO; (iii) pdxJ encodes a 26,384-Da polypeptide whose coding region is preceded by a PDX box, and dpj probably encodes a basic, 14,052-Da polypeptide; (iv) mini-Mud insertions in dpj and pdxJ, which are polar on dpj, severely limit E. coli growth; and (v) three classes of suppressors, including mutations in lon and suppressors of lon, that allow faster growth of pdxJ::mini-Mud mutants can be isolated. A model to account for the action of dpj suppressors is presented, and aspects of this genetic analysis are related to the pyridoxal 5'-phosphate biosynthetic pathway.

Location and characterization of genes involved in binding of starch to the surface of Bacteroides thetaiotaomicron.

Previous studies of starch utilization by the gram-negative anaerobe Bacteroides thetaiotaomicron have demonstrated that the starch-degrading enzymes are cell associated rather than extracellular, indicating that the first step in starch utilization is binding of the polysaccharide to the bacterial surface. Five transposon-generated mutants of B. thetaiotaomicron which were defective in starch binding (Ms-1 through Ms-5) had been isolated, but initial attempts to identify membrane proteins missing in these mutants were not successful. We report here the use of an immunological approach to identify four maltose-inducible membrane proteins, which were missing in one or more of the starch-binding mutants of B. thetaiotaomicron. Three of the maltose-inducible proteins were outer membrane proteins (115, 65, and 43 kDa), and one was a cytoplasmic membrane protein (80 kDa). The genes encoding these proteins were shown to be clustered in an 8.5-kbp segment of the B. thetaiotaomicron chromosome. Two other loci defined by transposon insertions, which appeared to contain regulatory genes, were located within 7 kbp of the cluster of membrane protein genes. The 115-kDa outer membrane protein was essential for utilization of maltoheptaose (G7), whereas loss of the other proteins affected growth on starch but not on G7. Not all of the proteins missing in the mutants were maltose regulated. We also detected two constitutively produced proteins (32 and 50 kDa) that were less prominent in all of the mutants than in the wild type. Both of these were outer membrane proteins.

The effects of IL-1 on mitogen-activated protein kinases in rabbit articular chondrocytes.

IL-1-activated chondrocytes express a large number of genes which contribute to cartilage degradation. The signaling pathways activated in response to IL-1 in these cells are not well-defined. We examined the effects of IL-1 and other stimuli on the mitogen activated protein kinase (MAPK) pathways in rabbit articular chondrocytes. We demonstrate that IL-1 activates three MAPKs, ERK, JNK and p38, in a time and dose-dependent manner. Activation is maximal by 15 minutes and returns to baseline levels by 1 hour. Maximal activation of ERK and p38 occurs with 1 ng/ml IL-1 whereas activation of JNK requires 10-fold higher levels. In contrast to IL-1, the PKC activator, PDBu preferentially activates ERK while TNF alpha preferentially activates JNK. LPS and TGF beta fail to stimulate any of the kinases examined. These results suggest that activation of the various MAPK pathways is important in the response of chondrocytes to IL-1, cytokines and growth factors.