Biomedical subjects

Mark G Kirchhof

Publications and source records attributed to Mark G Kirchhof.

6 recordsLinked to original sources

PubMed2006

Stationary phase expression of the arginine biosynthetic operon argCBH in Escherichia coli.

BACKGROUND: Arginine biosynthesis in Escherichia coli is elevated in response to nutrient limitation, stress or arginine restriction. Though control of the pathway in response to arginine limitation is largely modulated by the ArgR repressor, other factors may be involved in increased stationary phase and stress expression. RESULTS: In this study, we report that expression of the argCBH operon is induced in stationary phase cultures and is reduced in strains possessing a mutation in rpoS, which encodes an alternative sigma factor. Using strains carrying defined argR, and rpoS mutations, we evaluated the relative contributions of these two regulators to the expression of argH using operon-lacZ fusions. While ArgR was the main factor responsible for modulating expression of argCBH, RpoS was also required for full expression of this biosynthetic operon at low arginine concentrations (below 60 microM L-arginine), a level at which growth of an arginine auxotroph was limited by arginine. When the argCBH operon was fully de-repressed (arginine limited), levels of expression were only one third of those observed in deltaargR mutants, indicating that the argCBH operon is partially repressed by ArgR even in the absence of arginine. In addition, argCBH expression was 30-fold higher in deltaargR mutants relative to levels found in wild type, fully-repressed strains, and this expression was independent of RpoS. CONCLUSION: The results of this study indicate that both derepression and positive control by RpoS are required for full control of arginine biosynthesis in stationary phase cultures of E. coli.

Arginine↗

PubMed2006

A molecular perspective of CTLA-4 function.

Within the paradigm of the two-signal model of lymphocyte activation, the interest in costimulation has witnessed a remarkable emergence in the past few years with the discovery of a large array of molecules that can serve this role, including some with an inhibitory function. Interest has been further enhanced by the realization of these molecules' potential as targets to modulate clinical immune responses. Although the therapeutic translation of mechanistic knowledge in costimulatory molecules has been relatively straightforward, the capacity to target their inhibitory counterparts has remained limited. This limited capacity is particularly apparent in the case of the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), a major negative regulator of T cell responses. Because there have been several previous comprehensive reviews on the function of this molecule, we focus here on the physiological implications of its structural features. Such an exercise may ultimately help us to design immunotherapeutic agents that target CTLA-4.

Amino Acid Sequence↗

PubMed2005

Hierarchical regulation of CTLA-4 dimer-based lattice formation and its biological relevance for T cell inactivation.

CTLA-4 is an activation-induced, homodimeric inhibitory receptor in T cells. Recent crystallographic reports have suggested that it may form lattice-like arrays on the cell surface upon binding B7.1/B7.2 (CD80, CD86) molecules. To test the biological relevance of these CTLA-4-B7 lattices, we introduced a C122A point mutation in human CTLA-4, because this residue was shown to be essential for dimerization in solution. Surprisingly, we found that up to 35% of C122A CTLA-4 dimerized in human T lymphocytes. Moreover, C122A CTLA-4 partitioned within lipid rafts, colocalized with the TCR in the immunological synapse, and inhibited T cell activation. C122-independent dimerization of CTLA-4 involved N-glycosylation, because further mutation of the N78 and N110 glycosylation sites abrogated dimerization. Despite being monomeric, the N78A/N110A/C122A triple mutant CTLA-4 localized in the immunological synapse and inhibited T cell activation. Such functionality correlated with B7-induced dimerization of these mutant molecules. Based on these data, we propose a model of hierarchical regulation of CTLA-4 oligomerization by which B7 binding ultimately determines the formation of dimer-dependent CTLA-4 lattices that may be necessary for triggering B7-dependent T cell inactivation.

Alanine↗

PubMed2005

Polycationic lipids inhibit the pro-inflammatory response to LPS.

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. As such, it signals monocytes, macrophages and neutrophils to up-regulate phagocytic functions and to release pro-inflammatory cytokines. Despite the established role of CD14 as the main LPS receptor, the precise nature of the LPS signalling complex and its compartmentalization remain unknown. Interactions of LPS with other cell surface molecules such as TLR-4 and MD-2, and its subsequent internalization are required for LPS signalling. Here, we show that the polycationic lipid LipoFectamine causes inhibition of the LPS-induced MAPK activation and lack of pro-inflammatory cytokine production, despite proper localization of CD14 within lipid rafts and massive LPS internalization. The ability of LipoFectamine to inhibit LPS induced pro-inflammatory responses may be due to uncoupling of CD14 from TLR-4/MD-2 in the LPS signalling complex of mouse macrophages/microglial cells, as suggested by inhibition of LPS-induced concomitant internalization of these surface molecules. Thus, LipoFectamine may be a useful tool to dissect the molecular interactions leading to LPS signalling, and identifies a potential therapeutic strategy for LPS clearance.

Animals↗

PubMed2004

RpoS-regulated genes of Escherichia coli identified by random lacZ fusion mutagenesis.

RpoS is a conserved alternative sigma factor that regulates the expression of many stress response genes in Escherichia coli. The RpoS regulon is large but has not yet been completely characterized. In this study, we report the identification of over 100 RpoS-dependent fusions in a genetic screen based on the differential expression of an operon-lacZ fusion bank in rpoS mutant and wild-type backgrounds. Forty-eight independent gene fusions were identified, including several in well-characterized RpoS-regulated genes, such as osmY, katE, and otsA. Many of the other fusions mapped to genes of unknown function or to genes that were not previously known to be under RpoS control. Based on the homology to other known bacterial genes, some of the RpoS-regulated genes of unknown functions are likely important in nutrient scavenging.

Bacterial Proteins↗

PubMed2003

Regulation of T-cell activation by phosphodiesterase 4B2 requires its dynamic redistribution during immunological synapse formation.

Stimulation of T cells through their antigen receptors (TCRs) causes a transient increase in the intracellular concentration of cyclic AMP (cAMP). However, sustained high levels of cAMP inhibit T-cell responses, suggesting that TCR signaling is coordinated with the activation of cyclic nucleotide phosphodiesterases (PDEs). The molecular basis of such a pathway is unknown. Here we show that TCR-dependent signaling activates PDE4B2 and that this enhances interleukin-2 production. Such an effect requires the regulatory N terminus of PDE4B2 and correlates with partitioning within lipid rafts, early targeting of this PDE to the immunological synapse, and subsequent accumulation in the antipodal pole of the T cell as activation proceeds.

3',5'-Cyclic-AMP Phosphodiesterases↗