Cytokines are critical in the often fatal cascade of events that cause septic shock. One regulatory system that is likely to be important in controlling inflammatory responses is the neuroendocrine axis. The pituitary, for example, is ideally situated to integrate central and peripheral stimuli, and initiates the increase in systemic glucocorticoids that accompanies host stress responses. To assess further the contribution of the pituitary to systemic inflammatory processes, we examined the secretory profile of cultured pituitary cells and whole pituitaries in vivo after stimulation with bacterial lipopolysaccharide (LPS). Here we identify macrophage migration inhibitory factor (MIF) as a major secreted protein release by anterior pituitary cells in response to LPS stimulation. Serum analysis of control, hypophysectomized and T-cell-deficient (nude) mice suggests that pituitary-derived MIF contributes to circulating MIF present in the post-acute phase of endotoxaemia. Recombinant murine MIF greatly enhances lethality when co-injected with LPS and anti-MIF antibody confers full protection against lethal endotoxaemia. We conclude that MIF plays a central role in the toxic response to endotoxaemia and possibly septic shock.

Histone deacetylases (HDACs) play a key role in regulating eukaryotic gene expression. The HDAC domain, homologous to the yeast repressors RPD3 and HDA1, is considered necessary and sufficient for enzymatic activity. Here, we show that the catalytic domain of HDAC4 interacts with HDAC3 via the transcriptional corepressor N-CoR/SMRT. All experimental conditions leading to the suppression of HDAC4 binding to SMRT/N-CoR and to HDAC3 result in the loss of enzymatic activity associated with HDAC4. In vitro reconstitution experiments indicate that HDAC4 and other class II HDACs are inactive in the context of the SMRT/N-CoR-HDAC3 complex and do not contribute to its enzymatic activity. These observations indicate that class II HDACs regulate transcription by bridging the enzymatically active SMRT/N-CoR-HDAC3 complex and select transcription factors independently of any intrinsic HDAC activity.

Pancreatic amyloid is found in more than 95 % of type II diabetes patients. Pancreatic amyloid is formed by the aggregation of islet amyloid polypeptide (hIAPP or amylin), which is a 37-residue peptide. Because pancreatic amyloid is cytotoxic, it is believed that its formation is directly associated with the development of the disease. We recently showed that hIAPP amyloid formation follows the nucleation-dependent polymerization mechanism and proceeds via a conformational transition of soluble hIAPP into aggregated beta-sheets. Here, we report that the penta- and hexapeptide sequences, hIAPP(23-27) (FGAIL) and hIAPP(22-27) (NFGAIL) of hIAPP are sufficient for the formation of beta-sheet-containing amyloid fibrils. Although these two peptides differ by only one amino acid residue, they aggregate into completely different fibrillar assemblies. hIAPP(23-27) (FGAIL) fibrils self-assemble laterally into unusually broad ribbons, whereas hIAPP(22-27) (NFGAIL) fibrils coil around each other in a typical amyloid fibril morphology. hIAPP(20-27) (SNNFGAIL) also aggregates into beta-sheet-containing fibrils, whereas no amyloidogenicity is found for hIAPP(24-27) (GAIL), indicating that hIAPP(23-27) (FGAIL) is the shortest fibrillogenic sequence of hIAPP. Insoluble amyloid formation by the partial hIAPP sequences followed kinetics that were consistent with a nucleation-dependent polymerization mechanism. hIAPP(22-27) (NFGAIL), hIAPP(20-27) (SNNFGAIL), and also the known fibrillogenic sequence, hIAPP(20-29) (SNNFGAILSS) exhibited significantly lower kinetic and thermodynamic solubilities than the pentapeptide hIAPP(23-27) (FGAIL). Fibrils formed by all short peptide sequences and also by hIAPP(20-29) were cytotoxic towards the pancreatic cell line RIN5fm, whereas no cytotoxicity was observed for the soluble form of the peptides, a notion that is consistent with hIAPP cytotoxicity. Our results suggest that a penta- and hexapeptide sequence of an appropriate amino acid composition can be sufficient for beta-sheet and amyloid fibril formation and cytotoxicity and may assist in the rational design of inhibitors of pancreatic amyloid formation or other amyloidosis-related diseases.

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The cytokine macrophage migration inhibitory factor (MIF) has been identified to be secreted by the pituitary gland and the monocyte/macrophage and to play an important role in endotoxic shock. Despite the recent molecular cloning of a human T-cell MIF, characterization of the biochemical and biological properties of this protein has remained incomplete because substantial quantities of purified, recombinant, or native MIF have not been available. We describe the cloning of mouse MIF from anterior pituitary cells (AtT-20) and the purification of native MIF from mouse liver by sequential ion exchange and reverse-phase chromatography. For comparison purposes, human MIF was cloned from the Jurkat T-cell line and also characterized. Mouse and human MIF were highly homologous (90% identity over 115 amino acids). Recombinant mouse and human MIF were expressed in Escherichia coli and purified in milligram quantities by a simple two-step procedure. The molecular weight of native mouse MIF (12.5 kDa monomer) was identical with that of recombinant mouse MIF as assessed by gel electrophoresis and mass spectroscopy. No significant post-translational modifications were detected despite the presence of two potential N-linked glycosylation sites. Recombinant MIF inhibited monocyte migration in a dose-dependent fashion, and both recombinant and native MIF-exhibited comparable biological activities. MIF induced the secretion of tumor necrosis factor-alpha and stimulated nitric oxide production by macrophages primed with interferon-gamma. Circular dichroism spectroscopy revealed that bioactive mouse and human MIF exhibit a highly ordered, three-dimensional structure with a significant percentage of beta-sheet and alpha-helix conformation. Guanidine hydrochloride-induced unfolding experiments demonstrated that MIF is of low to moderate thermodynamic stability. These studies establish the biochemical identity of native and recombinant MIF and provide a first insight into the three-dimensional structural properties of this critical inflammatory mediator.