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.

Cytokines are multifunctional mediators that classically modulate immune activity by receptor-mediated pathways. Macrophage migration inhibitory factor (MIF) is a cytokine that has a critical role in several inflammatory conditions but that also has endocrine and enzymatic functions. The molecular targets of MIF action have so far remained unclear. Here we show that MIF specifically interacts with an intracellular protein, Jab1, which is a coactivator of AP-1 transcription that also promotes degradation of the cyclin-dependent kinase inhibitor p27Kip1 (ref. 10). MIF colocalizes with Jab1 in the cytosol, and both endogenous and exogenously added MIF following endocytosis bind Jab1. MIF inhibits Jab1- and stimulus-enhanced AP-1 activity, but does not interfere with the induction of the transcription factor NFkappaB. Jab1 activates c-Jun amino-terminal kinase (JNK) activity and enhances endogenous phospho-c-Jun levels, and MIF inhibits these effects. MIF also antagonizes Jab1-dependent cell-cycle regulation by increasing p27Kip1 expression through stabilization of p27Kip1 protein. Consequently, Jab1-mediated rescue of fibroblasts from growth arrest is blocked by MIF. Amino acids 50-65 and Cys 60 of MIF are important for Jab1 binding and modulation. We conclude that MIF may act broadly to negatively regulate Jab1-controlled pathways and that the MIF-Jab1 interaction may provide a molecular basis for key activities of MIF.

The detection of double-stranded (ds) DNA by SYBR Green I (SG) is important in many molecular biology methods including gel electrophoresis, dsDNA quantification in solution and real-time PCR. Biophysical studies at defined dye/base pair ratios (dbprs) were used to determine the structure-property relationships that affect methods applying SG. These studies revealed the occurrence of intercalation, followed by surface binding at dbprs above approximately 0.15. Only the latter led to a significant increase in fluorescence. Studies with poly(dA)* poly(dT) and poly(dG)* poly(dC) homopolymers showed sequence-specific binding of SG. Also, salts had a marked impact on SG fluorescence. We also noted binding of SG to single-stranded (ss) DNA, although SG/ssDNA fluorescence was at least approximately 11-fold lower than with dsDNA. To perform these studies, we determined the structure of SG by mass spectrometry and NMR analysis to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]. For comparison, the structure of PicoGreen (PG) was also determined and is [2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]+. These structure-property relationships help in the design of methods that use SG, in particular dsDNA quantification in solution and real-time PCR.

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SummaryCell wall dynamics in Candida albicans , the most common fungal pathogen in man, underlie regulatory processes during the yeast-to-hyphae transition. To analyse this regulation at the transcriptional level, we have established a DNA microarray representing genes implicated in cell wall biogenesis. Using these microarrays, we were able to identify YWP1 and HWP2 that are specifically transcribed in the yeast or hyphal growth form respectively. Cluster analysis revealed at least two major clusters of genes: cluster I comprised genes that were upregulated under at least one hyphae-inducing condition. Three as yet not further characterized genes were attributed to cluster II. These genes were transcribed in the yeast form of C. albicans and were downregulated in an EFG1 -dependent manner under specific hyphae-inducing conditions. We show further that, in contrast to CPH1 , EFG1 plays a major role in the transcriptional regulation of cell wall proteins under the conditions investigated. EFG1 was essential for the transcription of both hyphae-specific genes such as HWP1 and HWP2 as well as the yeast form-specific gene YWP1 . Moreover, we found that, under various conditions, EFG1 also can act as a strong repressor for the transcription of RBE1 , another not yet characterized cell wall protein. Overall, our data show that EFG1 plays a major role in the induction and repression of cell wall genes, not only in the hyphal form but also in the yeast form of C. albicans .

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