Recent studies have implicated the amyloid Abeta peptide and its ability to self-assemble as key factors in the pathogenesis of Alzheimer's disease. Relatively little is known about the structure of soluble Abeta or its oligomeric state, and the existing data are often contradictory. In this study, we used intrinsic fluorescence of wild type Abeta-(1-40), fluorescence resonance energy transfer (FRET), and gel filtration chromatography to examine the structure of Abeta-(1-40) in solution. We synthesized a series of mono-substituted fluorescent Abeta-(1-40) derivatives to use as donors and acceptors in FRET experiments. We selected fluorescent peptides that exhibit aggregation properties comparable to wild type Abeta for analysis in donor-acceptor pairs; two labeled with 5-(2-((iodoacetyl)amino)ethyl)aminonaphthylene-1-sulfonic acid at Cys-25 or Cys-34 and fluorescein maleimide at Cys-4 or Cys-7. Another peptide containing a Trp substitution at position 10 was used as an acceptor for the intrinsic Tyr fluorescence of wild type Abeta-(1-40). Equilibrium studies of the denaturation of Abeta-(1-40) by increasing concentrations of dimethyl sulfoxide (Me2SO) were conducted by monitoring fluorescence, with a midpoint value for the unfolding transition of both the substituted and wild type peptides at among 40 and 50% Me2SO. Abeta-(1-40) is well solvated and largely monomeric in Me2SO as evidenced by a lack of FRET. When donor and acceptor Abeta derivatives are mixed together in Me2SO and then diluted 10-fold into aqueous Tris-HCl buffer at pH 7.4, efficient FRET is observed immediately for all pairs of fluorescent peptides, indicating that donor-acceptor dimers exist in solution. FRET is abolished by the addition of an excess of unlabeled Abeta-(1-40), demonstrating that the fluorescent peptides interact with wild type Abeta-(1-40) to form heterodimers that do not exhibit FRET. The Abeta-(1-40) dimers appear to be very stable, because no subunit exchange is observed after 24 h between fluorescent homodimers. Gel filtration confirms that nanomolar concentrations of 14C-labeled Abeta-(1-40) and fluorescein-labeled Abeta-(1-40) elute at the same dimeric position as wild type Abeta-(1-40), suggesting that soluble Abeta-(1-40) is also dimeric at more physiologically plausible concentrations.

The mechanism for apical growth during hyphal morphogenesis in Candida albicans is unknown. Studies from Saccharomyces cerevisiae indicate that cell morphogenesis may involve cell cycle regulation by cyclin-dependent kinase. To examine whether this is the mechanism for hyphal morphogenesis, the temporal appearance of different spindle pole body and spindle structures, the cell cycle-regulated rearrangements of the actin cytoskeleton, and the phosphorylation state of the conserved Tyr19 of Cdc28 during the cell cycle were compared and found to be similar between yeast and serum-induced hyphal apical cells. These data suggest that hyphal elongation is not mediated by altering cell cycle progression or through phosphorylation of Tyr19 of Cdc28. We have also shown that germ tubes can evaginate before spindle pole body duplication, chitin ring formation, and DNA replication. Similarly, tip-associated actin polarization in each hypha occurs before the events of the G 1 /S transition and persists throughout the cell cycle, whereas cell cycle-regulated actin assemblies come and go. We have also shown that cells in phases other than G 1 can be induced to form hyphae. Hyphae induced from G 1 cells have no constrictions, and the first chitin ring is positioned in the germ tube at various distances from the base. Hyphae induced from budded cells have a constriction and a chitin ring at the bud neck, beyond which the hyphae continue to elongate with no further constrictions. Our data suggest that hyphal elongation and cell cycle morphogenesis programs are uncoupled, and each contributes to different aspects of cell morphogenesis. INTRODUCTIONCandida albicans is a polymorphic fungal pathogen that undergoes reversible morphogenetic transitions among budding, pseudohyphal, and hyphal growth forms (Odds, 1985). Its ability to switch between yeast and hyphal growth forms is directly related to its virulence, because mutants defective in hyphal growth are less virulent in mouse models than are their wild-type counterparts (Leberer et al., 1997;Lo et al., 1997;Gale et al., 1998). Hyphae may be suited to breach barriers in the host, whereas the yeast form is more easily disseminated within the host. Therefore, understanding the mechanisms for this morphogenetic switch should provide insight into the pathogenicity of this fungus.During hyphal growth in C. albicans, cell surface expansion is restricted to a small region at the hyphal tip. This apical growth zone is active during the entire hyphal growth period (Staebell and Soll, 1985). In contrast, yeast-form cells expand from a small area in a mostly apical manner only at the initial stage of budding. When the bud has reached a critical size, apical growth shuts down and general (isotropic) expansion takes place (Staebell and Soll, 1985). The localization of the actin cytoskeleton in yeast and hyphal cells reflects these differences in morphogenesis. Polarization of the actin cytoskeleton to the hyphal tip is observed in all hyphal cells (Anderson and Soll, 1986). However, in yeast-...

Candida albicans undergoes a dramatic morphological transition in response to various growth conditions. This ability to switch from a yeast form to a hyphal form is required for its pathogenicity. The intractability of Candida to traditional genetic approaches has hampered the study of the molecular mechanism governing this developmental switch. Our approach is to use the more genetically tractable yeast Saccharomyces cerevisiae to yield clues about the molecular control of filamentation for further studies in Candida. G 1 cyclins Cln1 and Cln2 have been implicated in the control of morphogenesis in S. cerevisiae. We show that C. albicans CLN1 (CaCLN1) has the same cell cycle-specific expression pattern as CLN1 and CLN2 of S. cerevisiae. To investigate whether G 1 cyclins are similarly involved in the regulation of cell morphogenesis during the yeast-to-hypha transition of C. albicans, we mutated CaCLN1. Cacln1/Cacln1 cells were found to be slower than wild-type cells in cell cycle progression. The Cacln1/Cacln1 mutants were also defective in hyphal colony formation on several solid media. Furthermore, while mutant strains developed germ tubes under several hypha-inducing conditions, they were unable to maintain the hyphal growth mode in a synthetic hypha-inducing liquid medium and were deficient in the expression of hypha-specific genes in this medium. Our results suggest that CaCln1 may coordinately regulate hyphal development with signal transduction pathways in response to various environmental cues.

Alzheimer's disease is characterized by the presence of insoluble, fibrous deposits composed principally of amyloid ␤ (A␤) peptide. A number of studies have provided information on the fibril structure and on the factors affecting fiber formation, but the details of the fibril structure are not known. We used fluorescence quenching to investigate the solvent accessibility and surface charge of the soluble A␤(1-40) dimer and amyloid fibrils. Analogs of A␤(1-40) containing a single tryptophan were synthesized by substituting residues at positions 4, 10, 34, and 40 with tryptophan. Quenching measurements in the dimeric state indicate that the amino-terminal analogs (A␤F4W and A␤Y10W) are accessible to polar quenchers, and the more carboxyl-terminal analog A␤V34W is less accessible. A␤V40W, on the other hand, exhibits a low degree of quenching, indicating that this residue is highly shielded from the solvent in the dimeric state. Correcting for the effect of reduced translational and rotational diffusion, fibril formation was associated with a selective increase in solvent exposure of residues 34 and 40, suggesting that a conformation change may take place in the carboxyl-terminal region coincident with the dimer to fibril transition.

The possibility of using reverse micelles to stabilize monomers prior to formation of dimeric triosephosphate isomerase (TPI) from rabbit muscle was studied. TPI denatured with guanidine hydrochloride undergoes reactivation in reverse micelles formed with n-octane, hexanol, cetyltrimethylammonium bromide, and water. Reactivation of around 80% is observed at TPI concentrations of about 2 micrograms/mL of reverse micelles and water concentrations above 4.0%. With 3.0% water, reactivation is about 10%. If denatured TPI is incubated for a few seconds in reverse micelles with 5.0% water (or higher) followed by incubation in 3.0% water, reactivation is between 35% and 50%. That is, a brief exposure of denatured TPI to reverse micelles with a relatively high water concentration yielded a significant amount of structures competent for formation of catalytically active dimers. As evidenced by kinetic data, these structures correspond to monomers of TPI [Garza-Ramos, G. Tuena de Gómez-Puyou, M., Gómez-Puyou, A., & Gracy R. W. (1992) Eur. J. Biochem. 208, 389-395]. After a 5-2.0% water transition, competent monomers were stabilized for at least 30 min; a subsequent rise in water concentration led to dimerization and appearance of activity. By changes in the amount of water, it was possible to determine in reverse micelles the amount of water required for monomer folding and dimerization; i.e., less water was required in the dimerization step. Experiments with a model system, trypsin and the soybean inhibitor, showed that, in reverse micelles with 2.0% water, protein-protein interactions readily take place.(ABSTRACT TRUNCATED AT 250 WORDS)