YAC-1 MHC class I variants reveal an association between decreased NK sensitivity and increased H-2 expression after interferon treatment or in vivo passage

Two H-2 negative variants of the YAC-1 lymphoma were selected by mutagenization and sequential in vitro selections and compared with wild-type cells for changes in NK sensitivity and H-2 expression after interferon treatment or in vivo passage. The H-2 negative variants and the low H-2 expressor YAC-1 wild-type cells had similar NK sensitivity. However, IFN-beta or recombinant IFN-gamma pretreatments increased the H-2 expression of YAC-1 and protected them from NK lysis, whereas the H-2 variants, which remained H-2 negative, were not protected and often more sensitive to NK lysis. The H-2 variants were similarly susceptible as wild-type cells to three other cellular effects of interferon: protection from virus infection, modulation of Con A capping, and inhibition of cell proliferation. Thus, the only interferon-mediated effect that distinguished the H-2 negative variants from wild-type cells was the inability of the former to increase their H-2 expression and decrease their NK sensitivity. The wild-type YAC-1 line showed increased H-2 expression and decreased NK sensitivity after in vivo passage. In contrast, in vivo passaged H-2 variants showed no reexpression of H-2, and remained NK sensitive. The altered responses to interferon and in vivo passage were specific for loss or down-regulation of H-2, because Thy-1 loss (H-2 positive) YAC-1 variants behaved as the wild-type cells in all respects. This study supports the hypothesis that NK cells may function in vivo to eliminate host cells that fail to express H-2 after interferon stimulation during an immune response; such cells are a potential threat because they may escape recognition by T lymphocytes despite the expression of viral or tumor-associated antigens.     

Adaptations of Photosynthetic Electron Transport,Carbon Assimilation,and Carbon Partitioning in Transgenic Nicotiana plumbaginifolia Plants to Changes in Nitrate Reductase Activity

Transgenic Nicotiana plumbaginifolia plants that express either a 5-fold increase or a 20-fold decrease in nitrate reductase (NR) activity were used to study the relationships between carbon and nitrogen metabolism in leaves. Under saturating irradiance the maximum rate of photosynthesis, per unit surface area, was decreased in the low NR expressors but was relatively unchanged in the high NR expressors compared with the wild-type controls. However, when photosynthesis was expressed on a chlorophyll (Chl) basis the low NR plants had comparable or even higher values than the wild-type plants. Surprisingly, the high NR expressors showed very similar rates of photosynthesis and respiration to the wild-type plants and contained identical amounts of leaf Chl, carbohydrate, and protein. These plants were provided with a saturating supply of nitrate plus a basal level of ammonium during all phases of growth. Under these conditions overexpression of NR had little impact on leaf metabolism and did not stimulate growth or biomass production. Large differences in photochemical quenching and nonphotochemical quenching components of Chl a fluorescence, as well as the ratio of variable to maximum fluorescence, (FV/FM), were apparent in the low NR expressors in comparison with the wild-type controls. Light intensity-dependent increases in nonphotochemical quenching and decreases in FV/FM were greatest in the low NR expressors, whereas photochemical quenching decreased uniformly with increasing irradiance in all plant types. Nonphotochemical quenching was increased at all except the lowest irradiances in the low NR expressors, allowing photosystem II to remain oxidized on its acceptor side. The relative contributions of photochemical and nonphotochemical quenching of Chl a fluorescence with changing irradiance were virtually identical in the high NR expressors and the wild-type controls. Zeaxanthin was present in all leaves at high irradiances; however, at high irradiance leaves from the low NR expressors contained considerably more zeaxanthin and less violaxanthin than wild-type controls or high NR expressors. The leaves of the low NR expressors contained less Chl, protein, and amino acids than controls but retained more carbohydrate (starch and sucrose) than the wild type or high NR expressors. Sucrose phosphate synthase activities were remarkably similar in all plant types regardless of the NR activity. In contrast phosphoenolpyruvate carboxylase activities were increased on a Chl or protein basis in the low NR expressors compared with the wild-type controls or high NR expressors. We conclude that large decreases in NR have profound repercussions for photosynthesis and carbon partitioning within the leaf but that increases in NR have negligible effects.     

Changes in the fibronectin-specific integrin expression pattern modify the migratory behavior of sarcoma S180 cells in vitro and in the embryonic environment

The molecules that mediate cell-matrix recognition, such as fibronectins (FN) and integrins, modulate cell behavior. We have previously demonstrated that FN and the beta 1-integrins are used during neural crest cell (NCC) migration in vitro as well as in vivo, and that the FN cell-binding domains I and II exhibit functional specificity in controlling either NCC attachment, spreading, or motility in vitro. In the present study, we have analyzed the effect of changes in the integrin expression patterns on migratory cell behavior in vivo. We have generated, after stable transfection, S180 cells expressing different levels of alpha 4 beta 1 or alpha 5 beta 1 integrins, two integrins that recognize distinct FN cell-binding domains. Murine S180 cells were chosen because they behave similarly to NCC after they are grafted into the NCC embryonic pathways in the chicken embryo. Thus, they provide a model system with which to investigate the mechanisms controlling in vitro and in vivo migratory cell behavior. We have observed that either the overexpression of alpha 5 beta 1 integrin or the induction of alpha 4 beta 1 expression in transfected S180 cells enhances their motility on FN in vitro. These genetically modified S180 cells also exhibit different migratory properties when grafted into the early trunk NCC migratory pathways. We observe that alpha 5 and low alpha 4 expressors migrate in both the ventral and dorsolateral paths simultaneously, in contrast to the parental S180 cells or the host NCC, which are delayed by 24 h in their invasion of the dorsolateral path. Moreover, the alpha 4 expressors exhibit different migratory properties according to their level of alpha 4 expression at the cell surface. Cells of the low alpha 4 expressor line invade both the ventral and dorsolateral pathways. In contrast, the high expressors remain as an aggregate at the graft site, possibly the result of alpha 4 beta 1-dependent homotypic aggregation. Thus, changes in the repertoire of FN-specific integrins enable the S180 cells to exploit different pathways in the embryo and regulate the speed with which they disperse in vivo and in culture. Our studies correlate well with known changes in integrin expression during neural crest morphogenesis and strongly suggest that neural crest cells that migrate into the dorsolateral path, i.e., melanoblasts, do so only after they have upregulated the expression of FN receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genotypic and developmental regulation of transient expression of a reporter gene in soybean zygotic cotyledons

Processes involved in transformation of regenerable soybean (Glycine max (L.) Merrill) immature zygotic cotyledons were studied by assaying the transient expression of the -glucuronidase gene driven by the 35S promoter and terminated at the 3 end by the soybean 7S storage protein gene. The plasmid containing the chimeric gene was delivered to the cotyledons via particle bombardment 900 PSI. Zygotic cotyledons from six soybean varieties were tested for transient expression of the -glucuronidase gene. The level of reporter gene expression differed between genotypes. The genotypes could be classified as high, fair or poor expressors. Cotyledons from different genotypes were then bombarded at 650, 900 or 1100 PSI. GUS expression varied among genotypes independently of the pressure of bombardment. Finally, the ability of cotyledons to express the reporter gene depended on the developmental stage of the seed from which it was excised with the younger stage being the least responsive. However, genotypic specific expression remained after controlling for developmental stage of the cotyledons.Abbreviations ANOVA analysis of variance - GUS -glucuronidase - PSI pounds per square inch - TEUs transient expression units - MSO3 MS media with 3% sucrose     

The bithorax complex—A model for cell determination in Drosophila

The bithorax complex is postulated to be the structural gene for an allosteric protein whose function is to translate a position in the developing egg into the expression of a unique set of genes characterizing the determined state of cells arising in that region of the egg. This is accomplished through the binding of inducer molecules, which exist in an anterior-posterior gradient in the egg, to the allosteric bithorax protein. Depending upon the nature of the inducer binding sites occupied, an expressor binding site on the bithorax protein is activated to one of a number of possible states. Depending upon the state of the expressor binding site, the bithorax protein binds to one of a number of classes of expressors. Binding of the bithorax protein to an expressor leads to the expression of those genes controlled by the expressor.     

Properties of some variants of human beta2-microglobulin and amyloidogenesis

Three variants of human beta(2)-microglobulin (beta(2)-m) were compared with wild-type protein. For two variants, namely the mutant R3Abeta(2)-m and the form devoid of the N-terminal tripeptide (DeltaN3beta(2)-m), a reduced unfolding free energy was measured compared with wild-type beta(2)-m, whereas an increased stability was observed for the mutant H31Ybeta(2)-m. The solution structure could be determined by (1)H NMR spectroscopy and restrained modeling only for R3Abeta(2)-m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Ybeta(2)-m and DeltaN3beta(2)-m. Precipitation and unfolding were observed over time periods shorter than 4-6 weeks with all the variants and, sometimes, with wild-type protein. The rate of structured protein loss from solution as a result of precipitation and unfolding always showed pseudo-zeroth order kinetics. This and the failure to observe an unfolded species without precipitation suggest that a nucleated conformational conversion scheme should apply for beta(2)-m fibrillogenesis. The mechanism is consistent with the previous and present results on beta(2)-m amyloid transition, provided a nucleated oligomeric species be considered the stable intermediate of fibrillogenesis, the monomeric intermediate being the necessary transition step along the pathway from the native protein to the nucleated oligomer.     

Identification and recovery of minor HIV-1 variants using the heteroduplex tracking assay and biotinylated probes

We describe a method to identify and recover minor human immunodeficiency virus type 1 (HIV-1) sequence variants from a complex population. The original heteroduplex tracking assay (HTA) was modified by incorporating a biotin tag into the probe to allow for direct sequence determination of the query strand. We used this approach to recover sequences from minor HIV-1 variants in the V3 region of the env gene, and to identify minor drug-resistant variants in pro. The biotin-HTA targeting of the V3 region of env allowed us to detect minor V3 variants, of which 45% were classified as CXCR4-using viruses. In addition, the biotin-protease HTA was able to detect mixtures of wild-type sequence and drug-resistance mutations in four subjects that were not detected by bulk sequence analysis. The biotin-HTA is a robust assay that first separates genetic variants then allows direct sequence analysis of major and minor variants.     

Syndecan-4 deficiency leads to high mortality of lipopolysaccharide-injected mice

Syndecan-4 is a transmembrane heparan sulfate proteoglycan belonging to the syndecan family. Following intraperitoneal injection of lipopolysaccharide (LPS), syndecan-4-deficient mice exhibited high mortality compared with wild-type controls. Severe endotoxin shock was observed in the deficient mice: systolic blood pressure and left ventricular fractional shortening were lower in the deficient mice than in the wild-type controls 9 h after LPS injection. Although histological examinations revealed no apparent differences between two groups, the plasma level of interleukin (IL)-1beta was higher in the deficient mice than in the wild-type controls 9 h after LPS injection. Consistent with the regulatory roles of syndecan-4, its expression in monocytes and endothelial cells of microvasculature increased in the wild-type mice after LPS administration. Although IL-1beta was produced to the same extent by macrophages from syndecan-4-deficient and wild-type mice after LPS stimulation, inhibition of its production by transforming growth factor-beta1 was impaired in the syndecan-4-deficient macrophages. These results indicate that syndecan-4 could be involved in prevention of endotoxin shock, at least partly through the inhibitory action of transforming growth factor-beta1 on IL-1beta production.     

Sialylation of beta1 integrins blocks cell adhesion to galectin-3 and protects cells against galectin-3-induced apoptosis

In previous studies, we determined that beta1 integrins from human colon tumors have elevated levels of alpha2-6 sialylation, a modification added by beta-galactosamide alpha-2,6-sialyltranferase I (ST6Gal-I). Intriguingly, the beta1 integrin is thought to be a ligand for galectin-3 (gal-3), a tumor-associated lectin. The effects of gal-3 are complex; intracellular forms typically protect cells against apoptosis through carbohydrate-independent mechanisms, whereas secreted forms bind to cell surface oligosaccharides and induce apoptosis. In the current study, we tested whether alpha2-6 sialylation of the beta1 integrin modulates binding to extracellular gal-3. Herein we report that SW48 colonocytes lacking alpha2-6 sialylation exhibit beta1 integrin-dependent binding to gal-3-coated tissue culture plates; however, binding is attenuated upon forced expression of ST6Gal-I. Removal of alpha2-6 sialic acids from ST6Gal-I expressors by neuraminidase treatment restores gal-3 binding. Additionally, using a blot overlay approach, we determined that gal-3 binds directly and preferentially to unsialylated, as compared with alpha2-6-sialylated, beta1 integrins. To understand the physiologic consequences of gal-3 binding, cells were treated with gal-3 and monitored for apoptosis. Galectin-3 was found to induce apoptosis in parental SW48 colonocytes (unsialylated), whereas ST6Gal-I expressors were protected. Importantly, gal-3-induced apoptosis was inhibited by function blocking antibodies against the beta1 subunit, suggesting that beta1 integrins are critical transducers of gal-3-mediated effects on cell survival. Collectively, our results suggest that the coordinate up-regulation of gal-3 and ST6Gal-I, a feature that is characteristic of colon carcinoma, may confer tumor cells with a selective advantage by providing a mechanism for blockade of the pro-apoptotic effects of secreted gal-3.     

Expression of Murine 5-Aminolevulinate Synthase Variants Causes Protoporphyrin IX Accumulation and Light-Induced Mammalian Cell Death

5-Aminolevulinate synthase (ALAS; EC 2.3.1.37) catalyzes the first committed step of heme biosynthesis in animals. The erythroid-specific ALAS isozyme (ALAS2) is negatively regulated by heme at the level of mitochondrial import and, in its mature form, certain mutations of the murine ALAS2 active site loop result in increased production of protoporphyrin IX (PPIX), the precursor for heme. Importantly, generation of PPIX is a crucial component in the widely used photodynamic therapies (PDT) of cancer and other dysplasias. ALAS2 variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. In order to assess the prospective utility of ALAS2 variants in PPIX production for PDT, K562 human erythroleukemia cells and HeLa human cervical carcinoma cells were transfected with expression plasmids for ALAS2 variants with greater enzymatic activity than the wild-type enzyme. The levels of accumulated PPIX in ALAS2-expressing cells were analyzed using flow cytometry with fluorescence detection. Further, cells expressing ALAS2 variants were subjected to white light treatments (21–22 kLux) for 10 minutes after which cell viability was determined. Transfection of HeLa cells with expression plasmids for murine ALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatments revealed that ALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid (ALA) treatment producing a similar amount of PPIX. The delivery of stable and highly active ALAS2 variants has the potential to expand and improve upon current PDT regimes.     

Profiling of membrane protein variants in a baculovirus system by coupling cell-surface detection with small-scale parallel expression

Production of structure-grade mammalian membrane proteins in substantial quantities has been hindered by a lack of methods for effectively profiling multiple constructs expression in higher eukaryotic systems such as insect or mammalian cells. To address this problem, a specialized small-scale eukaryotic expression platform by Thomson Instrument Company (Vertiga-IM) was developed and used in tandem with a Guava EasyCyte microcapillary 96-well cytometer to monitor cell density and health and evaluate membrane protein expression. Two proof of concept experiments were conducted using the human beta(2)-adrenergic receptor (beta(2)AR) and the gap junction protein connexin26 (Cx26) in a baculovirus expression system. First, cell surface expression was used to assess the expression levels of 14 beta(2)AR truncation variants expressed using the Vertiga-IM shaker. Three of these variants were then compared to wild-type beta(2)AR using three metrics: cell surface expression, saturation ligand binding and protein immunoblot analysis of dodecylmaltoside extracted material. Second, a series of systematic Cx26 truncation variants were evaluated for expression by protein immunoblot analysis. The cumulative results for these two systems show that the Vertiga-IM instrument can be used effectively in the parallel insect cell microexpression of membrane protein variants, and that the expression of cell surface molecules as monitored with the Guava EasyCyte instrument can be used to rapidly assess the production of properly folded proteins in the baculovirus expression system. This approach expedites the in vitro evaluation of a large number of mammalian membrane protein variants.     

Substitutions of the Conserved Gly47 Affect the CF1 Inhibitor and Proton Gate Functions of the Chloroplast ATP Synthase ε Subunit

The conserved residue Gly47 of the chloroplast ATP synthase ε subunit was substituted with Leu, Arg, Ala and Glu by site-directed mutagenesis. This process generated the mutants εG47L, εG47R, εG47A and εG47E, respectively. All the ε variants showed lower inhibitory effects on the soluble CF1(-ε) Ca^2 -ATPase compared with wild-type ε. In reduced conditions, εG47E and εG47R had a lower inhibitory effect on the oxidized CF1(-ε) Ca^2 -ATPase compared with wild-type ε. In contrast, εG47L and εG47Aincreased the Ca^2 -ATPase activity of soluble oxidized CF1(-ε). The replacement of Gly47 significantly impaired the interaction between the subunit ε and γ in an in vitro binding assay. Further study showed that all ε variants were more effective in blocking proton leakage from the thylakoid membranes. This enhanced ATP synthesis of the chloroplast and restored ATP synthesis activity of the reconstituted membranes to a level that was more efficient than that achieved by wild-type ε. These results indicate that the conserved Gly47 residue of the ε subunit is very important for maintaining the structure and function of the ε subunitand may affect the interaction between the ε subunit, β subunit of CF1 and subunit Ⅲ of CF0, therebyregulating the ATP hydrolysis and synthesis, as well as the proton translocation role of the subunit Ⅲ of CF0.     

A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance.

Systemic acquired resistance (SAR) is a nonspecific defense response in plants that is associated with an increase in the endogenous level of salicylic acid (SA) and elevated expression of pathogenesis-related (PR) genes. To identify mutants involved in the regulation of PR genes and the onset of SAR, we transformed Arabidopsis with a reporter gene containing the promoter of a beta-1,3-glucanase-encoding PR gene (BGL2) and the coding region of beta-glucuronidase (GUS). The resulting transgenic line (BGL2-GUS) was mutagenized, and the M2 progeny were scored for constitutive GUS activity. We report the characterization of one mutant, cpr1 (constitutive expressor of PR genes), that was identified in this screen and shown by RNA gel blot analysis also to have elevated expression of the endogenous PR genes BGL2, PR-1, and PR-5. Genetic analyses indicated that the phenotype conferred by cpr1 is caused by a single, recessive nuclear mutation and is suppressed in plants producing a bacterial salicylate hydroxylase, which inactivates SA. Furthermore, biochemical analysis showed that the endogenous level of SA is elevated in the mutant. Finally, the cpr1 plants were found to be resistant to the fungal pathogen Peronospora parasitica NOCO2 and the bacterial pathogen Pseudomonas syringae pv maculicola ES4326, which are virulent in wild-type BGL2-GUS plants. Because the cpr1 mutation is recessive and associated with an elevated endogenous level of SA, we propose that the CPR1 gene product acts upstream of SA as a negative regulator of SAR.     

CYP3A5 genotype predicts renal CYP3A activity and blood pressure in healthy adults.

A single-nucleotide polymorphism (A6986G) in the cytochrome p-450 3A5 (CYP3A5) gene distinguishes an expressor (*1) and a reduced-expressor (*3) allele and largely predicts CYP3A5 content in liver and intestine. CYP3A5 is the prevailing CYP3A isoform in kidney. We report that, among renal microsomes from 21 organ donors, those from *1/*3 individuals had at least eightfold higher mean kidney microsomal CYP3A5 content and 18-fold higher mean CYP3A catalytic activity than did those from *3/*3 individuals (P = 0.0001 and P = 0.0137, respectively). We also report significant associations between the A6986G polymorphism and systolic blood pressure (P = 0.0007), mean arterial pressure (P = 0.0075), and creatinine clearance (P = 0.0035) among 25 healthy African-American adults. These associations remained significant when sex, age, and body mass index were taken into account. The mean systolic blood pressure of homozygous CYP3A5 expressors (*1/*1) exceeded that of homozygous nonexpressors (*3/*3) by 19.3 mmHg. We speculate whether a high CYP3A5 expressor allele frequency among African-Americans may contribute to a high prevalence of sodium-sensitive hypertension in this population.     

Delineation of three subsets of class I human T antigens (HTA) on Molt-4 cells: serologic and regulatory relationship to HLA class I antigens

Three subsets of class I human T antigens (HTA) were serologically identified on the surface of the Molt-4 T lymphoma cell line. The HTA 1 subset is defined by NAI/34, D47, or 10H3.9 cross-reactive m.Ab. and by BL6 m.Ab. The HTA 2 and HTA 3 subsets are defined by M241 and 4A7.6 m.Ab., respectively. We obtained no evidence of any additional HTA subset. The different HTA antigens share only few epitopes with human leukocyte antigens (HLA-A, -B, and -C). Interestingly, these epitopes all belong to the same cluster defined on HLA class I molecules, but differ from one HTA subset to another. These results would therefore suggest that HTA and HLA class I antigens display a limited structural homology, but have a conserved epitopic area whose detailed structure differs for each HTA subset. Furthermore, the cell surface expression of each HTA class I molecule type is differently enhanced by natural interferon (IFN)-alpha or -gamma. This result additionally supports the serologic delineation of HTA subsets, and suggests that the corresponding genes in Molt-4 cells, are subjected to distinct regulations.     

Motility of fibronectin receptor-deficient cells on fibronectin and vitronectin: collaborative interactions among integrins.

Cells are capable of adhering to and migrating on protein components of the extracellular matrix. These cell-matrix interactions are thought to be mediated largely through a family of cell surface receptors termed integrins. However, the manner in which individual integrins are involved in cell adhesion and motility has not been fully determined. To explore this issue, we previously selected a series of CHO variants that are deficient in expression of the integrin alpha 5 beta 1, the "classical" fibronectin receptor. Two sets of subclones of these variants were defined which respectively express approximately 20% or 2% of fibronectin receptor on the cell surface when compared to wild-type cells (Schreiner, C. L., J. S. Bauer, Y. N. Danilov, S. Hussein, M. M. Sczekan, and R. L. Juliano. 1989. J. Cell Biol. 109:3157-3167). In the current study, the variant clones were tested for haptotactic motility on substrata coated with fibronectin or vitronectin. Data from assays using fibronectin show that cellular motility of the 20% variants was substantially decreased (30-75% of wild type), while the motility of the 2% variants was nearly abolished (2-20% of wild type). Surprisingly, a similar pattern was seen for haptotactic motility of both 2% and 20% variants when vitronectin was used (approximately 20-30% of wild type). The reduced haptotactic motility of the fibronectin receptor-deficient variant clones on vitronectin was shown not to be due to reduced vitronectin receptor (alpha v beta 3) expression nor to a failure of these variants to adhere to vitronectin substrata. Transfection of the deficient variants with a cDNA for the human alpha 5 subunit resulted in normal levels of fibronectin receptor expression (as a human alpha 5/hamster beta 1 chimera) and restored the motility of the CHO variants on fibronectin and vitronectin. This indicates that expression of the alpha 5 subunit is required for normal haptotactic motility on vitronectin substrata and suggests that the fibronectin receptor (alpha 5 beta 1) plays a cooperative role with vitronectin receptors in cell motility.     

A systematic study of the effect of physiological factors on beta2-microglobulin amyloid formation at neutral pH

Beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit in the musculo-skeletal system in patients undergoing long-term hemodialysis. How beta(2)m self-assembles in vivo is not understood, since the monomeric wild-type protein is incapable of forming fibrils in isolation in vitro at neutral pH, while elongation of fibril-seeds made from recombinant protein has only been achieved at low pH or at neutral pH in the presence of detergents or cosolvents. Here we describe a systematic study of the effect of 11 physiologically relevant factors on beta(2)m fibrillogenesis at pH 7.0 without denaturants. By comparing the results obtained for the wild-type protein with those of two variants (DeltaN6 and V37A), the role of protein stability in fibrillogenesis is explored. We show that DeltaN6 forms low yields of amyloid-like fibrils at pH 7.0 in the absence of seeds, suggesting that this species could initiate fibrillogenesis in vivo. By contrast, high yields of amyloid-like fibrils are observed for all proteins when assembly is seeded with fibril-seeds formed from recombinant protein at pH 2.5 stabilized by the addition of heparin, serum amyloid P component (SAP), apolipoprotein E (apoE), uremic serum, or synovial fluid. The results suggest that the conditions within the synovium facilitate fibrillogenesis of beta(2)m and show that different physiological factors may act synergistically to promote fibril formation. By comparing the behavior of wild-type beta(2)m with that of DeltaN6 and V37A, we show that the physiologically relevant factors enhance fibrillogenesis by stabilizing fibril-seeds, thereby allowing fibril extension by rare assembly competent species formed by local unfolding of native monomers.