Low levels of asparagine deamidation can have a dramatic effect on aggregation of amyloidogenic peptides: Implications for the study of amyloid formation

The polypeptide hormone amylin forms amyloid deposits in Type 2 diabetes mellitus and a 10-residue fragment of amylin (amylin(20-29)) is commonly used as a model system to study this process. Studies of amylin(20-29) and several variant peptides revealed that low levels of deamidation can have a significant effect on the secondary structure and aggregation behavior of these molecules. Results obtained with a variant of amylin(20-29), which has the primary sequence SNNFPAILSS, are highlighted. This peptide is particularly interesting from a technical standpoint. In the absence of impurities the peptide does not spontaneously aggregate and is not amyloidogenic. This peptide can spontaneously deamidate, and the presence of less than 5% of deamidation impurities leads to the formation of aggregates that have the hallmarks of amyloid. In addition, small amounts of deamidated material can induce amyloid formation by the purified peptide. These results have fundamental implications for the definition of an amyloidogenic sequence and for the standards of purity of peptides and proteins used for studies of amyloid formation.     

Temperature-dependent Hammond behavior in a protein-folding reaction: analysis of transition-state movement and ground-state effects

Characterization of the transition-state ensemble and the nature of the free-energy barrier for protein folding are areas of intense activity and some controversy. A key issue that has emerged in recent years is the width of the free-energy barrier and the susceptibility of the transition state to movement. Here we report denaturant-induced and temperature-dependent folding studies of a small mixed α-β protein, the N-terminal domain of L9 (NTL9). The folding of NTL9 was determined using fluorescence-detected stopped-flow fluorescence measurements conducted at seven different temperatures between 11 and 40 °C. Plots of the log of the observed first-order rate constant versus denaturant concentration, “chevron plots,” displayed the characteristic V shape expected for two-state folding. There was no hint of deviation from linearity even at the lowest denaturant concentrations. The relative position of the transition state, as judged by the Tanford β parameter, β_T, shifts towards the native state as the temperature is increased. Analysis of the temperature dependence of the kinetic and equilibrium m values indicates that the effect is due to significant movement of the transition state and also includes a contribution from temperature-dependent ground-state effects. Analysis of the Leffler plots, plots of ΔG^‡versus ΔG°, and their cross-interaction parameters confirms the transition-state movement. Since the protein is destabilized at high temperature, the shift represents a temperature-dependent Hammond effect. This provides independent confirmation of a recent theoretical prediction. The magnitude of the temperature-denaturant cross-interaction parameter is larger for NTL9 than has been reported for the few other cases studied. The implications for temperature-dependent studies of protein folding are discussed.     

Cassette-like variation of restriction enzyme genes in Escherichia coli C and relatives

A surprising result of comparative bacterial genomics has been the large amount of DNA found to be present in one strain but not in another of the same species. We examine in detail one location where gene content varies extensively, the restriction cluster in Escherichia coli. This region is designated the Immigration Control Region (ICR) for the density and variability of restriction functions found there. To better define the boundaries of this variable locus, we determined the sequence of the region from a restrictionless strain, E.coli C. Here we compare the 13.7 kb E.coli C sequence spanning the site of the ICR with corresponding sequences from five E.coli strains and Salmonella typhimurium LT2. To discuss this variation, we adopt the term ‘framework’ to refer to genes that are stable components of genomes within related lineages, while ‘migratory’ genes are transient inhabitants of the genome. Strikingly, seven different migratory DNA segments, encoding different sets of genes and gene fragments, alternatively occupy a single well-defined location in the seven strains examined. The flanking framework genes, yjiS and yjiA, display approximately normal patterns of conservation. The patterns observed are consistent with the action of a site-specific recombinase. Since no nearby gene codes for a likely recombinase of known families, such a recombinase must be of a new family or unlinked.     

Confidence of paternity and paternal care by eastern bluebirds

Male birds are often faced with low confidence of paternityin their mates' offspring, raising the question of how paternalcare covaries with confidence of paternity. We tested the hypothesisthat male eastern bluebirds (Sialia sialis) reduce care of nestlingsin response to experimentally decreased confidence of paternity.Actual paternity, as assessed by DNA fingerprinting, had noeffect on male feeding rates, nor did males reduce care whenconfidence of paternity was experimentally decreased. Malesthat had been removed for 2 days while their mate was fertile(experimental group) fed nestlings at absolute rates similarto those of control males. The proportion of feeding trips providedby males was also similar for control and experimental nests.We found no difference in fledging success and nestling growthbetween experimental and control broods. Seven original residentmales were displaced by previously unbanded males. Althoughthese replacement males appeared to feed nestlings at normalrates, the nests attended by replacement males suffered reducedfledging success compared to control and experimental nests.Overall, we found no evidence that males reduce feeding effortwhen confidence of paternity is experimentally decreased. Malesmay tolerate some reduction in confidence of paternity withoutreducing care if paternal care is crucial to nestling survival.Alternatively, males may assess paternity within a brood usingcues other than their ability to guard their fertile mates.     

Apolipoprotein C3 SstI polymorphism and triglyceride levels in Asian Indians

Background  

A close association between Sst I polymorphism in the 3' untranslated region of the apolipoproteinC3 (APOC3 ) gene and levels of plasma triglycerides (TG) had been reported by different investigators. Hypertriglyceridemia(HTG) is a known risk factor for coronary artery disease (CAD) in the context of Asian Indians. We conducted a study on the relationship between APOC3 SstI polymorphism (S1S1, S1S2 and S2S2 genotypes) and plasma TG levels in a group of 139 male healthy volunteers from Northern India.     

Discovery and distribution of super-integrons among Pseudomonads

Until recently, integrons (systems for acquisition and expression of new genetic materials) have been associated generally with antibiotic resistance gene cassettes. The discovery of 'super-integrons' in Vibrionaceae suggests a greater impact of this gene acquisition mechanism on bacterial genome evolution than initially believed. Super-integrons may contain more than 100 gene cassettes and may encode other determinants, including biochemical functions or virulence factors. Here, we report the genetic organization of a super-integron from Pseudomonas alcaligenes ATCC 55044. This is the first evidence of a super-integron in a non-pathogenic bacterium, one which is widely distributed in a great number of ecological niches such as soil and aquatic habitats. Here, the sequence composition, open reading frame (ORF) content and organization of In55044 are described and found to have features intermediate between the multidrug-resistant integrons and the Vibrio cholerae super-integron. Similar structures are inferred to be present in several Pseudomonas species, based on polymerase chain reaction (PCR) experiments.     

Positive selection of nodulation-deficient Rhizobium phaseoli.

A high proportion of Rhizobium phaseoli mutants that survived infection with phage F1 were found to be nodulation deficient. Two that were examined in detail had internal defects in addition to the expected surface defects. One internal defect was in the enzyme phosphoglucose isomerase. The use of phages to select appropriate mutants should apply generally in any system in which surface components are involved.     

Intramolecular cyclization in irradiated nucleic acids: correlation between high-performance liquid chromatography and an immunochemical assay for 8,5'-cycloadenosine in irradiated poly(A)

A correlation between high-performance liquid chromatography (HPLC) analysis and an in situ enzyme-linked immunosorbent assay (ELISA) for 8,5'-cycloadenosine formation in irradiated poly(A) has been established. The correlation shows that the ELISA precisely reflects changes in the combined yield of R- and S-8,5'-cycloadenosine but that a correction factor must be applied to the ELISA values for accuracy. The HPLC analysis reveals that the intramolecular cyclization proceeds stereoselectively in irradiated poly(A) to preferentially produce the R isomer at pH 7.0 which is similar to the result for irradiated adenosine but in contrast to the result for 5'-AMP where the S isomer predominates at neutral pH. The HPLC analysis shows that two events originating in hydroxyl radical attack at the sugar phosphate backbone in poly(A); that is, adenine release and 8,5'-cycloadenosine formation have somewhat different dose-yield responses. The formation of 8-hydroxyadenosine was detected in the HPLC chromatograms of poly(A) irradiated under N2O at neutral pH, and the yield of this compound was similar to the yield observed in 5'-AMP or adenosine irradiated under similar conditions.