Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.      

大鼠胼胝体内神经肽Y免疫反应阳性纤维的发育

本实验用免疫组织化学ＡＢＣ法研究了大鼠胼胝体内神经肽Ｙ免疫反应阳性（ＮＰＹ－ＩＲ）纤维的生后发育。结果发现,许多ＮＰＹ－ＩＲ纤维在大鼠出生时便存在于胼胝体内。ＮＰＹ－ＩＲ胼胝体纤维的密度在生后１周内继续逐渐增高,在第２周内达到最高峰。之后,ＮＰＹ－ＩＲ胼胝体纤维的密度逐渐下降,至第３周末时接近成年时的水平,即仅有少量ＮＰＹ－ＩＲ纤维存在于胼胝体内。这些结果提示在大鼠早期生后发育过程中许多ＮＰＹ－ＩＲ胼胝体纤维是暂时性的,其作用可能与大脑皮质的机能发育有关。     

Purification and characterization of ferulate and p-coumarate decarboxylase from Bacillus pumilus.

Bacillus pumilus PS213 isolated from bovine ruminal fluid was able to transform ferulic acid and p-coumaric acid to 4-vinylguaiacol and 4-vinylphenol, respectively, by nonoxidative decarboxylation. The enzyme responsible for this activity has been purified and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extract from a culture induced by ferulic acid or p-coumaric acid shows three bands that are not present in the crude extract of an uninduced culture, while the purified enzyme shows a single band of 23 kDa; the molecular mass calculated by size exclusion chromatography is 45 kDa. Enzyme activity is optimal at 37 degrees C and pH 5.5 and is not enhanced by any cation. Kinetic studies indicated a Km of 1.03 mM and a Vmax of 0.19 mmol.min-1/mg.liter-1 for ferulic acid and a Km of 1.38 mM and a Vmax of 0.22 mmol.min-1/mg.liter-1 for p-coumaric acid.     

The distribution of residues in a polypeptide sequence is a determinant of aggregation optimized by evolution

It has been shown that the propensity of a protein to form amyloid-like fibrils can be predicted with high accuracy from the knowledge of its amino acid sequence. It has also been suggested, however, that some regions of the sequences are more important than others in determining the aggregation process. Here, we have addressed this issue by constructing a set of “sequence scrambled” variants of the first 29 residues of horse heart apomyoglobin (apoMb_1-29), in which the sequence was modified while maintaining the same amino acid composition. The clustering of the most amyloidogenic residues in one region of the sequence was found to cause a marked increase of the elongation rate (k_agg) and a remarkable shortening of the lag phase (t_lag) of the fibril growth, as determined by far-UV circular dichroism and thioflavin T fluorescence. We also show that taking explicitly into consideration the presence of aggregation-promoting regions in the predictive methods results in a quantitative agreement between the theoretical and observed k_agg and t_lag values of the apoMb_1-29 variants. These results, together with a comparison between homologous segments from the family of globins, indicate the existence of a negative selection against the clustering of highly amyloidogenic residues in one or few regions of polypeptide sequences.     

Limited proteolysis of bovine alpha-lactalbumin: isolation and characterization of protein domains

The partly folded states of alpha-lactalbumin (alpha-LA) exposed to acid solution at pH 2.0 (A-state) or at neutral pH upon EDTA-mediated removal of the single protein-bound calcium ion (apo form) have been probed by limited proteolysis experiments. These states are nowadays commonly considered to be molten globules and thus protein-folding intermediates. Pepsin was used for proteolysis at acid pH, while proteinase K and chymotrypsin at neutral pH. The expectations were that these proteolytic probes would detect sites and/or chain regions in the partly folded states of alpha-LA sufficiently dynamic, or even unfolded, capable of binding and adaptation to the specific stereochemistry of the protease's active site. A time-course analysis of the proteolytic events revealed that the fast, initial proteolytic cuts of the 123-residue chain of alpha-LA in its A-state or apo form by the three proteases occur at the same chain region 39-54, the actual site(s) of cleavage depending upon the protease employed. This region in native alpha-LA encompasses the beta-sheets of the protein. Subsequent cleavages occur mostly at chain regions 31-35 and 95-105. Four fragment species of alpha-LA have been isolated by reverse-phase high-performance liquid chromatography, and their conformational properties examined by circular dichroism and fluorescence emission spectroscopy. The single chain fragment 53-103, containing all the binding sites for calcium in native alpha-LA and cross-linked by two disulfide bridges, maintains in aqueous buffer and in the presence of calcium ions a folded structure characterized by the same content of alpha-helix of the corresponding chain segment in native alpha-LA. Evidence for some structure was also obtained for the two-chain species 1-40 and 104-123, as well as 1-31 and 105-123, both systems being covalently linked by two disulfide bonds. In contrast, the protein species given by fragment 1-34 connected to fragment 54-123 or 57-123 via four disulfide bridges adopts in solution a folded structure with the helical content expected for a native-like conformation. Of interest, the proteolytic fragment species herewith isolated correspond to the structural domains and subdomains of alpha-LA that can be identified by computational analysis of the three-dimensional structure of native alpha-LA (Siddiqui AS, Barton GI, 1995, Protein Sci 4:872-884). The fast, initial cleavages at the level of the beta-sheet region of native alpha-LA indicate that this region is highly mobile or even unfolded in the alpha-LA molten globule(s), while the rest of the protein chain maintains sufficient structure and rigidity to prevent extensive proteolysis. The subsequent cleavages at chain segment 95-105 indicate that also this region is somewhat mobile in the A-state or apo form of the protein. It is concluded that the overall domain topology of native alpha-LA is maintained in acid or at neutral pH upon calcium depletion. Moreover, the molecular properties of the partly folded states of alpha-LA deduced here from proteolysis experiments do correlate with those derived from previous NMR and other physicochemical measurements.     

Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis

The SH3 domains are small protein modules of 60-85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The SH3 domain of the p85alpha subunit of bovine phosphatidylinositol 3'-kinase (PI3-SH3) under acidic solution adopts a compact denatured state from which amyloid fibrils are readily formed. This aggregation process has been found to be modulated substantially by solution conditions. Here, we have analyzed the conformational features of the native and acid denatured states of PI3-SH3 by limited proteolysis experiments using proteinase K and pepsin, respectively. Moreover, we have analyzed the propensity of PI3-SH3 to be hydrolyzed by pepsin at different stages in the process of aggregation and amyloid formation at pH 1.2 and 2.0 and compared the sites of proteolysis under these conditions with the conformational features of both native and aggregated PI3-SH3. The results demonstrate that the denatured state of PI3-SH3 formed at low pH is relatively resistant to proteolysis, indicating that it is partially folded. The long loop connecting beta-strands b and c in the native protein is the region in this structure most susceptible to proteolysis. Remarkably, aggregates of PI3-SH3 that are formed initially from this denatured state in acid solution display enhanced susceptibility to proteolysis of the long loop, suggesting that the protein becomes more unfolded in the early stages of aggregation. By contrast, the more defined amyloid fibrils that are formed over longer periods of time are completely resistant to proteolysis. We suggest that the protein aggregates formed initially are relatively dynamic species that are able readily to reorganize their interactions to enable formation of very well ordered fibrillar structures. In addition, the disordered and non-native character of the polypeptide chains in the early aggregates could be important in determining the high cytotoxicity that has been revealed in previous studies of these species.     

An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis

The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site.