Skin and hair pigmentation variation in Island Melanesia

Skin and hair pigmentation are two of the most easily visible examples of human phenotypic variation. Selection-based explanations for pigmentation variation in humans have focused on the relationship between melanin and ultraviolet radiation, which is largely dependent on latitude. In this study, skin and hair pigmentation were measured as the melanin (M) index, using narrow-band reflectance spectroscopy for 1,135 individuals from Island Melanesia. Overall, the results show remarkable pigmentation variation, given the small geographic region surveyed. This variation is discussed in terms of differences between males and females, among islands, and among neighborhoods within those islands. The relationship of pigmentation to age, latitude, and longitude is also examined. We found that male skin pigmentation was significantly darker than females in 5 of 6 islands examined. Hair pigmentation showed a negative, but weak, correlation with age, while skin pigmentation showed a positive, but also weak, correlation with age. Skin and hair pigmentation varied significantly between islands as well as between neighborhoods within those islands. Bougainvilleans showed significantly darker skin than individuals from any other island considered, and are darker than a previously described African-American population. These findings are discussed in relation to prevailing hypotheses about the role of natural selection in shaping pigmentation variation in the human species, as well as the role of demographic processes such as admixture and drift in Island Melanesia.     

Species cohesion of an extremophyte (Carex angustisquama,Cyperaceae) in solfatara fields maintained under interspecific natural hybridization

Background and AimsHybridization is the main driver of plant diversification, and gene flow via hybridization has multifaceted effects on plant evolution. Carex angustisquama is an extremophyte that grows on soils heavily acidified by volcanism. Despite its habitat distinct from that of other species, this species is known to form interspecific hybrids, implying interspecific gene flow. It is crucial to verify the extent and direction of interspecific gene flow between C. angustisquama and closely related species to understand the evolutionary process of an extremophyte in solfatara fields.MethodsIn this study, expressed sequence tag–simple sequence repeat markers were utilized to infer the extent and direction of interspecific gene flow between C. angustisquama and closely related species.Key ResultsBayesian clustering and simulation analyses revealed that all individuals of the three hybrid species were classified into the first hybrid generation or first backcross to C. angustisquama; therefore, current interspecific gene flow is limited. Moreover, in the Bayesian inference of historical gene flow based on multispecies samples, the model that assumed no interspecific gene flow was the most strongly supported across all species pairs, including phylogenetically close but ecologically distinctive species pairs.ConclusionsOur results revealed that interspecific gene flow between C. angustisquama and its related species has been limited both currently and historically. Moreover, our results of Bayesian inference of historical gene flow indicated that extrinsic, rather than intrinsic, factors probably act as isolating barriers between Carex species, with hybrid breakdown via microhabitat segregation being the probable potential barrier. Overall, our findings provide insights into the evolutionary process of an extremophyte in solfatara fields and offer an important example of the mechanisms of diversification of the speciose genus Carex.     

Sequential four-state folding/unfolding of goat α-lactalbumin and its N-terminal variants

Equilibria and kinetics of folding/unfolding of α-lactalbumin and its two N-terminal variants were studied by circular dichroism spectroscopy. The two variants were wild-type recombinant and Glu1-deletion (E1M) variants expressed in Escherichia coli. The presence of an extra methionine at the N terminus in recombinant α-lactalbumin destabilized the protein by 2 kcal/mol, while the stability was recovered in the E1M variant in which Glu1 was replaced by Met1. Kinetic folding/unfolding reactions of the proteins, induced by stopped-flow concentration jumps of guanidine hydrochloride, indicated the presence of a burst-phase in refolding, and gave chevron plots with significant curvatures in both the folding and unfolding limbs. The folding-limb curvature was interpreted in terms of accumulation of the burst-phase intermediate. However, there was no burst phase observed in the unfolding kinetics to interpret the unfolding-limb curvature. We thus assumed a sequential four-state mechanism, in which the folding from the burst-phase intermediate takes place via two transition states separated by a high-energy intermediate. We estimated changes in the free energies of the burst-phase intermediate and two transition states, caused by the N-terminal variations and also by the presence of stabilizing calcium ions. The Φ values at the N terminus and at the Ca(2+)-binding site thus obtained increased successively during folding, demonstrating the validity of the sequential mechanism. The stability and the folding behavior of the E1M variant were essentially identical to those of the authentic protein, allowing us to use this variant as a pseudo-wild-type α-lactalbumin in future studies.     

Fibrillogenic propensity of the GroEL apical domain: a Janus-faced minichaperone

The chaperonin GroEL plays an essential role in promoting protein folding and in protecting against misfolding and aggregation in the cellular environment. In this study, we report that both GroEL and its isolated apical domain form amyloid-like fibrils under physiological conditions, and that the fibrillation of the apical domain is accelerated under acidic conditions. We also found, however, that despite its fibrillation propensity, the apical domain exhibits a pronounced inhibitory effect on the fibril growth of β(2)-microglobulin. Thus, the analysis of the behaviour of the apical domain reveals how aggregation and chaperone-mediated anti-aggregation processes can be closely related.     

P19 embryonal carcinoma cells exhibit high sensitivity to botulinum type C and D/C mosaic neurotoxins

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release at peripheral nerve terminals. They are serologically classified from A to G, C/D and D/C mosaic neurotoxins forming further subtypes of serotypes C and D. Cultured primary neurons, as well as neuronal cell lines such as PC12 and Neuro-2a, are often utilized in cell-based experiments on the toxic action of botulinum toxins. However, there are very few reports of the use of neural cell lines for studying BoNTs/C and D. In addition, the differentiated P19 neuronal cell line, which possesses cholinergic properties, has yet to be tested for its susceptibility to BoNTs. Here, the responsiveness of differentiated P19 cells to BoNT/C and BoNT/DC is reported. Both BoNT/C and BoNT/DC were shown to effectively bind to, and be internalized by, neurons derived from P19 cells. Subsequently, the intracellular substrates for BoNT/C and BoNT/DC were cleaved by treatment of the cells with the toxins in a ganglioside-dependent manner. Moreover, P19 neurons exhibited high sensitivity to BoNT/C and BoNT/DC, to the same extent as cultured primary neurons. These findings suggest that differentiated P19 cells possess full sensitivity to BoNT/C and BoNT/DC, thus making them a novel susceptible cell line for research into BoNTs.     

Interaction between polypeptide 3ABC and the 5'-terminal structural elements of the genome of Aichi virus: implication for negative-strand RNA synthesis

Secondary structural elements at the 5' end of picornavirus genomic RNA function as cis-acting replication elements and are known to interact specifically with viral P3 proteins in several picornaviruses. In poliovirus, ribonucleoprotein complex formation at the 5' end of the genome is required for negative-strand synthesis. We have previously shown that the 5'-end 115 nucleotides of the Aichi virus genome, which are predicted to fold into two stem-loops (SL-A and SL-C) and one pseudoknot (PK-B), act as a cis-acting replication element and that correct folding of these structures is required for negative-strand synthesis. In this study, we investigated the interaction between the 5'-terminal 120 nucleotides of the genome and the P3 proteins, 3AB, 3ABC, 3C, and 3CD, by gel shift assay and Northwestern analysis. The results showed that 3ABC and 3CD bound to the 5'-terminal region specifically. The binding of 3ABC was observed on both assays, while that of 3CD was detected only on Northwestern analysis. No binding of 3AB or 3C was observed. Binding assays using mutant RNAs demonstrated that disruption of the base pairings of the stem of SL-A and one of the two stem segments of PK-B (stem-B1) abolished the 3ABC binding. In addition, the specific nucleotide sequence of stem-B1 was responsible for the efficient 3ABC binding. These results suggest that the interaction of 3ABC with the 5'-terminal region of the genome is involved in negative-strand synthesis. On the other hand, the ability of 3CD to interact with the 5'-terminal region did not correlate with the RNA replication ability.     

Phorbol ester-dependent phosphorylation regulates the association of p57/coronin-1 with the actin cytoskeleton

The p57/coronin-1 protein is a member of the coronin family of actin-binding proteins, which are characterized by the presence of WD (tryptophan/aspartic acid) repeats and a coiled-coil motif in the molecule. It is selectively expressed in immune cells and has been suggested to play crucial roles in leukocyte functions, including cell migration and phagocytosis. In this study we examined the effects of p57/coronin-1 phosphorylation on the association of the protein with actin. Treatment of HL60 human leukemic cells or p57/coronin-1-transfected HEK293 cells with phorbol 12-myristate 13-acetate (PMA) reduced the association of p57/coronin-1 with the actin cytoskeleton, as indicated by cell fractionation experiments and by fluorescence microscopic observation. Two-dimensional gel electrophoresis of HL60 cell lysate revealed that p57/coronin-1 was phosphorylated upon PMA stimulation of the cells, giving two major and two minor spots of phosphorylated forms, each with distinct isoelectric points. The p57/coronin-1 molecules associated with the cytoskeleton in PMA-treated HL60 cells were phosphorylated at lower levels than those recovered in the cytosolic fraction. In addition, p57/coronin-1 co-sedimented with F-actin polymerized in vitro had lower phosphorylation levels than the molecules remaining in the supernatant. By affinity chromatographic analysis using anti-p57/coronin-1 antibody-conjugated Sepharose, p57/coronin-1 derived from PMA-treated HL60 cells showed lower affinity for actin than that from untreated cells. Finally, recovery of p57/coronin-1 in the actin cytoskeleton-rich fraction from neutrophil-like differentiated HL60 cells decreased during phagocytosis, concomitant with enhanced phosphorylation of p57/coronin-1. These results strongly suggest that the phosphorylation of p57/coronin-1 down-regulates its association with actin and modulates the reorganization of actin-containing cytoskeleton.     

Gene cloning and enzymatic characteristics of a novel gamma-cyclodextrin-specific cyclodextrinase from alkalophilic Bacillus clarkii 7364

A new gene, cda, was found in the downstream region of the cgt gene encoding cyclodextrin (CD) glucanotransferase from Bacillus clarkii 7364. Cda encoded by the cda was a cyclodextrinase that has extremely high specificity for gamma-CD. The rates of hydrolysis toward alpha- and beta-CD, maltooctaose and polysaccharides were less than 4% of that toward gamma-CD. Cda also has a transglycosylation activity, by which the maltotriose moiety was transferred from maltohexaose and maltopentaose. The comparison of the amino acid sequences between Cda and CD-degrading enzymes revealed the sequence of Cda has unique features. One of them is Gly247 next to the catalytic nucleophile Asp246. Most enzymes in GH family 13 have more bulky amino acids at this position. Other features in Cda are the lack of the N-domain in CD-degrading enzymes involving in the dimerization contributing to the preference of CDs and the existence of a long extra sequence in the C-terminus. Despite the lack of N-domain, Cda showed a dodecameric structure. The long extra sequence in the C-terminus might contribute to the oligomerization of Cda through a new mechanism. These unique features indicate that Cda is a novel type of CD-degrading enzyme.