The Barrett's antigen anterior gradient-2 silences the p53 transcriptional response to DNA damage

The esophageal epithelium is subject to damage from bile acid reflux that promotes normal tissue injury resulting in the development of Barrett's epithelium. There is a selection pressure for mutating p53 in this preneoplastic epithelium, thus identifying a physiologically relevant model for discovering novel regulators of the p53 pathway. Proteomic technologies were used to identify such p53 regulatory factors by identifying proteins that were overexpressed in Barrett's epithelium. A very abundant polypeptide selectively expressed in Barrett's epithelium was identified as anterior gradient-2. Immunochemical methods confirmed that anterior gradient-2 is universally up-regulated in Barrett's epithelium, relative to normal squamous tissue derived from the same patient. Transfection of the anterior gradient-2 gene into cells enhances colony formation, similar to mutant oncogenic p53 encoded by the HIS175 allele, suggesting that anterior gradient-2 can function as a survival factor. Deletion of the C-terminal 10 amino acids of anterior gradient-2 neutralizes the colony enhancing activity of the gene, suggesting a key role for this domain in enhancing cell survival. Constitutive overexpression of anterior gradient-2 does not alter cell-cycle parameters in unstressed cells, suggesting that this gene is not directly modifying the cell cycle. However, cells overexpressing anterior gradient-2 attenuate p53 phosphorylation at both Ser(15) and Ser(392) and silence p53 transactivation function in ultraviolet (UV)-damaged cells. Deletion of the C-terminal 10 amino acids of anterior gradient-2 permits phosphorylation at Ser(15) in UV-damaged cells, suggesting that the C-terminal motif promoting colony survival also contributes to suppression of the Ser(15) kinase pathway. These data identify anterior gradient-2 as a novel survival factor whose study may shed light on cellular pathways that attenuate the tumor suppressor p53.     

Insulin at pH 2: structural analysis of the conditions promoting insulin fibre formation

When insulin solutions are subjected to acid, heat and agitation, the normal pattern of insulin assembly (dimers-->tetramers-->hexamers) is disrupted; the molecule undergoes conformational changes allowing it to follow an alternative aggregation pathway (via a monomeric species) leading to the formation of insoluble amyloid fibres. To investigate the effect of acid pH on the conformation and aggregation state of the protein, the crystal structure of human insulin at pH 2.1 has been determined to 1.6 A resolution. The structure reveals that the native fold is maintained at low pH, and that the molecule is still capable of forming dimers similar to those found in hexameric insulin structures at higher pH. Sulphate ions are incorporated into the molecule and the crystal lattice where they neutralise positive charges on the protein, stabilising its structure and facilitating crystallisation. The sulphate interactions are associated with local deformations in the protein, which may indicate that the structure is more plastic at low pH. Transmission electron microscopy analysis of insulin fibres reveals that the appearance of the fibres is greatly influenced by the type of acid employed. Sulphuric acid produces distinctive highly bunched, truncated fibres, suggesting that the sulphate ions have a sophisticated role to play in fibre formation, rather as they do in the crystal structure. Analytical ultracentrifugation studies show that in the absence of heating, insulin is predominantly dimeric in mineral acids, whereas in acetic acid the equilibrium is shifted towards the monomer. Hence, the effect of acid on the aggregation state of insulin is also complex. These results suggest that acid conditions increase the susceptibility of the molecule to conformational change and dissociation, and enhance the rate of fibrillation by providing a charged environment in which the attractive forces between the protein molecules is increased.     

Evolution and virulence of serogroup 6 pneumococci on a global scale

To study the evolution and virulence of pneumococcal populations, we used multilocus sequence typing to identify the major clones among 212 carriage and invasive isolates expressing capsular serogroup 6 from 39 countries. The global population consisted of 8 major complexes and 6 minor complexes of related clones and 32 clones of diverse origin. Surprisingly, serotype 6A clones evolved by mutation nearly as often as by recombination, whereas serotype 6B clones evolved almost exclusively by recombination (P = 0.0029). This is the first report of population genetic differences among serotypes of this species. The largest clonal complex was associated with invasive disease (P = 0.019) and included a common ancestor for five previously identified drug-resistant clones. The putative ancestors of the major clonal complexes were represented by a greater proportion of carriage isolates than were their descendents (P = 0.001), and the ancestors tended to be less virulent than their descendents in a mouse model of infection. These data suggested that virulent serogroup 6 clones have evolved multiple times from less-virulent ancestral clones.     

Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists,imiquimod and resiquimod

The immune response modifiers, imiquimod and resiquimod, are TLR7 agonists that induce type I interferon in numerous species, including humans. Recently, it was shown that plasmacytoid dendritic cells (pDC) are the primary interferon-producing cells in the blood in response to viral infections. Here, we characterize the activation of human pDC with the TLR7 agonists imiquimod and resiquimod. Results indicate that imiquimod and resiquimod induce IFN-alpha and IFN-omega from purified pDC, and pDC are the principle IFN-producing cells in the blood. Resiquimod-stimulated pDC also produce a number of other cytokines including TNF-alpha and IP-10. Resiquimod enhances co-stimulatory marker expression, CCR7 expression, and pDC viability. Resiquimod was compared throughout the study to the pDC survival factors, IL-3 and IFN-alpha; resiquimod more effectively matures pDC than either IL-3 or IFN-alpha alone. These results demonstrate that imidazoquinoline molecules directly induce pDC maturation as determined by cytokine induction, CCR7 and co-stimulatory marker expression and prolonging viability.     

Limited hybridization between Quercus lobata and Quercus douglasii (Fagaceae) in a mixed stand in central coastal California

Many oak species are interfertile, and morphological and genetic evidence for hybridization is widespread. Here we use DNA microsatellite markers to characterize hybridization between two closely related oak species in a mixed stand in central coastal California, Quercus lobata (valley oak) and Q. douglasii (blue oak) (Fagaceae). Genotypes from four microsatellite loci indicate that many alleles are shared between the two species. However, each species harbors unique alleles, and allele frequencies differ significantly. A Bayesian analysis of genetic structure in the stand identified two highly differentiated genetic clusters, essentially corresponding to species assignment based on morphology. Data from the four loci were sufficient to assign all 135 trees to one of the two species. In addition, five putative hybrid individuals having intermediate morphologies could be assigned genetically to one or the other species, and all but one had low probability of hybrid ancestry. Overally, only six (4.6%) trees showed >0.05 probability of hybrid ancestry, in all cases their probabilities for nonhybrid ancestry were substantially higher. We conclude that adult hybrids of Q. douglasii × Q. lobata are rare at this site and plasticity in morphological characters may lead to overestimates of hybridization among Quercus species.     

Sialylation is modulated through maturation in hemocytes from Macrobrachium rosenbergii

In this work we identified in adult and juvenile freshwater prawn, Macrobrachium rosenbergii, three major type of circulating hemocytes: fusiform; rounded; and large ovoid hemocytes. Rounded and large hemocytes represent the first defense line, since this type of cells exerts phagocytic activity as well as lectin synthesis. Considering that glycosylation plays important roles in cell communication and as a target for pathogenic microorganisms, in this report was also described the main glycosidic modifications that occur in the large and rounded hemocytes from the freshwater prawn during maturation as determined with lectins. Neu5Acalpha2,6Gal, was identified homogeneously distributed in the membrane in 90% of hemocytes from juvenile organisms. Maturation of the freshwater prawn induced a decrease or complete loss of Neu5Acalpha2,6Gal residues that were replaced with Neu5Acalpha2,3 molecules in practically all hemocytes from adult organisms. This change was paralleled by a diminution in 9-O-acetyl-neuraminic acid (Neu5,9Ac(2)) expression. T and Tn antigens (Galbetal,3 GalNAcalpha1-0-Ser/Thr or GalNAcalpha1-0-Ser/Thr, respectively), as well as N-glycosidically linked glycans, seem to be highly conserved throughout maturation. Our results show that sialylation of freshwater prawn hemocytes is modulated throughout the maturation process.     

Microevolution in island rodents

We perform a meta-analysis on morphological data from four island rodent populations exhibiting microevolution (>100 years). Data consisting of incidences of skeletal variants, cranial, and external measurements are from house mice (Mus musculus) on one Welsh and one Scottish island, black rats (Rattus rattus) on two Galapagos islands, and deer mice (Peromyscus maniculatus) on three California Channel islands. We report extremely high rates of microevolution for many traits; 60% of all mensural traits measured changed at a rate of 600 d or greater (max. 2682 d). The proportion of all mensural traits evolving at 600–800 d (23%) was idiosyncratic and departed from an expected negative exponential distribution. We argue that selection, rather than founder events, is largely responsible for the substantial shifts in morphology seen among insular rodents. Examining individual traits, there is a trend towards the nose becoming longer and wider, while the skull becomes shallower, shown by both rats and mice on five different islands. We found a significant correlation between island size and degree of skeletal variant evolution and between island distance from the mainland (or nearest island) and degree of cranial and external character evolution. Thus, microevolution of rodents is greater on smaller and more remote islands.     

Population genetic structure of the lemon shark (Negaprion brevirostris) in the western Atlantic: DNA microsatellite variation

DNA microsatellite markers were used to characterize the population genetic structure of the lemon shark, Negaprion brevirostris, in the western Atlantic. This study demonstrates for the first time the usefulness of microsatellites to study population genetic structure and mating systems in the Chondricthyes. Lemon sharks (mostly juveniles) were sampled non-destructively from four locations, Gullivan Bay and Marquesas Key in Florida, Bimini, Bahamas, and Atol das Rocas, Brazil. At least 545 individuals were genotyped at each of four dinucleotide loci. The number of alleles per locus ranged from 19 to 43, and expected heterozygosities ranged from 0.69 to 0.90. Relatively little genetic structure was found in the western Atlantic, with small but significant values for estimators of F(ST) and R(ST) among populations, theta (0.016) and rho (0.026), respectively. No sharp discontinuities were found between the Caribbean sites and Brazil, and most alleles were found at all four sites, indicating that gene flow occurs throughout the western Atlantic with no evidence for distinct stocks.     

MSH4 acts in conjunction with MLH1 during mammalian meiosis.

MSH4 is a meiosis-specific MutS homolog. In yeast, it is required for reciprocal recombination and proper segregation of homologous chromosomes at meiosis I. MLH1 (MutL homolog 1) facilitates both mismatch repair and crossing over during meiosis in yeast. Germ-line mutations in the MLH1 human gene are responsible for hereditary nonpolyposis cancer, but the analysis of MLH1-deficient mice has revealed that MLH1 is also required for reciprocal recombination in mammals. Here we show that hMSH4 interacts with hMLH1. The two proteins are coimmunoprecipitated regardless of the presence of DNA or ATP, suggesting that the interaction does not require the binding of MSH4 to DNA. The domain of hMSH4 responsible for the interaction is in the amino-terminal part of the protein whereas the region that contains the ATP binding site and helix-turn-helix motif does not bind to hMLH1. Immunolocalization analysis shows that MSH4 is present at sites along the synaptonemal complex as soon as homologous chromosomes synapse. The number of MSH4 foci decreases gradually as pachynema progresses. During this transition, MLH1 foci begin to appear and colocalize with MSH4. These results suggest that MSH4 is first required for chromosome synapsis and that this MutS homologue is involved later with MLH1 in meiotic reciprocal recombination.