Haplotypes in the dystrophin DNA segment point to a mosaic origin of modern human diversity

Although Africa has played a central role in human evolutionary history, certain studies have suggested that not all contemporary human genetic diversity is of recent African origin. We investigated 35 simple polymorphic sites and one T(n) microsatellite in an 8-kb segment of the dystrophin gene. We found 86 haplotypes in 1,343 chromosomes from around the world. Although a classical out-of-Africa topology was observed in trees based on the variant frequencies, the tree of haplotype sequences reveals three lineages accounting for present-day diversity. The proportion of new recombinants and the diversity of the T(n) microsatellite were used to estimate the age of haplotype lineages and the time of colonization events. The lineage that underwent the great expansion originated in Africa prior to the Upper Paleolithic (27,000-56,000 years ago). A second group, of structurally distinct haplotypes that occupy a central position on the tree, has never left Africa. The third lineage is represented by the haplotype that lies closest to the root, is virtually absent in Africa, and appears older than the recent out-of-Africa expansion. We propose that this lineage could have left Africa before the expansion (as early as 160,000 years ago) and admixed, outside of Africa, with the expanding lineage. Contemporary human diversity, although dominated by the recently expanded African lineage, thus represents a mosaic of different contributions.     

Macrophage recognition of externalized phosphatidylserine and phagocytosis of apoptotic Jurkat cells--existence of a threshold

Phosphatidylserine (PS) is predominantly confined to the inner leaflet of plasma membrane in cells, but it is externalized on the cell surface during apoptosis. This externalized PS is required for effective phagocytosis of apoptotic cells by macrophages. Because PS trans-bilayer asymmetry is not absolute in different types of nonapoptotic cells, we hypothesized that the amounts of externalized PS may be critical for macrophage discrimination between apoptotic and nonapoptotic cells. We developed a sensitive electron paramagnetic resonance method to quantify the amounts of externalized PS based on specific binding of paramagnetic annexin V-microbead conjugates with PS on cell surfaces. Using this technique, we found that nonapoptotic Jurkat cells externalize 0.9 pmol of endogenous PS/10(6) Jurkat cells. For cells with different amounts of integrated exogenous PS on their surface, no phagocytic response was observed at PS levels <5 pmol/10(6) Jurkat cells; at higher PS concentrations, phagocytosis increased in a concentration-dependent manner. Apoptosis in Jurkat cells caused externalization of approximately 240 pmol PS/10(6) Jurkat cells; these amounts of externalized PS are manyfold higher than the threshold amounts of PS required for phagocytosis. Thus, macrophages have a sensitivity threshold for PS externalized on the cell surface that provides for reliable recognition and distinction between normal cells with low contents of externalized PS and apoptotic cells with remarkably elevated PS levels.     

Cloning,expression, and assembly of sericin-like protein

Recombinant sericin proteins of different molecular masses (17.4, 31.9, and 46.5 kDa), based on the 38-amino acid repetitive motif of native sericin, were cloned, expressed, and purified. The recombinant sericin self-assembled during dialysis (starting concentration of 2.5 mg/ml) forming twisted fibers. Circular dichroism and Fourier transform infrared spectroscopy studies demonstrated protein conformational transitions occurred from random coil to beta-sheets during the dialysis. Congo red-stained recombinant sericin fibrils exhibited apple-green birefringence, indicating long-range order in the array of beta-sheets. Biosynthetic sericin has a high content of polar amino acids (e.g. > 40 mol % serine), leading to a beta-sheet conformation formed by hydrogen bonding via polar zipper interactions. Analysis of recombinant sericin sequence using Mandel-Gutfreund's (Mandel-Gutfreund, Y., and Gregoret, L. M. (2002) J. Mol. Biol. 323, 453-461) definition of polar and non-polar amino acids showed that the hydrophobicity pattern resembles the most frequent pattern of amyloidogenic proteins, polar amino acid aggregates (PPPPP). Many beta-proteins and peptides are designed to study amyloidogenesis using a polar/non-polar alternating pattern (PNPNPN). Sericin-like proteins or peptides provide an alternative model in terms of hydrophobicity pattern with which to explore questions related to beta-sheet formation and amyloidogenesis. The glue-like property of sericin is attributed to the hydrogen bonding between serine residues of sericin with serine residues in the fibroin structural components of silk fiber.     

Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells

Low energy visible light (LEVL) irradiation has been shown to exert some beneficial effects on various cell cultures. For example, it increases the fertilizing capability of sperm cells, promotes cell proliferation, induces sprouting of neurons, and more. To learn about the mechanism of photobiostimulation, we studied the relationship between increased intracellular calcium ([Ca2+]i) and reactive oxygen species production following LEVL illumination of cardiomyocytes. We found that visible light causes the production of O2. and H2O2 and that exogenously added H2O2 (12 microm) can mimic the effect of LEVL (3.6 J/cm2) to induce a slow and transient increase in [Ca2+]i. This [Ca2+]i elevation can be reduced by verapamil, a voltage-dependent calcium channel inhibitor. The kinetics of [Ca2+]i elevation and morphologic damage following light or addition of H2O2 were found to be dose-dependent. For example, LEVL, 3.6 J/cm2, which induced a transient increase in [Ca2+]i, did not cause any cell damage, whereas visible light at 12 J/cm2 induced a linear increase in [Ca2+]i and damaged the cells. The linear increase in [Ca2+]i resulting from high energy doses of light could be attenuated into a non-linear small rise in [Ca2+]i by the presence of extracellular catalase during illumination. We suggest that the different kinetics of [Ca2+]i elevation following various light irradiation or H2O2 treatment represents correspondingly different adaptation levels to oxidative stress. The adaptive response of the cells to LEVL represented by the transient increase in [Ca2+]i can explain LEVL beneficial effects.     

Chromosome instability in colorectal tumor cells is associated with defects in microtubule plus-end attachments caused by a dominant mutation in APC

The attachment of microtubule plus ends to kinetochores and to the cell cortex is essential for the fidelity of chromosome segregation. Here, we characterize the causes underlying the high rates of chromosome instability (CIN+) observed in colorectal tumor cells. We show that CIN+ tumor cells exhibit inefficient microtubule plus-end attachments during mitosis, accompanied by impairment of chromosome alignment in metaphase. The mitotic abnormalities associated with CIN+ tumor cells correlated with status of adenomatous polyposis coli (APC). Importantly, we have shown that a single truncating mutation in APC, similar to mutations found in tumor cells, acts dominantly to interfere with microtubule plus-end attachments and to cause a dramatic increase in mitotic abnormalities. We propose that APC functions to modulate microtubule plus-end attachments during mitosis, and that a single mutant APC allele predisposes cells to increased mitotic abnormalities, which may contribute to tumor progression.     

Conversion of monophasic to recurrent autoimmune disease by autoreactive T cell subsets

Autoimmune uveitis has been elicited in susceptible rodents by several ocular-specific Ags. In most of these animal models the induced uveitis is acute and monophasic. Because recurrent uveitis poses the highest risk for blinding ocular complications in human disease, a spontaneous relapsing animal model would be most helpful in understanding the disease pathogenesis. In our current study we have observed that the adoptive transfer of interphotoreceptor retinoid-binding protein residues 1177-1191-specific T cells to naive Lewis rats induced a chronic relapsing disease, in contrast to the monophasic disease induced by immunization with interphotoreceptor retinoid-binding protein residues 1177-1191 emulsified in CFA. The chronic relapsing uveitis induced by autoreactive T cell subsets is dependent on the number of autoreactive T cells generated as well as their activation status. Our study documented a spontaneous model of recurrent uveitis in the rat, which should assist us in the study of disease pathogenesis and the design of specific therapy.     

CpG-containing oligodeoxynucleotide 1826 converts the weak uveitogenic rat interphotoreceptor retinoid-binding protein peptide 1181-1191 into a strong uveitogen

Aberrant activation of autoreactive T cells is one of the major causes of autoimmune disease. Autoantigens are sequestered and in many cases weak immunogens. For example, in experimental autoimmune uveitis, immunization of naive rats with autologous interphotoreceptor retinoid-binding protein (IRBP) fails to induce intraocular inflammation or a strong T cell response, whereas bovine IRBP is a strong inducer of experimental autoimmune uveitis. Such observations challenge the view that the autoantigen alone is responsible for the development of autoimmunity. Here, we demonstrate that autologous rat IRBP is converted to a strong immunogen in the presence of a small dose of CpG-containing oligodeoxynucleotides. Our results indicate that specific CpG-containing oligodeoxynucleotides may play an important role in the activation and expansion of autoreactive T cells in vivo, leading to autoimmune disease.     

Enhanced photosynthesis and growth of transgenic plants that express ictB, a gene involved in HCO3- accumulation in cyanobacteria

Transgenic Arabidopsis thaliana and Nicotiana tabacum plants that express ictB, a gene involved in HCO3- accumulation within the cyanobacterium Synechococcus sp. PCC 7942, exhibited significantly faster photosynthetic rates than the wild-types under limiting but not under saturating CO2 concentrations. Under conditions of low relative humidity, growth of the transgenic A. thaliana plants was considerably faster than the wild-type. This enhancement of growth was not observed under humid conditions. There was no difference in the amount of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) detected in the wild-types and their respective transgenic plants. Following activation in vitro, the activities of RubisCO from either low- or high-humidity-grown transgenic plants were similar to those observed in the wild-types. In contrast, the in vivo RubisCO activity, i.e. without prior activation, in plants grown under low humidity was considerably higher in ictB-expressing plants than in their wild-types. The CO2 compensation point in the transgenic plants that express ictB was lower than in the wild-types, suggesting that the concentration of CO2 in close proximity to RubisCO was higher. This may explain the higher activation level of RubisCO and enhanced photosynthetic activities and growth in the transgenic plants. These data indicated a potential use of ictB for the stimulation of crop yield.