The exon A (C77G) mutation is a common cause of abnormal CD45 splicing in humans

The leukocyte common (CD45) Ag is essential for normal T lymphocyte function and alternative splicing at the N terminus of the gene is associated with changes in T cell maturation and differentiation. Recently, a statistically significant association was reported in a large series of human thymus samples between phenotypically abnormal CD45 splicing and the presence of the CC chemokine receptor 5 deletion 32 (CCR5del32) allele, which confers resistance to HIV infection in homozygotes. We show here that abnormal splicing in these thymus samples is associated with the presence of the only established cause of CD45 abnormal splicing, a C77G transversion in exon A. In addition we have examined 227 DNA samples from peripheral blood of healthy donors and find no association between the exon A (C77G) and CCR5del32 mutations. Among 135 PBMC samples, tested by flow cytometric analysis, all those exhibiting abnormal splicing of CD45 also showed the exon A C77G transversion. We conclude that the exon A (C77G) mutation is a common cause of abnormal CD45 splicing and that further disease association studies of this mutation are warranted.     

Genetic evidence for the proto-Austronesian homeland in Asia: mtDNA and nuclear DNA variation in Taiwanese aboriginal tribes.

Previous studies of mtDNA variation in indigenous Taiwanese populations have suggested that they held an ancestral position in the spread of mtDNAs throughout Southeast Asia and Oceania (Melton et al. 1995; Sykes et al. 1995), but the question of an absolute proto-Austronesian homeland remains. To search for Asian roots for indigenous Taiwanese populations, 28 mtDNAs representative of variation in four tribal groups (Ami, Atayal, Bunun, and Paiwan) were sequenced and were compared with each other and with mtDNAs from 25 other populations from Asia and Oceania. In addition, eight polymorphic Alu insertion loci were analyzed, to determine if the pattern of mtDNA variation is concordant with nuclear DNA variation. Tribal groups shared considerable mtDNA sequence identity (P>.90), where gene flow is believed to have been low, arguing for a common source or sources for the tribes. mtDNAs with a 9-bp deletion have considerable mainland-Asian diversity and have spread to Southeast Asia and Oceania through a Taiwanese bottleneck. Only four Taiwanese mtDNA haplotypes without the 9-bp deletion were shared with any other populations, but these shared types were widely dispersed geographically throughout mainland Asia. Phylogenetic and principal-component analyses of Alu loci were concordant with conclusions from the mtDNA analyses; overall, the results suggest that the Taiwanese have temporally deep roots, probably in central or south China, and have been isolated from other Asian populations in recent history.     

Myeloperoxidase-derived reactive chlorinating species from human monocytes target plasmalogens in low density lipoprotein

A role for myeloperoxidase (MPO) in atherosclerosis has received considerable attention recently. To identify potential chlorinated lipid products in human low density lipoprotein (LDL), studies were designed to demonstrate that MPO-derived reactive chlorinating species (RCS) target the plasmalogen pool of LDL isolated from peripheral human blood in vitro. The vinyl ether bond of LDL plasmalogens was targeted by MPO-derived RCS, resulting in the release of the 16- and 18-carbon-containing alpha-chloro fatty aldehydes, 2-chlorohexadecanal and 2-chlorooctadecanal, respectively, from the plasmalogen glycerol backbone. Targeting of the LDL plasmalogen vinyl ether bond was dependent on the presence of MPO-derived RCS. Electrospray ionization mass spectrometric analysis of MPO-treated LDL demonstrated that a novel population of unsaturated lysophosphatidylcholine molecular species was produced by a phospholipase A2-independent mechanism. Unsaturated lysophosphatidylcholine molecular species elicited cyclic AMP response element binding protein phosphorylation in RAW 264.7 cells. Additionally, MPO-mediated targeting of both monocyte and LDL plasmalogen pools was demonstrated in phorbol myristate acetate-stimulated human monocytes, resulting in the production of both 2-chlorohexadecanal and 2-chlorooctadecanal. In contrast, alpha-chloro fatty aldehydes were not produced in phorbol myristate acetate-stimulated mouse monocytes. Collectively, the present studies demonstrate a novel MPO-specific mechanism that mediates the production of a novel group of unsaturated lysophosphatidylcholine molecular species and chlorinated aldehydes from both LDL and monocyte plasmalogen pools that may have important effects during inflammatory reactions mediated by monocytes, most notably atherosclerosis.     

Genetic basis for rapidly evolved tolerance in the wild: adaptation to toxic pollutants by an estuarine fish species

Atlantic killifish (Fundulus heteroclitus) residing in some urban and industrialized estuaries of the US eastern seaboard demonstrate recently evolved and extreme tolerance to toxic aryl hydrocarbon pollutants, characterized as dioxin‐like compounds (DLCs). Here, we provide an unusually comprehensive accounting (69%) through quantitative trait locus (QTL) analysis of the genetic basis for DLC tolerance in killifish inhabiting an urban estuary contaminated with PCB congeners, the most toxic of which are DLCs. Consistent with mechanistic knowledge of DLC toxicity in fish and other vertebrates, the aryl hydrocarbon receptor (ahr2) region accounts for 17% of trait variation; however, QTL on independent linkage groups and their interactions have even greater explanatory power (44%). QTL interpreted within the context of recently available Fundulus genomic resources and shared synteny among fish species suggest adaptation via interacting components of a complex stress response network. Some QTL were also enriched in other killifish populations characterized as DLC‐tolerant and residing in distant urban estuaries contaminated with unique mixtures of pollutants. Together, our results suggest that DLC tolerance in killifish represents an emerging example of parallel contemporary evolution that has been driven by intense human‐mediated selection on natural populations.     

Identification of interacting amino acids at the histone 2A--2B binding site.

Histones 2A and 2B of calf thymus were cross-linked within intact nuclei by UV irradiation. This procedure induces the formation of covalent cross-links between noncovalently interacting residues in the histones of native chromatin. Tryptic peptide and partial sequence analysis of the cross-linked product has shown that the covalent linkage is between tyrosine-37, -40, or -42 (we have not yet determined which) of H2B and proline-26 of H2A. We conclude that these residues constitute part of the hydrophobic H2A--H2B binding domain within the nucleosomes of native chromatin.     

Structure of nucleosome core particles containing uH2A (A24)

We have purified uH2A (A24) and reconstituted it, in place of H2A, into high molecular weight nucleohistone containing the core histones and DNA. uH2A-containing core particles were then prepared by nuclease digestion and studies on these particles were carried out. We show that two uH2A molecules can be accommodated within a core particle. We also show that the presence of two uH2A molecules in a core particle does not alter significantly either the pattern or the rate of DNase I digestion as compared to both reconstituted and native core particles. Finally, we show that HMG proteins 14 and 17 can bind to uH2A-containing core particles. We conclude that uH2A has little influence on structure at the level of individual nucleosomes.     

The effect of LIGHT in inducing maturation of monocyte-derived dendritic cells from MDS patients

LIGHT is a recently cloned novel cytokine belonging to the TNF family that is selectively expressed on immature dendritic cells (iDCs) generated from monocytes isolated from human PBMCs. In these studies, we demonstrate that exogenous soluble LIGHT or soluble CD40 ligand (CD40L) can promote monocyte-derived dendritic cell maturation in vitro by the up-regulation of CD86, CD80, CD83, and HLA-DR antigen expression. Immature dendritic cells differentiated from monocytes of MDS patients displayed lower levels of costimulatory and HLA-DR molecules compared with iDCs differentiated from monocytes of normal subjects. However, upon induction of maturation by LIGHT or CD40L, the expression of costimulatory and HLA-DR molecules is comparable between DCs from MDS and normal subjects. Exogenous LIGHT- and CD40L-stimulated mature DCs (mDCs) also displayed increased antigen presentation to autologous T lymphocytes (tetanus toxin) or allogeneic T lymphocytes in mixed lymphocyte reactions. DCs matured by LIGHT showed increased secretion of IL-6, IL-12p75, and TNF-, but not IL-1. We conclude that both LIGHT and CD40L are immunoregulating factors that induce monocyte-derived iDCs from MDS patients to undergo maturation resulting in increased antigen presentation and T-cell activation. Monocyte-derived DCs can be stimulated to undergo phenotypic and functional changes with LIGHT that might be applied in the development of a DC-based vaccine for MDS treatment.     

A scalable system for production of functional pancreatic progenitors from human embryonic stem cells

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.     

Dendritic cells from HIV-1 infected individuals are less responsive to toll-like receptor (TLR) ligands

We compared TLR responsiveness in PBMC from HIV-1-infected and uninfected individuals using the TLR agonists: TLR7 (3M-001), TLR8 (3M-002), and TLR7/8 (3M-011). Activation and maturation of plasmacytoid dendritic cells (pDC) were measured by evaluating CD86, CD40, and CD83 expression and myeloid dendritic cell (mDC) activation was measured by evaluating CD40 expression. All agonists tested induced activation and maturation of pDC in PBMC cultures of cells from HIV+ and HIV- individuals. The TLR7 agonist induced significantly less pDC maturation in cells from HIV+ individuals. Quantitative assessment of secreted IFN-alpha and pro-inflammatory cytokines at the single cell level showed that pDC from HIV+ individuals stimulated with TLR7 and TLR7/8 induced IFN-alpha. TLR8 and TLR7/8 agonists induced IL-12 and COX-2 expression in mDC from HIV+ and HIV- individuals. Understanding pDC and mDC activation and maturation in HIV-1 infection could lead to more rational development of immunotherapeutic strategies to stimulate the adaptive immune response to HIV-1.     

The DNase I sensitive state of "active" globin gene chromatin resists trypsin treatments which disrupt chromatin higher order structure

Active genes in higher eukaryotes reside in chromosomal domains which are more sensitive to digestion by DNase I than the surrounding inactive chromatin. Although it is widely assumed that some modification of higher order structure is important to the preferential DNase I sensitivity of active chromatin, this has so far not been tested. Here we show that the structural distinction between DNase I sensitive and resistant chromatin is remarkably stable to digestion by trypsin. Chick embryonic red blood cell nuclei were subjected to increasing levels of trypsin digestion and then assayed in the following three ways: (1) by gel electrophoresis for histone cleavage, (2) by sedimentation and nuclease digestion for loss of higher order structure, and (3) by dot-blot hybridization to globin and ovalbumin probes for disappearance of preferential DNase I sensitivity. We have found that chromatin higher order structure is lost concomitantly with the cleavage of histones H1, H5, and H3. In contrast, the preferential sensitivity of the globin domain to DNase I persists until much higher concentrations of trypsin, and indeed is not completely abolished even by the highest levels of trypsin we have used. We therefore conclude that the structural distinction of active chromatin, recognized by DNase I, does not reside at the level of higher order structure.