Torrey Pines Institute for Molecular Studies

About: Torrey Pines Institute for Molecular Studies is a nonprofit organization based out in San Diego, California, United States. It is known for research contribution in the topics: Antigen & T cell. The organization has 2323 authors who have published 2217 publications receiving 112618 citations.

Replication and subnuclear location dynamics of the immunoglobulin heavy-chain locus in B-lineage cells.

Abstract: In Saccharomyces cerevisiae, the initiation of DNA replication is sequence specific (27; for a review, see reference 23). In mammalian cells, however, the mechanism underlying this process is still being defined. Although specific origins of replication have been mapped, initiation regions as large as 50 kb have also been described (for reviews, see references 20 and 22), making the potential role of sequence specificity less clear. In mammalian cells, the origins of DNA replication are activated in different intervals of S phase and the timing of the activation of these origins is precisely regulated. The regulation of the replication timing of a specific gene appears to reflect the transcriptional activity of surrounding sequences (for a review, see reference 58). Evidence that shows that the localization of some genes or even of an entire chromosome within the nucleus is nonrandom and is linked both to chromatin structure and replication timing has been presented (9, 16, 41; for a review, see reference 14). Thus, early- and late-replication foci tend to occupy different subnuclear domains in interphase cells (for a review, see reference 28). Recent observations suggest that nuclear structure might play an important role in defining when and where DNA replication initiates (for a review, see reference 21). The immunoglobulin heavy-chain (Igh) locus (consisting of, in the 5′-to-3′ direction, 15 heavy-chain variable [Vh] gene families, 12 heavy-chain diversity [Dh] segments, 4 heavy-chain joining [Jh] segments, an intronic enhancer, 8 constant-region [Ch] genes, and the 3′ regulatory region) is an important model system for the study of mammalian DNA replication. The Igh locus shows successive changes in transcription and DNA rearrangements during B-cell development (Fig. ​(Fig.1).1). V(D)J joining occurs at the pro-B-cell stage, preceded by transcriptional activation of Vh genes. Class switching occurs later in B-cell development. These processes result in unique combinations of V, D, J, and C segments encoding and transcribing individual heavy-chain (and light-chain) genes in individual B cells (for a review, see reference 45). B-cell lines representing different stages of B-cell development are available for the study of the replication and nuclear localization of the Igh locus. The cell lines used in the present study are listed in Table ​Table1.1. The mouse Igh locus is very large (approximately 3 Mb) but has been physically mapped and cloned in yeast artificial chromosome and bacterial artificial chromosome (BAC) arrays (13) and is being sequenced. The information derived from these arrays greatly facilitated the present study. FIG. 1. Rearrangements of the Igh locus during B-cell development.The germ line configuration of the Igh locus on murine chromosome 12 is shown (44, 57). In the 63-12 RAG2−/− pro-B-cell line, the entire locus is unrearranged and in germ line configuration, ... TABLE 1. Characteristics of B-lineage cell lines used in this study Previous studies using bromodeoxyuridine (BrdU) S-phase fractionation have shown that the replication timing of the Igh locus in non-B-cell lines, such as the murine erythroleukemia (MEL) cell line, is different from that in pre-B-cell lines (11, 34, 35). In MEL cells, an ∼400-kb sequence forms a transition region between an early-replication domain downstream of the 3′ regulatory region and a late-replication domain containing Vh genes (triphasic-replication pattern) (Fig. ​(Fig.2A).2A). Sequences within this transition region replicate 3′ to 5′ progressively later during S. A previous study using neutral-alkaline two-dimensional (N/A 2D) gel electrophoresis has shown that the transition region is formed by a single replicon through which replication forks progress in the 3′-to-5′ direction (25). In contrast, the entire Igh locus is replicated within the first hour of S in two pre-B-cell lines. Previous studies also demonstrated that part of the Igh-C region replicates early in S in plasma cell lines (34). FIG. 2. Replication timing of the murine Igh locus in B-lineage cell lines. The time in S phase when a particular segment is replicated is expressed in terms of the C value (y axis) and is plotted versus its distance in kilobases from the Cα gene (x axis), ... One explanation for the dramatically different replication programs detected in non-B cells and pre-B cells is that the DNA replication program is linked to the transcriptional status of the Igh locus. In MEL cells, the Igh locus is transcriptionally silent, while in pre-B-cell lines, this locus is actively transcribed. Since the pattern of transcription along the locus changes during B-cell development, we decided to examine the replication of the Igh locus in B-cell lines at different stages of B-cell development. The experiments presented here reveal two distinct programs of replication for the Igh locus in B-lineage cells. In pro- and pre-B-cell lines, including those derived from RAG2−/− mice, the entire Igh locus is replicated early in S phase. The temporal pattern of Igh replication in other cell lines representing late stages of B-cell development (an immature-B-cell line and plasma cell lines) resembles that in non-B cells. The temporal-transition regions in the different cell lines are similar in length but encompass different sequences, reflecting individual DNA rearrangement events associated with VDJ and class switch recombination. The replication timing of the Igh locus in ex vivo-expanded pro-B cells and lipopolysaccharide (LPS)-stimulated spleen B cells is consistent with the data obtained from the cell lines. In this study, we address the potential mechanism for this dramatic difference in replication programs. N/A 2D gel electrophoresis demonstrates that replication forks progress in both directions through the examined Igh-C genes in cell lines in which the entire locus replicates early in S. Our results suggest that the early replication of the Igh locus is achieved through the activation of one or more replication origins. In contrast, in B cells in which the Igh locus replicates through a triphasic pattern, replication forks progress in a single 3′-to-5′ direction (opposite to the transcriptional orientation) through all the constant-region genes we examined. Our observations that the directions of replication fork progression can differ in B-lineage cell lines raise the possibility that changes in Igh replication are involved in processes of Igh rearrangement and expression. We have found that, associated with the changes in replication, the entire Igh locus is located away from the nuclear periphery when it is replicated early in S phase but is located near the nuclear periphery when it is replicated in a triphasic pattern.

The prince and the pauper. A tale of anticancer targeted agents

Abstract: Cancer rates are set to increase at an alarming rate, from 10 million new cases globally in 2000 to 15 million in 2020. Regarding the pharmacological treatment of cancer, we currently are in the interphase of two treatment eras. The so-called pregenomic therapy which names the traditional cancer drugs, mainly cytotoxic drug types, and post-genomic era-type drugs referring to rationally-based designed. Although there are successful examples of this newer drug discovery approach, most target-specific agents only provide small gains in symptom control and/or survival, whereas others have consistently failed in the clinical testing. There is however, a characteristic shared by these agents: -their high cost-. This is expected as drug discovery and development is generally carried out within the commercial rather than the academic realm. Given the extraordinarily high therapeutic drug discovery-associated costs and risks, it is highly unlikely that any single public-sector research group will see a novel chemical "probe" become a "drug". An alternative drug development strategy is the exploitation of established drugs that have already been approved for treatment of non-cancerous diseases and whose cancer target has already been discovered. This strategy is also denominated drug repositioning, drug repurposing, or indication switch. Although traditionally development of these drugs was unlikely to be pursued by Big Pharma due to their limited commercial value, biopharmaceutical companies attempting to increase productivity at present are pursuing drug repositioning. More and more companies are scanning the existing pharmacopoeia for repositioning candidates, and the number of repositioning success stories is increasing. Here we provide noteworthy examples of known drugs whose potential anticancer activities have been highlighted, to encourage further research on these known drugs as a means to foster their translation into clinical trials utilizing the more limited public-sector resources. If these drug types eventually result in being effective, it follows that they could be much more affordable for patients with cancer; therefore, their contribution in terms of reducing cancer mortality at the global level would be greater.

The cytoplasmic tail slows the folding of human immunodeficiency virus type 1 Env from a late prebundle configuration into the six-helix bundle.

Abstract: Effects of the cytoplasmic tail (CT) of human immunodeficiency virus type 1 Env on the process of membrane fusion were investigated. Full-length Env (wild type [WT]) and Env with its CT truncated (ΔCT) were expressed on cell surfaces, these cells were fused to target cells, and the inhibition of fusion by peptides that prevent Env from folding into a six-helix bundle conformation was measured. For both X4-tropic and R5-tropic Env proteins, ΔCT induced faster fusion kinetics than did the WT, and peptides were less effective at inhibiting ΔCT-induced fusion. We tested the hypothesis that the inhibitory peptides were less effective at inhibiting ΔCT-induced fusion because ΔCT folds more quickly into a six-helix bundle. Early and late intermediates of WT- and ΔCT-induced fusion were captured, and the ability of peptides to block fusion when added at the intermediate stages was quantified. When added at the early intermediate, the peptides were still less effective at inhibiting ΔCT-induced fusion but they were equally effective at preventing WT- and ΔCT-induced fusion when added at the late intermediate. We conclude that for both X4-tropic and R5-tropic Env proteins, the CT facilitates conformational changes that allow the trimeric coiled coil of prebundles to become optimally exposed. But once Env does favorably expose its coiled coil to inhibitory peptides, the CT hinders subsequent folding into a six-helix bundle. Because of this facilitation of maximal exposure and hindrance of bundle formation, the coiled coil is optimally exposed for a longer time for WT than for ΔCT. This accounts for the greater peptide inhibition of WT-induced fusion.

Regulation of Immunity by a Novel Population of Qa-1-Restricted CD8αα+TCRαβ+ T Cells

Abstract: Regulatory mechanisms involving CD8 + T cells (CD8 regulatory T cells (Tregs)) are important in the maintenance of immune homeostasis. However, the inability to generate functional CD8 Treg clones with defined Ag specificity has precluded a direct demonstration of CD8 Treg-mediated regulation. In the present study, we describe the isolation of functional lines and clones representing a novel population of TCRαβ + Tregs that control activated Vβ8.2 + CD4 T cells mediating experimental autoimmune encephalomyelitis. They express exclusively the CD8αα homodimer and recognize a peptide from a conserved region of the TCR Vβ8.2 chain in the context of the Qa-1a (CD8αα Tregs). They secrete type 1 cytokines but not IL-2. CD8αα Tregs kill activated Vβ8.2 + but not Vβ8.2 − or naive T cells. The CD8αα Tregs prevent autoimmunity upon adoptive transfer or following in vivo activation. These findings reveal an important negative feedback regulatory mechanism targeting activated T cells and have implications in the development of therapeutic strategies for autoimmune diseases and transplantation.