Variations among cell lines in the synthesis of sphingolipids in de novo and recycling pathways

There are several pathways for the incorporation of sugars into glycosphingolipids (GSL). Sugars can be added to ceramide that contains sphinganine (dihydrosphingosine) synthesized de novo (pathway 1), to ceramide synthesized from sphingoid bases produced by hydrolysis of sphingolipids (pathway 2), and into GSL recycling from the endosomal pathway through the Golgi (pathway 3). We reported previously the surprising observation that SW13 cells, a human adrenal carcinoma cell line, synthesize most of their GSL in pathway 2. We now present data on the synthesis of GSL in four additional cell lines. Approximately 90% of sugar incorporation took place in pathway 2, and 10% or less in pathway 1, in human foreskin fibroblasts and NB41A3 neuroblastoma cells. In contrast, approximately 50-90% of sugar incorporation took place in pathway 1 in C2C12 myoblasts. The C2C12 cells divide more rapidly and synthesize 10-14 times as much GSL as the other three cell lines. In C6 glioma cells, approximately 30% of sugar incorporation occurred in pathway 1 and 60% in pathway 2. There was no relation between the utilization of pathways for GSL and sphingomyelin synthesis in foreskin fibroblasts and C2C12 cells. In both cells pathways 1 and 2 each accounted for 50% of incorporation of choline into sphingomyelin. In five of the six cell lines that we have studied, most GSL synthesis takes place in pathway 2. We suggest that when the need for synthesis is relatively low, as in slowly dividing cells, GSL are synthesized predominantly from sphingoid bases salvaged from the hydrolytic pathway. When cells are dividing more rapidly, the need for increased synthesis is met by upregulating the de novo pathway.      

Non-native interactions play an effective role in protein folding dynamics

Systematic Monte Carlo simulations of simple lattice models show that the final stage of protein folding is an ordered process where native contacts get locked (i.e., the residues come into contact and remain in contact for the duration of the folding process) in a well‐defined order. The detailed study of the folding dynamics of protein‐like sequences designed as to exhibit different contact energy distributions, as well as different degrees of sequence optimization (i.e., participation of non‐native interactions in the folding process), reveals significant differences in the corresponding locking scenarios—the collection of native contacts and their average locking times, which are largely ascribable to the dynamics of non‐native contacts. Furthermore, strong evidence for a positive role played by non‐native contacts at an early folding stage was also found. Interestingly, for topologically simple target structures, a positive interplay between native and non‐native contacts is observed also toward the end of the folding process, suggesting that non‐native contacts may indeed affect the overall folding process. For target models exhibiting clear two‐state kinetics, the relation between the nucleation mechanism of folding and the locking scenario is investigated. Our results suggest that the stabilization of the folding transition state can be achieved through the establishment of a very small network of native contacts that are the first to lock during the folding process.     

Statistical modeling of biomedical corpora: mining the Caenorhabditis Genetic Center Bibliography for genes related to life span

Background  

The statistical modeling of biomedical corpora could yield integrated, coarse-to-fine views of biological phenomena that complement discoveries made from analysis of molecular sequence and profiling data. Here, the potential of such modeling is demonstrated by examining the 5,225 free-text items in the Caenorhabditis Genetic Center (CGC) Bibliography using techniques from statistical information retrieval. Items in the CGC biomedical text corpus were modeled using the Latent Dirichlet Allocation (LDA) model. LDA is a hierarchical Bayesian model which represents a document as a random mixture over latent topics; each topic is characterized by a distribution over words.     

The shape of human gene family phylogenies

Background  

The shape of phylogenetic trees has been used to make inferences about the evolutionary process by comparing the shapes of actual phylogenies with those expected under simple models of the speciation process. Previous studies have focused on speciation events, but gene duplication is another lineage splitting event, analogous to speciation, and gene loss or deletion is analogous to extinction. Measures of the shape of gene family phylogenies can thus be used to investigate the processes of gene duplication and loss. We make the first systematic attempt to use tree shape to study gene duplication using human gene phylogenies.     

CD4+ T cells pass through an effector phase during the process of in vivo tolerance induction

An important process in the generation of tolerance to peripheral self-Ags is the induction of unresponsiveness in mature specific T cells. Although the end stage of this process, termed anergy, is well defined, the pathway by which naive T cells become anergic remains to be elucidated. Using an in vivo self-tolerance model, we demonstrate that CD4(+) T cells pass through a significant effector stage on their way to an anergic state. This stage is characterized by production of effector cytokines, provision of help for CD8(+) T cells, and induction of in vivo pathology within organs that express cognate Ag. These results suggest that the initial activation stage in T cell tolerance is similar to that seen in memory induction. They also suggest that autoimmune pathology can result during the natural process of tolerance induction rather than requiring that tolerance be broken.     

Immunotherapy of established tumors using bone marrow transplantation with antigen gene--modified hematopoietic stem cells

A major focus of cancer immunotherapy is to develop strategies to induce T-cell responses through presentation of tumor antigens by dendritic cells (DCs). Current vaccines are limited in their ability to efficiently transfer antigens to DCs in vivo. Ex vivo-generated DCs can be efficiently loaded with antigen but after reinjection, few DCs traffic to secondary lymphoid organs, the critical sites for antigen presentation. To enhance efficiency and durability of antigen presentation by DCs, we transduced hematopoietic stem-progenitor cells (HSCs) with a model tumor antigen and then transplanted the gene-modified cells into irradiated recipient mice, which resulted in efficient expression of the transgene in a large proportion of donor derived DCs in lymphoid organs. The combination of bone marrow transplantation (BMT) using transduced HSCs, systemic agents that generate and activate DCs, and mature T-cell infusion resulted in substantial expansion and activation of antigen-specific T cells. This tripartite strategy provided potent antigen-specific immunotherapy for an aggressive established tumor.     

Stat3 activity in melanoma cells affects migration of immune effector cells and nitric oxide-mediated antitumor effects

Infiltration of immune effector cells in tumors is critical for antitumor immune responses. However, what regulates immune cell infiltration of tumors remains to be identified. Stat3 is constitutively activated with high frequency in diverse cancers, promoting tumor cell growth and survival. Blocking Stat3 signaling in tumors in vivo results in tumor growth inhibition that involves killing of nontransfected tumor cells and infiltration of immune effector cells, suggesting that Stat3 activity in tumor cells might affect immune cell recruitment. However, dying tumor cells can also attract immune cells. In this study, we show in isogenic murine melanomas that natural Stat3 activity is associated with tumor growth and reduction of T cell infiltration. Blocking Stat3 signaling in the melanoma cells containing high Stat3 activity results in expression of multiple chemoattractants, leading to increased migration of lymphocytes, NK cells, neutrophils, and macrophages. In addition, blocking Stat3 triggers tumor cells to produce soluble factors capable of activating macrophage production of NO in vitro and in vivo. TNF-alpha and IFN-beta, which are secreted by Stat3-inhibited tumor cells, are able to activate macrophage NO production, whereas neutralizing TNF-alpha in the tumor supernatant from Stat3-blocked tumor cells abrogates nitrite production. Moreover, interrupting Stat3 signaling in tumor cells leads to macrophage-mediated, nitrite-dependent cytostatic activity against nontransduced tumor cells. These results suggest that tumor Stat3 activity affects recruitment of diverse immune effectors and it can be manipulated to activate the effector phase of innate immune responses.     

Quality determination and the repair of poor quality spots in array experiments

Background  

A common feature of microarray experiments is the occurence of missing gene expression data. These missing values occur for a variety of reasons, in particular, because of the filtering of poor quality spots and the removal of undefined values when a logarithmic transformation is applied to negative background-corrected intensities. The efficiency and power of an analysis performed can be substantially reduced by having an incomplete matrix of gene intensities. Additionally, most statistical methods require a complete intensity matrix. Furthermore, biases may be introduced into analyses through missing information on some genes. Thus methods for appropriately replacing (imputing) missing data and/or weighting poor quality spots are required.