Reduced neurogenesis in the rat hippocampus following high fructose consumption

In this study, we investigated how prolonged consumption of sugar solution affects hippocampal neurogenesis. We gave rats sucrose or fructose solution for four weeks and observed a 40% reduction in BrdU/NeuN-immunoreactive cells in the hippocampal dentate gyrus. This reduction in hippocampal neurogenesis was accompanied by increased apoptosis in the hippocampus and increased circulating levels of TNF-α. Therefore, we hypothesize that the reduction in hippocampal neurogenesis may be due to the increased apoptosis induced by TNF-α. Our results suggest that chronic ingestion of fructose is detrimental to the survival of newborn hippocampal neurones. The results presented in the present study add to the list of harmful effects associated with prolonged and excessive consumption of sugary beverages and soft drinks.     

Discovery and optimization of potent and selective benzonaphthyridinone analogs as small molecule mTOR inhibitors with improved mouse microsome stability

Starting from small molecule mTOR inhibitor Torin1, replacement of the piperazine ring with a phenyl ring resulted in a new series of mTOR inhibitors (as exemplified by 10) that showed superior potency and selectivity for mTOR, along with significantly improved mouse liver microsome stability and a longer in vivo half-life.     

Microenvironmental influence on pre-clinical activity of polo-like kinase inhibition in multiple myeloma: implications for clinical translation

Polo-like kinases (PLKs) play an important role in cell cycle progression, checkpoint control and mitosis. The high mitotic index and chromosomal instability of advanced cancers suggest that PLK inhibitors may be an attractive therapeutic option for presently incurable advanced neoplasias with systemic involvement, such as multiple myeloma (MM). We studied the PLK 1, 2, 3 inhibitor BI 2536 and observed potent (IC50<40 nM) and rapid (commitment to cell death <24 hrs) in vitro activity against MM cells in isolation, as well as in vivo activity against a traditional subcutaneous xenograft mouse model. Tumor cells in MM patients, however, don't exist in isolation, but reside in and interact with the bone microenvironment. Therefore conventional in vitro and in vivo preclinical assays don't take into account how interactions between MM cells and the bone microenvironment can potentially confer drug resistance. To probe this question, we performed tumor cell compartment-specific bioluminescence imaging assays to compare the preclinical anti-MM activity of BI 2536 in vitro in the presence vs. absence of stromal cells or osteoclasts. We observed that the presence of these bone marrow non-malignant cells led to decreased anti-MM activity of BI 2536. We further validated these results in an orthotopic in vivo mouse model of diffuse MM bone lesions where tumor cells interact with non-malignant cells of the bone microenvironment. We again observed that BI 2536 had decreased activity in this in vivo model of tumor-bone microenvironment interactions highlighting that, despite BI 2536's promising activity in conventional assays, its lack of activity in microenvironmental models raises concerns for its clinical development for MM. More broadly, preclinical drug testing in the absence of relevant tumor microenvironment interactions may overestimate potential clinical activity, thus explaining at least in part the gap between preclinical vs. clinical efficacy in MM and other cancers.     

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.     

Spreading of Heterochromatin Is Limited to Specific Families of Maize Retrotransposons

Transposable elements (TEs) have the potential to act as controlling elements to influence the expression of genes and are often subject to heterochromatic silencing. The current paradigm suggests that heterochromatic silencing can spread beyond the borders of TEs and influence the chromatin state of neighboring low-copy sequences. This would allow TEs to condition obligatory or facilitated epialleles and act as controlling elements. The maize genome contains numerous families of class I TEs (retrotransposons) that are present in moderate to high copy numbers, and many are found in regions near genes, which provides an opportunity to test whether the spreading of heterochromatin from retrotransposons is prevalent. We have investigated the extent of heterochromatin spreading into DNA flanking each family of retrotransposons by profiling DNA methylation and di-methylation of lysine 9 of histone 3 (H3K9me2) in low-copy regions of the maize genome. The effects of different retrotransposon families on local chromatin are highly variable. Some retrotransposon families exhibit enrichment of heterochromatic marks within 800–1,200 base pairs of insertion sites, while other families exhibit very little evidence for the spreading of heterochromatic marks. The analysis of chromatin state in genotypes that lack specific insertions suggests that the heterochromatin in low-copy DNA flanking retrotransposons often results from the spreading of silencing marks rather than insertion-site preferences. Genes located near TEs that exhibit spreading of heterochromatin tend to be expressed at lower levels than other genes. Our findings suggest that a subset of retrotransposon families may act as controlling elements influencing neighboring sequences, while the majority of retrotransposons have little effect on flanking sequences.     

Molecular and Genetic Analyses of Four Nonfunctional S Haplotype Variants Derived from a Common Ancestral S Haplotype Identified in Sour Cherry (Prunus cerasus L.)

Tetraploid sour cherry (Prunus cerasus) has an S-RNase-based gametophytic self-incompatibility (GSI) system; however, individuals can be either self-incompatible (SI) or self-compatible (SC). Unlike the situation in the Solanaceae, where self-compatibility accompanying polyploidization is often due to the compatibility of heteroallelic pollen, the genotype-dependent loss of SI in sour cherry is due to the compatibility of pollen containing two nonfunctional S haplotypes. Sour cherry individuals with the S₄S₆S_36aS_36b genotype are predicted to be SC, as only pollen containing both nonfunctional S_36a and S_36b haplotypes would be SC. However, we previously found that individuals of this genotype were SI. Here we describe four nonfunctional S₃₆ variants. Our molecular analyses identified a mutation that would confer loss of stylar S function for one of the variants, and two alterations that might cause loss of pollen S function for all four variants. Genetic crosses showed that individuals possessing two nonfunctional S₃₆ haplotypes and two functional S haplotypes have reduced self-fertilization due to a very low frequency of transmission of the one pollen type that would be SC. Our finding that the underlying mechanism limiting successful transmission of genetically compatible gametes does not involve GSI is consistent with our previous genetic model for Prunus in which heteroallelic pollen is incompatible. This provides a unique case in which breakdown of SI does not occur despite the potential to generate SC pollen genotypes.GAMETOPHYTIC self-incompatibility (GSI) is a widespread mechanism in flowering plants that prevents self-fertilization and promotes out-crossing (De Nettancourt 2001). In GSI plants, pollen tube growth is arrested if there is a match between the genes at the S-locus that control pollen and stylar specificity. The gene controlling stylar specificity in the Solanaceae, Rosaceae, and Plantaginaceae is known to encode a ribonuclease (S-RNase) (for a review see McClure 2009), while the gene controlling pollen specificity encodes an F-box protein [S haplotype-specific F-box protein (SFB) or S-locus F-box protein (SLF)] (Lai et al. 2002; Entani et al. 2003; Ushijima et al. 2003; Sijacic et al. 2004). As these two specificity genes are tightly linked and recombination between these two genes has never been observed (Ikeda et al. 2005), these two S-locus specificity genes are collectively termed the S haplotype.Characterization of the S haplotype is most advanced in Prunus (Rosaceae) due to the small physical size of the S haplotype region and the close proximity of the stylar S (S-RNase) and pollen S (SFB) genes (Entani et al. 2003; Ushijima et al. 2003; Yamane et al. 2003b; Ikeda et al. 2005). Within Prunus, sweet cherry (Prunus avium) and sour cherry (P. cerasus) represent a model diploid–tetraploid series that has been used to investigate the effects of polyploidy on GSI. Tetraploid sour cherry is considered to have arisen through hybridization between sweet cherry and tetraploid ground cherry (P. fruticosa) (Olden and Nybom 1968). Like sweet cherry, sour cherry exhibits an S-RNase-based GSI system (Yamane et al. 2001; Hauck et al. 2002; Tobutt et al. 2004) and interspecific crossing studies have demonstrated that sour cherry shares eight sweet cherry S haplotypes: S₁, S₄, S₆, S₉, S₁₂, S₁₃, S₁₄, and S₁₆ (Bošković et al. 2006; Hauck et al. 2006a,b; Tsukamoto et al. 2006, 2008). However, in contrast to sweet cherry, natural sour cherry selections include both self-incompatible (SI) and self-compatible (SC) types. A genetic model demonstrating that the genotype-dependent loss of SI in sour cherry is due to the accumulation of a minimum of two nonfunctional S haploytpes within a single individual was developed and validated (Hauck et al. 2006b). These nonfunctional S haplotypes were characterized as either pollen-part mutants or stylar-part mutants, depending on whether the pollen S or stylar S specificity was disrupted. In Prunus, pollen-part and stylar-part mutants are denoted by a prime symbol “′” or a subscribed “m,” respectively, following the S haplotype number (Tsukamoto et al. 2006). Molecular characterizations of five of the nonfunctional S haplotypes from sour cherry characterized to date support the genetic results because mutations were identified that affected the S-RNase and/or SFB. These changes in coding or regulatory regions included mutations within the S-RNase and/or SFB causing premature stop codons, transposable element insertions within SFB and upstream of the S-RNase, and a 23-bp deletion in a conserved region of the S-RNase (Yamane et al. 2003a; Hauck et al. 2006a; Tsukamoto et al. 2006).According to the genetic model, termed the “one-allele-match model,” sour cherry pollen is rejected if one or both of the functional S haplotypes in the 2x pollen grain match an S haplotype in the style (Hauck et al. 2006b). Therefore, only pollen containing two nonfunctional S haplotypes would be SC; thus, a sour cherry genotype is SC if it has a minimum of two nonfunctional S haplotypes. We previously tested the one-allele-match model using 92 sour cherry selections from four progeny populations (Hauck et al. 2006b). For all the progeny except three, their S genotype correctly predicted whether they were SI or SC. The three progeny individuals that were the exception all had the same genotype: S₄S₆S_aS_d. These individuals were predicted to be SC as the S_a and S_d haplotypes were shown to be nonfunctional in genetic studies and therefore S_aS_d pollen should be SC. However, these progeny were classified as SI on the basis of observations of self-pollen tube growth in the styles. The S_a and S_d haplotypes were originally distinguished on the basis of different RFLP fragment sizes using an S-RNase probe; the HindIII fragment sizes for S_a and S_d differed by ∼200 bp, 6.4-kb and 6.2-kb, respectively (Yamane et al. 2001; Hauck et al. 2002). However, partial S-RNase and SFB sequences from the S_a and S_d haplotypes were identical (N. R. Hauck and A. F. Iezzoni, unpublished results), suggesting that S_a and S_d represented different mutations of the same S haplotype. Therefore, we hypothesized that the SI phenotype of the S₄S₆S_aS_d individuals resulted from complementary pistil S and pollen S mutations in the nonfunctional S_a and S_d haplotypes, thus behaving genetically as one functional S haplotype.We previously reported that heteroallelic sour cherry pollen containing two different functional pollen S haplotypes is incompatible (Hauck et al. 2006b). This finding is counter to the well-documented phenomenon in the Solanaceae where SC accompanying polyploidization is frequently due to the SC of heteroallelic pollen (Lewis 1943; Golz et al. 1999, 2001; Tsukamoto et al. 2005; Xue et al. 2009). Therefore, models explaining the molecular basis of self-recognition in Prunus and the Solanaceae must be consistent with these differing genetic expectations. Recently, Huang et al. (2008) reported competitive interaction in a SC selection of tetraploid P. pseudocerasus, raising the possibility that the SC mechanism between these two tetraploid Prunus species could be different. However, although the data in Huang et al. (2008) are consistent with heteroallelic pollen being SC, homoallelic pollen (e.g., S₁S₁, S₅S₅, or S₇S₇) was not shown to be successful in compatible crosses and unsuccessful in incompatible ones. Therefore, it is possible that the SC in P. pseudocerasus could be caused by mutations in other genes critical for the SI reaction. Because of the importance of these differing genetic expectations for understanding S-RNase-based GSI, we sought to investigate our previously identified exceptions to the one-allele-match model. Specifically, our objective was to test our prior hypothesis that the nonfunctional S_a and S_d haplotypes interact in a complementary manner and therefore behave together genetically as a single functional S haplotype. In this work, the S_a and S_d haplotypes were renamed S_36a and S_36b, respectively, following the order of previously published S haplotypes (Tsukamoto et al. 2008; Vaughan et al. 2008) for reasons explained in the results.     

Monocyte-Derived Dendritic Cells Exhibit Increased Levels of Lysosomal Proteolysis as Compared to Other Human Dendritic Cell Populations

Background

Fine control of lysosomal degradation for limited processing of internalized antigens is a hallmark of professional antigen presenting cells. Previous work in mice has shown that dendritic cells (DCs) contain lysosomes with remarkably low protease content. Combined with the ability to modulate lysosomal pH during phagocytosis and maturation, murine DCs enhance their production of class II MHC-peptide complexes for presentation to T cells.

Methodology/Principal Findings

In this study we extend these findings to human DCs and distinguish between different subsets of DCs based on their ability to preserve internalized antigen. Whereas DCs derived in vitro from CD34+ hematopoietic progenitor cells or isolated from peripheral blood of healthy donors are protease poor, DCs derived in vitro from monocytes (MDDCs) are more similar to macrophages (MΦs) in protease content. Unlike other DCs, MDDCs also fail to reduce their intralysosomal pH in response to maturation stimuli. Indeed, functional characterization of lysosomal proteolysis indicates that MDDCs are comparable to MΦs in the rapid degradation of antigen while other human DC subtypes are attenuated in this capacity.

Conclusions/Significance

Human DCs are comparable to murine DCs in exhibiting a markedly reduced level of lysosomal proteolysis. However, as an important exception to this, human MDDCs stand apart from all other DCs by a heightened capacity for proteolysis that resembles that of MΦs. Thus, caution should be exercised when using human MDDCs as a model for DC function and cell biology.     

Hindered diffusion through an aqueous pore describes invariant dye selectivity of Cx43 junctions

The permselectivity (permeance/conductance) of Cx43-comprised gap junctions is a variable parameter of junctional function. To ascertain whether this variability in junctional permselectivity is explained by heterogeneous charge or size selectivity of the comprising channels, the permeance of individual Cx43 gap junctions to combinations of two dyes differing in either size or charge was determined in four cell types: Rin43, NRKe, HeLa43, and cardiac myocytes. The results show that Cx43 junctions are size- but not charge-selective and that both selectivities are constant parameters of junctional function. The consistency of dye selectivities indicates that the large continuum of measured junctional permselectivities cannot be ascribed to an equivalent continuum of individual channel selectivities. Further, the relative dye permeance sequence of NBD-M-TMA approximately Alexa 350 > Lucifer yellow > Alexa 488 > Alexa 594 (Stokes radii of 4.3 A, 4.4 A, 4.9 A, 5.8 A, and 7.4 A, respectively) and the conductance sequence of KCl > TEACl approximately Kglutamate are well described by hindered diffusion through an aqueous pore with radius approximately 10 A and length 160 A. The permselectivity and dye selectivity data suggest the variable presence in Cx43-comprised junctions of conductive channels that are either dye-impermeable or dye-permeable.     

Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate

Sphingosine 1-phosphate (S1P) influences heart rate, coronary artery caliber, endothelial integrity, and lymphocyte recirculation through five related high affinity G-protein-coupled receptors. Inhibition of lymphocyte recirculation by non-selective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the embryonic lethality of the S1P(1) knock-out and the unavailability of selective agonists or antagonists. A potent, S1P(1)-receptor selective agonist structurally unrelated to S1P was found to activate multiple signals triggered by S1P, including guanosine 5'-3-O-(thio)triphosphate binding, calcium flux, Akt and ERK1/2 phosphorylation, and stimulation of migration of S1P(1)- but not S1P(3)-expressing cells in vitro. The agonist also alters lymphocyte trafficking in vivo. Use of selective agonism together with deletant mice lacking S1P(3) receptor reveals that agonism of S1P(1) receptor alone is sufficient to control lymphocyte recirculation. Moreover, S1P(1) receptor agonist plasma levels are causally associated with induction and maintenance of lymphopenia. S1P(3), and not S1P(1), is directly implicated in sinus bradycardia. The sustained bradycardia induced by S1P receptor non-selective immunosuppressive agonists in wild-type mice is abolished in S1P(3)-/- mice, whereas S1P(1)-selective agonist does not produce bradycardia. Separation of receptor subtype usage for control of lymphocyte recirculation and heart rate may allow the identification of selective immunosuppressive S1P(1) receptor agonists with an enhanced therapeutic window. S1P(1)-selective agonists will be of broad utility in understanding cell functions in vitro, and vascular physiology in vivo, and the success of the chemical approach for S1P(1) suggests that selective tools for the resolution of function across this broad lipid receptor family are now possible.