Estrogen Sulfotransferase/SULT1E1 Promotes Human Adipogenesis

Estrogen sulfotransferase (EST/SULT1E1) is known to catalyze the sulfoconjugation and deactivation of estrogens. The goal of this study is to determine whether and how EST plays a role in human adipogenesis. By using human primary adipose-derived stem cells (ASCs) and whole-fat tissues from the abdominal subcutaneous fat of obese and nonobese subjects, we showed that the expression of EST was low in preadipocytes but increased upon differentiation. Overexpression and knockdown of EST in ASCs promoted and inhibited differentiation, respectively. The proadipogenic activity of EST in humans was opposite to the antiadipogenic effect of the same enzyme in rodents. Mechanistically, EST promoted adipogenesis by deactivating estrogens. The proadipogenic effect of EST can be recapitulated by using an estrogen receptor (ER) antagonist or ERα knockdown. In contrast, activation of ER in ASCs inhibited adipogenesis by decreasing the recruitment of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ) onto its target gene promoters, whereas ER antagonism increased the recruitment of PPARγ to its target gene promoters. Linear regression analysis revealed a positive correlation between the expression of EST and body mass index (BMI), as well as a negative correlation between ERα expression and BMI. We conclude that EST is a proadipogenic factor which may serve as a druggable target to inhibit the turnover and accumulation of adipocytes in obese patients.     

Evolution of the Scarabaeini (Scarabaeidae: Scarabaeinae)

Abstract. A phylogenetic analysis of the Scarabaeini, based on 244 morphological characters, including 154 multistate and three biological characters, is presented. Tree topologies generated from unweighted data and some weighted algorithms are similar and support only two clades in the tribe representing the genera Scarabaeus L. and Pachylomerus Bertoloni. The only supported subordinate groups treated in this paper as subgenera are Kheper Kirby stat. nov. , Pachysoma MacLeay, Scarabaeolus Balthasar and Sceliages Westwood stat. nov. Drepanopodus Janssens syn. nov. is synonymised with Scarabaeus and six additional names, Madateuchus Paulian, Mnematidium Ritsema, Mnematium MacLeay, Neateuchus Gillet, Neomnematium Janssens and Neopachysoma Ferreira, remain synonyms. A single origin of flightlessness is supported with the subgenus Pachysoma, the most derived lineage in this clade. Rolling dung balls backwards is the ancestral behaviour and predominant mode of food relocation in Scarabaeini, although tunnelling, forward pushing, and carrying also are utilized by some lineages. Pushing food has evolved independently in Sceliages species and S. galenus (Westwood) and a novel mode of forward food relocation by dragging evolved in the subgenus Pachysoma. Feeding on wet dung is the plesiomorphic condition and maintained by the majority of species in the tribe. The most unusual feeding behaviours in the tribe are represented by the obligate millipede-feeding species of Sceliages and the dry dung pellet and/or detritus feeders of Pachysoma.     

Osmotic Balance Regulates Cell Fusion during Mating in Saccharomyces cerevisiae

Successful zygote formation during yeast mating requires cell fusion of the two haploid mating partners. To ensure that cells do not lyse as they remodel their cell wall, the fusion event is both temporally and spatially regulated: the cell wall is degraded only after cell–cell contact and only in the region of cell–cell contact. To understand how cell fusion is regulated, we identified mutants defective in cell fusion based upon their defect in mating to a fus1 fus2 strain (Chenevert, J., N. Valtz, and I. Herskowitz. 1994. Genetics 136:1287–1297). Two of these cell fusion mutants are defective in the FPS1 gene, which codes for a glycerol facilitator (Luyten, K., J. Albertyn, W.F. Skibbe, B.A. Prior, J. Ramos, J.M. Thevelein, and S. Hohmann. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:1360–1371). To determine whether inability to maintain osmotic balance accounts for the defect in cell fusion in these mutants, we analyzed the behavior of an fps1Δ mutant with reduced intracellular glycerol levels because of a defect in the glycerol-3-phosphate dehydrogenase (GPD1) gene (Albertyn, J., S. Hohmann, J.M. Thevelein, and B.A. Prior. 1994. Mol. Cell. Biol. 14:4135– 4144): deletion of GPD1 partially suppressed the cell fusion defect of fps1 mutants. In contrast, overexpression of GPD1 exacerbated the defect. The fusion defect could also be partially suppressed by 1 M sorbitol. These observations indicate that the fusion defect of fps1 mutants results from inability to regulate osmotic balance and provide evidence that the osmotic state of the cell can regulate fusion. We have also observed that mutants expressing hyperactive protein kinase C exhibit a cell fusion defect similar to that of fps1 mutants. We propose that Pkc1p regulates cell fusion in response to osmotic disequilibrium. Unlike fps1 mutants, fus1 and fus2 mutants are not influenced by expression of GPD1 or by 1 M sorbitol. Their fusion defect is thus unlikely to result from altered osmotic balance.     

D-BRAIN: Anatomically Accurate Simulated Diffusion MRI Brain Data

Diffusion Weighted (DW) MRI allows for the non-invasive study of water diffusion inside living tissues. As such, it is useful for the investigation of human brain white matter (WM) connectivity in vivo through fiber tractography (FT) algorithms. Many DW-MRI tailored restoration techniques and FT algorithms have been developed. However, it is not clear how accurately these methods reproduce the WM bundle characteristics in real-world conditions, such as in the presence of noise, partial volume effect, and a limited spatial and angular resolution. The difficulty lies in the lack of a realistic brain phantom on the one hand, and a sufficiently accurate way of modeling the acquisition-related degradation on the other. This paper proposes a software phantom that approximates a human brain to a high degree of realism and that can incorporate complex brain-like structural features. We refer to it as a Diffusion BRAIN (D-BRAIN) phantom. Also, we propose an accurate model of a (DW) MRI acquisition protocol to allow for validation of methods in realistic conditions with data imperfections. The phantom model simulates anatomical and diffusion properties for multiple brain tissue components, and can serve as a ground-truth to evaluate FT algorithms, among others. The simulation of the acquisition process allows one to include noise, partial volume effects, and limited spatial and angular resolution in the images. In this way, the effect of image artifacts on, for instance, fiber tractography can be investigated with great detail. The proposed framework enables reliable and quantitative evaluation of DW-MR image processing and FT algorithms at the level of large-scale WM structures. The effect of noise levels and other data characteristics on cortico-cortical connectivity and tractography-based grey matter parcellation can be investigated as well.     

Immunoaffinity purification and fluorescence studies of human adenosine deaminase

Human thymus adenosine deaminase was isolated by using a monoclonal antibody affinity column. The highly purified enzyme produced by this rapid, efficient procedure had a molecular weight of 44,000. Quenching of the intrinsic protein fluorescence by small molecules was used to probe the accessibility of tryptophan residues in the enzyme and enzyme-inhibitor complexes. The fluorescence emission spectrum of human adenosine deaminase at 295-nm excitation had a maximum at about 335 nm and a quantum yield of 0.03. Addition of polar fluorescence quenchers, iodide and acrylamide, shifted the peak to the blue, and the hydrophobic quencher trichloroethanol shifted the peak to the red, indicating that the emission spectrum is heterogeneous. The fluorescence quenching parameters obtained for these quenchers reveal that the tryptophan environments in the protein are relatively hydrophobic. Binding of both ground-state and transition-state analogue inhibitors caused decreases in the fluorescence intensity of the enzyme, suggesting that one or more tryptophans may be near the active site. The kinetics of the fluorescence decrease were consistent with a slow conformational alteration in the transition-state inhibitor complexes. Fluorescence quenching experiments using polar and nonpolar quenchers were also carried out for the enzyme-inhibitor complexes. The quenching parameters for all enzyme-inhibitor complexes differed from those for the uncomplexed enzyme, suggesting that inhibitor binding causes changes in the conformation of adenosine deaminase. For comparison, parallel quenching studies were performed for calf adenosine deaminase in the absence and presence of inhibitors. While significant structural differences between adenosine deaminase from the two sources were evident, our data indicate that both enzymes undergo conformational changes on binding ground-state and transition-state inhibitors.     

Detection of legumin gene DNA sequences in pea by in situ hybridization

In order to detect low copy number sequences in pea using biotin-labelled probes we optimised some aspects of the in situ hybridization technique. We found protoplast preparations to be superior to standard squashes in terms of their signal: noise ratio. Heat and alkali denaturation of chromosomal DNA were both more effective than acid denaturation. A comparison of antibody-fluorochrome and streptavidin-enzyme conjugates showed the streptavidin-alkaline phosphatase conjugate to be the most sensitive detection system. Using the optimised method, we were able to detect a single site for a 13.5 kb legumin gene clone.     

Phospholipase D1 Regulates Lymphocyte Adhesion via Upregulation of Rap1 at the Plasma Membrane

Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated. We report here that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as Rap1 and is translocated to the plasma membrane along with Rap1. Moreover, PLD activity is required for both translocation and activation of Rap1. Increased T-cell adhesion in response to stimulation of the antigen receptor depended on PLD1. C3G, a Rap1 guanine nucleotide exchange factor located in the cytosol of resting cells, translocated to the plasma membranes of stimulated T cells. Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane.Regulated adhesion of lymphocytes is required for immune function. The β2 integrin lymphocyte function-associated antigen 1 (LFA-1) mediates lymphocyte adhesion to endothelium, antigen-presenting cells, and virally infected target cells (14). These cell-cell adhesions enable lymphocyte trafficking in and out of lymphoid organs, T-cell activation, and cytotoxicity, respectively (2, 34). Thus, the regulation of LFA-1 adhesiveness is central to adaptive immunity.LFA-1 is a bidirectional receptor in that it mediates both outside-in and inside-out signaling (30). Outside-in signaling is analogous to signaling by conventional receptors and is defined as stimulation of intracellular signaling pathways as a consequence of ligation of LFA-1 with any of its extracellular ligands, such as intracellular adhesion molecule 1 (ICAM-1). Inside-out signaling refers to intracellular signaling events that result in a higher-affinity state of the ectodomain of LFA-1 for its cognate ligands. Regulatory events that mediate inside-out signaling converge on the cytoplasmic tails of the LFA-1 α and β chains, which transduce signals to their ectodomains (14). Signaling molecules implicated in inside-out signaling through LFA-1 include talin, Vav1, PKD1, several adaptor proteins (SLP-76, ADAP, and SKAP-55), the Ras family GTPase Rap1, and two of its effectors, RAPL and RIAM (26). How these proteins interact to activate LFA-1 remains poorly understood.Rap1 is a member of the Ras family of GTPases and has been implicated in growth control, protein trafficking, polarity, and cell-cell adhesion (6). The ability of activated Rap1 to promote LFA-1-mediated lymphocyte adhesion is well established (33). The physiologic relevance of this pathway is highlighted by leukocyte adhesion deficiency type III (LAD III), where immunocompromised patients have a congenital defect in GTP loading of Rap1 in leukocytes (24). LFA-1 is a plasma membrane protein, consistent with its role in cell-cell adhesion, which by definition is a cell surface phenomenon. Paradoxically, the bulk of Rap1 is expressed on intracellular vesicles. We have characterized these vesicles as recycling endosomes and have shown that the intracellular pool of Rap1 can be mobilized by exocytosis to augment the expression of Rap1 at the plasma membranes of lymphocytes, leading to increased adhesion (5). We used a fluorescent probe of activated Rap1 in live cells to show that only the pool of Rap1 at the plasma membrane becomes GTP bound upon lymphocyte activation. Thus, it appears that delivery of Rap1 via vesicular transport to the plasma membrane and activation of the GTPase on that compartment are linked. Among the signaling enzymes known to regulate vesicular trafficking is phospholipase D (PLD). Whereas PLD type 2 (PLD2) is expressed at the plasma membranes of lymphocytes, PLD1 is expressed on intracellular vesicles (29). We now show that PLD1 resides on the same vesicles as Rap1, is delivered along with Rap1 to the plasma membranes of stimulated T cells, and is required for Rap1 activation and T-cell adhesion.