Prostasomes from four different species are able to produce extracellular adenosine triphosphate (ATP)

Background

Prostasomes are extracellular vesicles. Intracellularly they are enclosed by another larger vesicle, a so called “storage vesicle” equivalent to a multivesicular body of late endosomal origin. Prostasomes in their extracellular context are thought to play a crucial role in fertilization.

Methods

Prostasomes were purified according to a well worked-out schedule from seminal plasmas obtained from human, canine, equine and bovine species. The various prostasomes were subjected to SDS-PAGE separation and protein banding patterns were compared. To gain knowledge of the prostasomal protein systems pertaining to prostasomes of four different species proteins were analyzed using a proteomic approach. An in vitro assay was employed to demonstrate ATP formation by prostasomes of different species.

Results

The SDS-PAGE banding pattern of prostasomes from the four species revealed a richly faceted picture with most protein bands within the molecular weight range of 10–150 kDa. Some protein bands seemed to be concordant among species although differently expressed and the number of protein bands of dog prostasomes seemed to be distinctly fewer. Special emphasis was put on proteins involved in energy metabolic turnover. Prostasomes from all four species were able to form extracellular adenosine triphosphate (ATP). ATP formation was balanced by ATPase activity linked to the four types of prostasomes.

Conclusion

These potencies of a possession of functional ATP-forming enzymes by different prostasome types should be regarded against the knowledge of ATP having a profound effect on cell responses and now explicitly on the success of the sperm cell to fertilize the ovum.

General significance

This study unravels energy metabolic relationships of prostasomes from four different species.     

Quantifying Missing Heritability at Known GWAS Loci

Recent work has shown that much of the missing heritability of complex traits can be resolved by estimates of heritability explained by all genotyped SNPs. However, it is currently unknown how much heritability is missing due to poor tagging or additional causal variants at known GWAS loci. Here, we use variance components to quantify the heritability explained by all SNPs at known GWAS loci in nine diseases from WTCCC1 and WTCCC2. After accounting for expectation, we observed all SNPs at known GWAS loci to explain more heritability than GWAS-associated SNPs on average (). For some diseases, this increase was individually significant: for Multiple Sclerosis (MS) () and for Crohn''s Disease (CD) (); all analyses of autoimmune diseases excluded the well-studied MHC region. Additionally, we found that GWAS loci from other related traits also explained significant heritability. The union of all autoimmune disease loci explained more MS heritability than known MS SNPs () and more CD heritability than known CD SNPs (), with an analogous increase for all autoimmune diseases analyzed. We also observed significant increases in an analysis of Rheumatoid Arthritis (RA) samples typed on ImmunoChip, with more heritability from all SNPs at GWAS loci () and more heritability from all autoimmune disease loci () compared to known RA SNPs (including those identified in this cohort). Our methods adjust for LD between SNPs, which can bias standard estimates of heritability from SNPs even if all causal variants are typed. By comparing adjusted estimates, we hypothesize that the genome-wide distribution of causal variants is enriched for low-frequency alleles, but that causal variants at known GWAS loci are skewed towards common alleles. These findings have important ramifications for fine-mapping study design and our understanding of complex disease architecture.     

Steroid control of gonadotropin-releasing hormone secretion: associated changes in pro-opiomelanocortin and preproenkephalin messenger RNA expression in the ovine hypothalamus

The endogenous opioid peptides have been implicated in mediating the actions of estrogen and progesterone on GnRH release. We used in situ hybridization histochemistry to determine whether steroid-induced changes in GnRH/LH release in the female sheep are associated with changes in the cellular mRNA content of the precursors for beta-endorphin (pro-opiomelanocortin; POMC) and met-enkephalin (pre-proenkephalin; PENK). Two specific hypotheses were tested. First, that the inhibitory actions of progesterone are associated with an increase in opioid gene expression in specific hypothalamic nuclei. Our data support this hypothesis. Thus, an increase in progesterone was associated with increased POMC gene expression in the arcuate nucleus and PENK in the paraventricular nucleus. Further, the increase in POMC was restricted to regions of the arcuate nucleus that contain steroid sensitive beta-endorphin neurons. Our second hypothesis, that gene expression for the two opioid precursors would decrease prior to the start of the estradiol-stimulated GnRH surge, was not supported. Rather, POMC (but not PENK) gene expression in the arcuate nucleus was significantly higher in estradiol-treated animals than controls at the peak of the GnRH surge. These data suggest that beta-endorphin neurons in subdivisions of the arcuate nucleus and enkephalin neurons in the paraventricular nucleus are part of the neural network by which progesterone inhibits LH release. While enkephalin neurons may not play a role in estrogen positive feedback, increases in POMC mRNA in the arcuate nucleus at the time of the GnRH peak may be important for replenishing beta-endorphin stores and terminating estrous behavior.     

Sensitivity of yeast strains with long G-tails to levels of telomere-bound telomerase

The Saccharomyces cerevisiae Pif1p helicase is a negative regulator of telomere length that acts by removing telomerase from chromosome ends. The catalytic subunit of yeast telomerase, Est2p, is telomere associated throughout most of the cell cycle, with peaks of association in both G1 phase (when telomerase is not active) and late S/G2 phase (when telomerase is active). The G1 association of Est2p requires a specific interaction between Ku and telomerase RNA. In mutants lacking this interaction, telomeres were longer in the absence of Pif1p than in the presence of wild-type PIF1, indicating that endogenous Pif1p inhibits the active S/G2 form of telomerase. Pif1p abundance was cell cycle regulated, low in G1 and early S phase and peaking late in the cell cycle. Low Pif1p abundance in G1 phase was anaphase-promoting complex dependent. Thus, endogenous Pif1p is unlikely to act on G1 bound Est2p. Overexpression of Pif1p from a non-cell cycle-regulated promoter dramatically reduced viability in five strains with impaired end protection (cdc13–1, yku80Δ, yku70Δ, yku80–1, and yku80–4), all of which have longer single-strand G-tails than wild-type cells. This reduced viability was suppressed by deleting the EXO1 gene, which encodes a nuclease that acts at compromised telomeres, suggesting that the removal of telomerase by Pif1p exposed telomeres to further C-strand degradation. Consistent with this interpretation, depletion of Pif1p, which increases the amount of telomere-bound telomerase, suppressed the temperature sensitivity of yku70Δ and cdc13–1 cells. Furthermore, eliminating the pathway that recruits Est2p to telomeres in G1 phase in a cdc13–1 strain also reduced viability. These data suggest that wild-type levels of telomere-bound telomerase are critical for the viability of strains whose telomeres are already susceptible to degradation.     

Removal of virus from boar semen spiked with porcine circovirus type 2

The virus porcine circovirus type 2 (PCV2) is associated with different disease entities, including reproductive failure. The objective of this study was to investigate the use of a semen processing technique for the elimination of infectious PCV2 in semen. PCV2 was chosen as a model virus because of its small size, high resistance to inactivation and as a known risk factor for boar semen contamination. Aliquots of ejaculates were spiked with PCV2 and processed by a double processing technique, consisting of Single Layer Centrifugation on Androcoll?-P followed by a "swim-up" procedure. Samples were collected from the resulting fractions during the selection process and analyzed for the presence of infectious PCV2. Virus titres were determined by performing a 50% tissue culture infective dose assay (TCID(50)) by end point dilution and with the use of an indirect peroxidise monolayer assay technique. With an initial infectious virus titre of 3.25-3.82 (TCID(50))/50μL the two-step sperm selection method eliminated 2.92±0.23 logs of infectious PCV2, corresponding to more than 99% reduction. Sperm quality was not affected by the selection procedure.     

TGF-β1 modifications in nuclear matrix proteins of osteoblasts during differentiation

Nuclear matrix protein (NMP) composition of osteoblasts shows distinct two-dimensional gel electrophoretic profiles of labeled proteins as a function of stages of cellular differentiation. Because NMPs are involved in the control of gene expression, we examined modifications in the representation of NMPs induced by TGF-β1 treatment of osteoblasts to gain insight into the effects of TGF-β on development of the osteoblast phenotype. Exposure of proliferating fetal rat calvarial derived primary cells in culture to TGF-β1 for 48 h (day 4–6) modifies osteoblast cell morphology and proliferation and blocks subsequent formation of mineralized nodules. Nuclear matrix protein profiles were very similar between control and TGF-β–treated cultures until day 14, but subsequently differences in nuclear matrix proteins were apparent in TGF-β–treated cultures. These findings support the concept that TGF-β1 modifies the final stage of osteoblast mineralization and alters the composition of the osteoblast nuclear matrix as reflected by selective and TGF-β–dependent modifications in the levels of specific nuclear matrix proteins. The specific changes induced by TGF-β in nuclear matrix associated proteins may reflect specialized mechanisms by which TGF-β signalling mediates the alterations in cell organization and nodule formation and/or the consequential block in extracellular mineralization. J. Cell. Biochem. 69:291–303, 1998. © 1998 Wiley-Liss, Inc.     

Canopy interactions and physical stress gradients in subtidal communities

Species interactions are integral drivers of community structure and can change from competitive to facilitative with increasing environmental stress. In subtidal marine ecosystems, however, interactions along physical stress gradients have seldom been tested. We observed seaweed canopy interactions across depth and latitudinal gradients to test whether light and temperature stress structured interaction patterns. We also quantified interspecific and intraspecific interactions among nine subtidal canopy seaweed species across three continents to examine the general nature of interactions in subtidal systems under low consumer pressure. We reveal that positive and neutral interactions are widespread throughout global seaweed communities and the nature of interactions can change from competitive to facilitative with increasing light stress in shallow marine systems. These findings provide support for the stress gradient hypothesis within subtidal seaweed communities and highlight the importance of canopy interactions for the maintenance of subtidal marine habitats experiencing environmental stress.     

The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice

Previous studies have associated activation of canonical Wnt signaling in osteoblasts with elevated bone formation. Here we report that deletion of the murine Wnt antagonist, secreted frizzled-related protein (sFRP)-1, prolongs and enhances trabecular bone accrual in adult animals. sFRP-1 mRNA was expressed in bones and other tissues of +/+ mice but was not observed in -/- animals. Despite its broad tissue distribution, ablation of sFRP-1 did not affect blood and urine chemistries, most nonskeletal organs, or cortical bone. However, sFRP-1-/- mice exhibited increased trabecular bone mineral density, volume, and mineral apposition rate when compared with +/+ controls. The heightened trabecular bone mass of sFRP-1-/- mice was observed in adult animals between the ages of 13-52 wk, occurred in multiple skeletal sites, and was seen in both sexes. Mechanistically, loss of sFRP-1 reduced osteoblast and osteocyte apoptosis in vivo. In addition, deletion of sFRP-1 inhibited osteoblast lineage cell apoptosis while enhancing the proliferation and differentiation of these cells in vitro. Ablation of sFRP-1 also increased osteoclastogenesis in vitro, although changes in bone resorption were not observed in intact animals in vivo. Our findings demonstrate that deletion of sFRP-1 preferentially activates Wnt signaling in osteoblasts, leading to enhanced trabecular bone formation in adults.