CRHSP-24 phosphorylation is regulated by multiple signaling pathways in pancreatic acinar cells

Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112-2117, 2002). Investigation of the effects of phorbol ester (12-o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in 32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+ dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 microM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.     

Inactivation of the glycine transporter 1 gene discloses vital role of glial glycine uptake in glycinergic inhibition

The glycine transporter subtype 1 (GlyT1) is widely expressed in astroglial cells throughout the mammalian central nervous system and has been implicated in the regulation of N-methyl-D-aspartate (NMDA) receptor activity. Newborn mice deficient in GlyT1 are anatomically normal but show severe motor and respiratory deficits and die during the first postnatal day. In brainstem slices from GlyT1-deficient mice, in vitro respiratory activity is strikingly reduced but normalized by the glycine receptor (GlyR) antagonist strychnine. Conversely, glycine or the GlyT1 inhibitor sarcosine suppress respiratory activity in slices from wild-type mice. Thus, during early postnatal life, GlyT1 is essential for regulating glycine concentrations at inhibitory GlyRs, and GlyT1 deletion generates symptoms found in human glycine encephalopathy.     

Effects of transforming growth factor β1 expression in a rat colon carcinoma: growth inhibition, leukocyte infiltration and production of interleukin-10 and tumor necrosis factor α

The cytokine transforming growth factor β-1 (TGFβ1), was transfected into a TGFβ1-negative rat colon carcinoma. The growth of isografts of TGFβ1-expressing tumors was compared to that of vector control transfectants. The TGFβ1 transfectant grew significantly more slowly after intrahepatic isografting than did vector control and wild-type tumors. The TGFβ1-transfected tumor tissue had significantly greater infiltration of both CD4⁺ and CD8⁺ T lymphocytes than did the vector control tumor. The tumor-infiltrating leukocytes (TIL) from TGFβ1-transfected tumor secreted significantly more of the cytokines interleukin-10 (IL-10) and tumor necrosis factor α (TNFα) than did TIL from the vector control tumor. The TGFβ1 transfectant also demonstrated a significantly slower outgrowth in immunodeficient SCID mice, supporting a non-T-lymphocyte-dependent mechanism for the tumor retardation. In SCID mice, the TGFβ1-transfected tumor demonstrated significantly greater infiltration of both granulocytes and macrophages than did the vector control transfectant. We also demonstrated a direct inhibitory effect of rat TNFα on tumor proliferation in vitro. These results suggest that TGFβ1 induces a local secretion of immunomodulating cytokines and that this may influence monocytes, lymphocytes and granulocytes to retard tumor outgrowth. Received: 7 July 1999 / Accepted: 12 August 1999     

Protein phosphatase 2A holoenzyme and its subunits from Medicago sativa

We detected an about 200 kDa holoenzyme of protein phosphatase 2A (PP2A) in the crude extract of Medicago sativa microcallus cells by gel permeation chromatography. By polymerase chain reaction (PCR) we isolated two M. sativa cDNA fragments corresponding to the catalytic (C) subunit, and one each coding for the A and the B regulatory subunits of PP2A. The C subunit sequences were different from that published previously, indicating the existence of at least three different isoforms in M. sativa. Using the PCR fragments as probes, we obtained two distinct full-length clones for both the A and B subunits from an alfalfa cDNA library. Our results demonstrate that the components of the PP2A holoenzyme, namely the catalytic and regulatory subunits, are present in alfalfa in several isoforms and that their sequences are highly similar to their plant, yeast and animal counterparts. The distinct regulatory subunit genes are constitutively expressed during the cell cycle. Interestingly, two A-B subunit pairs had parallel mRNA steady-state levels in different plant tissues suggesting that not all of the possible isoform combinations are present in all tissues. The expression of the MsPP2A B subunit form was induced by abscisic acid indicating a specific function for this protein in the stress response.     

Biomanipulation as an Application of Food-Chain Theory: Constraints, Synthesis, and Recommendations for Temperate Lakes

The aim of this review is to identify problems, find general patterns, and extract recommendations for successful biomanipulation. An important conclusion is that the pelagic food chain from fish to algae may not be the only process affected by a biomanipulation. Instead, this process should be viewed as the “trigger” for secondary processes, such as establishment of submerged macrophytes, reduced internal loading of nutrients, and reduced resuspension of particles from the sediment. However, fish reduction also leads to a high recruitment of young-of-the-year (YOY) fish, which feed extensively on zooplankton. This expansion of YOY the first years after fish reduction is probably a major reason for less successful biomanipulations. Recent, large-scale biomanipulations have made it possible to update earlier recommendations regarding when, where, and how biomanipulation should be performed. More applicable recommendations include (1) the reduction in the biomass of planktivorous fish should be 75% or more; (2) the fish reduction should be performed efficiently and rapidly (within 1–3 years); (3) efforts should be made to reduce the number of benthic feeding fish; (4) the recruitment of YOY fish should be reduced; (5) the conditions for establishment of submerged macrophytes should be improved; and (6) the external input of nutrients (phosphorus and nitrogen) should be reduced as much as possible before the biomanipulation. Recent biomanipulations have shown that, correctly performed, the method also achieves results in large, relatively deep and eutrophic lakes, at least in a 5-year perspective. Although repeated measures may be necessary, the general conclusion is that biomanipulation is not only possible, but also a relatively inexpensive and attractive method for management of eutrophic lakes, and in particular as a follow-up measure to reduced nutrient load. Received 14 April 1998; accepted 31 August 1998     

Supression of hemin-mediated oxidation of low-density lipoprotein and subsequent endothelial reactions by hydrogen sulfide (H2S)

Heme-mediated oxidative modification of low-density lipoprotein (LDL) plays a crucial role in early atherogenesis. It has been shown that hydrogen sulfide (H₂S) produced by vascular smooth muscle cells is present in plasma at a concentration of about 50 µmol/L. H₂S is a strong reductant which can react with reactive oxygen species like superoxide anion and hydrogen peroxide. The current study investigated the effect of H₂S on hemin-mediated oxidation of LDL and oxidized LDL (oxLDL)-induced endothelial reactions. H₂S dose dependently delayed the accumulation of lipid peroxidation products—conjugated dienes, lipid hydroperoxides (LOOH), and thiobarbituric acid reactive substances—during hemin-mediated oxidation. Moreover, H₂S decreased the LOOH content of both oxidized LDL and lipid extracts derived from soft atherosclerotic plaque, which was accompanied by reduced cytotoxicity. OxLDL-mediated induction of the oxidative stress responsive gene, heme oxygenase-1, was also abolished by H₂S. Finally we have shown that H₂S can directly protect endothelium against hydrogen peroxide and oxLDL-mediated endothelial cytotoxicity. These results demonstrate novel functions of H₂S in preventing hemin-mediated oxidative modification of LDL, and consequent deleterious effects, suggesting a possible antiatherogenic action of H₂S.     

Molecular Characterization of Melanoma Cases in Denmark Suspected of Genetic Predisposition

Both environmental and host factors influence risk of cutaneous melanoma (CM), and worldwide, the incidence varies depending on constitutional determinants of skin type and pigmentation, latitude, and patterns of sun exposure. We performed genetic analysis of CDKN2A, CDK4, BAP1, MC1R, and MITFp.E318K in Danish high-risk melanoma cases and found CDKN2A germline mutations in 11.3% of CM families with three or more affected individuals, including four previously undescribed mutations. Rare mutations were also seen in CDK4 and BAP1, while MC1R variants were common, occurring at more than twice the frequency compared to Danish controls. The MITF p.E318K variant similarly occurred at an approximately three-fold higher frequency in melanoma cases than controls. To conclude, we propose that mutation screening of CDKN2A and CDK4 in Denmark should predominantly be performed in families with at least 3 cases of CM. In addition, we recommend that testing of BAP1 should not be conducted routinely in CM families but should be reserved for families with CM and uveal melanoma, or mesothelioma.     

Yield‐biodiversity trade‐off in patchy fields of Miscanthus × giganteus

Increasing crop productivity to meet rising demands for food and energy, but doing so in an environmentally sustainable manner, is one of the greatest challenges for agriculture to date. In Ireland, Miscanthus × giganteus has the potential to become a major feedstock for bioenergy production, but the economic feasibility of its cultivation depends on high yields. Miscanthus fields can have a large number of gaps in crop cover, adversely impacting yield and hence economic viability. Predominantly positive effects of Miscanthus on biodiversity reported from previous research might be attributable to high crop patchiness, particularly during the establishment phase. The aim of this research was to assess crop patchiness on a field scale and to analyse the relationship between Miscanthus yield and species richness and abundance of selected taxa of farmland wildlife. For 14 Miscanthus fields at the end of their establishment phase (4–5 years after planting), which had been planted either on improved grassland (MG) or tilled arable land (MT), we determined patchiness of the crop cover, percentage light penetration (LP) to the lower canopy, Miscanthus shoot density and height, vascular plants and epigeic arthropods. Plant species richness and noncrop vegetation cover in Miscanthus fields increased with increasing patchiness, due to higher levels of LP to the lower canopy. The species richness of ground beetles and the activity density of spiders followed the increase in vegetation cover. Plant species richness and activity density of spiders on both MT and MG fields, as well as vegetation cover and activity density of ground beetles on MG fields, were negatively associated with Miscanthus yield. In conclusion, positive effects of Miscanthus on biodiversity can diminish with increasing productivity. This matter needs to be considered when assessing the relative ecological impacts of developing biomass crops in comparison with other land use.