RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G₂/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G₂/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G₂/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling.     

Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation.     

Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins

The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane alpha-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.     

Spatial variation of the stable nitrogen isotope ratio of woody plants along a topoedaphic gradient in a subtropical savanna

Variation in the stable N isotope ratio (δ¹⁵N) of plants and soils often reflects the influence of environment on the N cycle. We measured leaf δ¹⁵N and N concentration ([N]) on all individuals of Prosopis glandulosa (deciduous tree legume), Condalia hookeri (evergreen shrub), and Zanthoxylum fagara (evergreen shrub) present within a belt transect 308 m long × 12 m wide in a subtropical savanna ecosystem in southern Texas, USA in April and August 2005. Soil texture, gravimetric water content (GWC), total N and δ¹⁵N were also measured along the transect. At the landscape scale, leaf δ¹⁵N was negatively related to elevation for all the three species along this topoedaphic sequence. Changes in soil δ¹⁵N, total N, and GWC appeared to contribute to this spatial pattern of leaf δ¹⁵N. In lower portions of the landscape, greater soil N availability and GWC are associated with relatively high rates of both N mineralization and nitrification. Both soil δ¹⁵N and leaf [N] were positively correlated with leaf δ¹⁵N of non-N₂ fixing plants. Leaf δ¹⁵N of P. glandulosa, an N₂-fixing legume, did not correlate with leaf [N]; the δ¹⁵N of P. glandulosa’s leaves were closer to atmospheric N₂ and significantly lower than those of C. hookeri and Z. fagara. Additionally, at smaller spatial scales, a proximity index (which reflected the density and distance of surrounding P. glandulosa trees) was negatively correlated with leaf δ¹⁵N of C. hookeri and Z. fagara, indicating the N₂-fixing P. glandulosa may be important to the N nutrition of nearby non-N₂-fixing species. Our results indicate plant ¹⁵N natural abundance can reflect the extent of N retention and help us better understand N dynamics and plant-soil interactions at ecosystem and landscape scales.     

TLR2 and TLR4 Mediate Differential Responses to Limb Ischemia through MyD88-Dependent and Independent Pathways

Introduction

The danger signal HMGB1 is released from ischemic myocytes, and mediates angiogenesis in the setting of hindlimb ischemia. HMGB1 is a ligand for innate immune receptors TLR2 and TLR4. While both TLR2 and TLR4 signal through myeloid differentiation factor 88 (MyD88), TLR4 also uniquely signals through TIR-domain-containing adapter-inducing interferon-β (TRIF). We hypothesize that TLR2 and TLR4 mediate ischemic myocyte regeneration and angiogenesis in a manner that is dependent on MyD88 signaling.

Methods

Mice deficient in TLR2, TLR4, MyD88 and TRIF underwent femoral artery ligation in the right hindlimb. Laser Doppler perfusion imaging was used to assess the initial degree of ischemia and the extent of perfusion recovery. Muscle regeneration, necrosis and fat replacement at 2 weeks post-ligation were assessed histologically and vascular density was quantified by immunostaining. In vitro, endothelial tube formation was evaluated in matrigel in the setting of TLR2 and TLR4 antagonism.

Results

While control and TLR4 KO mice demonstrated prominent muscle regeneration, both TLR2 KO and TRIF KO mice exhibited marked necrosis with significant inflammatory cell infiltrate. However, MyD88 KO mice had a minimal response to the ischemic insult with little evidence of injury. This observation could not be explained by differences in perfusion recovery which was similar at two weeks in all the strains of mice. TLR2 KO mice demonstrated abnormal vessel morphology compared to other strains and impaired tube formation in vitro.

Discussion

TLR2 and TRIF signaling are necessary for muscle regeneration after ischemia while MyD88 may instead mediate muscle injury. The absence of TLR4 did not affect muscle responses to ischemia. TLR4 may mediate inflammatory responses through MyD88 that are exaggerated in the absence of TLR2. Additionally, the actions of TLR4 through TRIF may promote regenerative responses that are required for recovery from muscle ischemia.     

Progressive defects observed in mouse sperm during the course of three generations of selenium deficiency

Three successive generations of mice were fed a Torula yeast based Se-deficient diet with or without 0.1 ppm Se in the drinking water. The Se-deficient mice, in the course of three generations, showed a decrease in body weight, testis weight, epididymal weight, and sperm production. The percentage of morphologically abnormal sperm increased in successive generations. The majority of sperm defects were found in the midpiece region of the tail. Many of these aberrant sperm were motile. A progressive decrease in fertility was noted during the first two generations of Se deficiency. This system confirms the essential role of Se in spermatogenesis and provides a model for the evaluation of the primary effect of Se deprivation on the structural development of sperm.     

Synergistic activation of ENaC by three membrane-bound channel-activating serine proteases (mCAP1, mCAP2, and mCAP3) and serum- and glucocorticoid-regulated kinase (Sgk1) in Xenopus Oocytes

Sodium balance is maintained by the precise regulation of the activity of the epithelial sodium channel (ENaC) in the kidney. We have recently reported an extracellular activation of ENaC-mediated sodium transport (I(Na)) by a GPI-anchored serine protease (mouse channel-activating protein, mCAP1) that was isolated from a cortical collecting duct cell line derived from mouse kidney. In the present study, we have identified two additional membrane-bound serine proteases (mCAP2 and mCAP3) that are expressed in the same cell line. We show that each of these proteases is able to increase I(Na) 6-10-fold in the Xenopus oocyte expression system. I(Na) and the number (N) of channels expressed at the cell surface (measured by binding of a FLAG monoclonal I(125)-radioiodinated antibody) were measured in the same oocyte. Using this assay, we show that mCAP1 increases I(Na) 10-fold (P < 0.001) but N remained unchanged (P = 0.9), indicating that mCAP1 regulates ENaC activity by increasing its average open probability of the whole cell (wcP(o)). The serum- and glucocorticoid-regulated kinase (Sgk1) involved in the aldosterone-dependent signaling cascade enhances I(Na) by 2.5-fold (P < 0.001) and N by 1.6-fold (P < 0.001), indicating a dual effect on N and wcP(o). Compared with Sgk1 alone, coexpression of Sgk1 with mCAP1 leads to a ninefold increase in I(Na) (P < 0.001) and 1.3-fold in N (P < 0.02). Similar results were observed for mCAP2 and mCAP3. The synergism between CAPs and Sgk1 on I(Na) was always more than additive, indicating a true potentiation. The synergistic effect of the two activation pathways allows a large dynamic range for ENaC-mediated sodium regulation crucial for a tight control of sodium homeostasis.     

STRUCTURALLY PRESERVED FOSSIL PLANTS FROM ANTARCTICA. I. ANTARCTICYCAS,GEN. NOV., A TRIASSIC CYCAD STEM FROM THE BEARDMORE GLACIER AREA

Silicified stems with typical cycadalean anatomy are described from specimens collected from the Fremouw Formation (Triassic) in the Transantarctic Mountains of Antarctica. Axes are slender with a large parenchymatous pith and cortex separated by a narrow ring of vascular tissue. Mucilage canals are present in both pith and cortex. Vascular tissue consists of endarch primary xylem, a narrow band of secondary xylem tracheids, cambial zone, and region of secondary phloem. Vascular bundles contain uni- to triseriate rays with larger rays up to 2 mm wide separating the individual bundles. Pitting on primary xylem elements ranges from helical to scalariform; secondary xylem tracheids exhibit alternate circular bordered pits. Traces, often accompanied by a mucilage canal, extend out through the large rays into the cortex where some assume a girdling configuration. A zone of periderm is present at the periphery of the stem. Large and small roots are attached to the stem and are conspicuous in the surrounding matrix. The anatomy of the Antarctic cycad is compared with that of other fossil and extant cycadalean stems.     

Phosphorus and carbon availability regulate structural composition and complexity of AM fungal mycelium

The regulation of the structural composition and complexity of the mycelium of arbuscular mycorrhizal (AM) fungi is not well understood due to their obligate biotrophic nature. The aim of this study was to investigate the structure of extraradical mycelium at high and low availability of carbon (C) to the roots and phosphorus (P) to the fungus. We used monoxenic cultures of the AM fungus Rhizophagus irregularis (formerly Glomus intraradices) with transformed carrot roots as the host in a cultivation system including a root-free compartment into which the extraradical mycelium could grow. We found that high C availability increased hyphal length and spore production and anastomosis formation within individual mycelia. High P availability increased the formation of branched absorbing structures and reduced spore production and the overall length of runner hyphae. The complexity of the mycelium, as indicated by its fractal dimensions, increased with both high C and P availability. The results indicate that low P availability induces a growth pattern that reflects foraging for both P and C. Low C availability to AM roots could still support the explorative development of the mycelium when P availability was low. These findings help us to better understand the development of AM fungi in ecosystems with high P input and/or when plants are subjected to shading, grazing or any management practice that reduces the photosynthetic ability of the plant.