It depends on the hinge: a structure-functional analysis of galectin-8, a tandem-repeat type lectin

Galectin-8, a member of the galectin family of mammalian lectins, is made of two carbohydrate-recognition domains (CRDs), joined by a "hinge" region. Ligation of integrins by galectin-8 induces a distinct cytoskeletal organization, associated with activation of the extracellular-regulated kinase (ERK) and phosphatidylinositol 3-kinase signaling cascades. We show that these properties of galectin-8 are mediated by the concerted action of its two CRDs and involve both protein-sugar and protein-protein interactions. Accordingly, the isolated N- or C-CRD domains of galectin-8 or galectin-8 mutated at selected residues implicated in sugar binding (E251Q; W85Y, W248Y, W[85,248]Y) exhibited reduced sugar binding, which was accompanied by severe impairment in the capacity of these mutants to promote the adhesive, spreading, and signaling functions of galectin-8. Other mutations that did not impair sugar binding (e.g. E88Q) still impeded the signaling and cell-adherence functions of galectin-8. Deletion of the "hinge" region similarly impaired the biological effects of galectin-8. These results provide evidence that cooperative interactions between the two CRDs and the "hinge" domain are required for the proper functioning of galectin-8.     

The molecular chaperone Hsp90α is required for meiotic progression of spermatocytes beyond pachytene in the mouse

The molecular chaperone Hsp90 has been found to be essential for viability in all tested eukaryotes, from the budding yeast to Drosophila. In mammals, two genes encode the two highly similar and functionally largely redundant isoforms Hsp90α and Hsp90β. Although they are co-expressed in most if not all cells, their relative levels vary between tissues and during development. Since mouse embryos lacking Hsp90β die at implantation, and despite the fact that Hsp90 inhibitors being tested as anti-cancer agents are relatively well tolerated, the organismic functions of Hsp90 in mammals remain largely unknown. We have generated mouse lines carrying gene trap insertions in the Hsp90α gene to investigate the global functions of this isoform. Surprisingly, mice without Hsp90α are apparently normal, with one major exception. Mutant male mice, whose Hsp90β levels are unchanged, are sterile because of a complete failure to produce sperm. While the development of the male reproductive system appears to be normal, spermatogenesis arrests specifically at the pachytene stage of meiosis I. Over time, the number of spermatocytes and the levels of the meiotic regulators and Hsp90 interactors Hsp70-2, NASP and Cdc2 are reduced. We speculate that Hsp90α may be required to maintain and to activate these regulators and/or to disassemble the synaptonemal complex that holds homologous chromosomes together. The link between fertility and Hsp90 is further supported by our finding that an Hsp90 inhibitor that can cross the blood-testis barrier can partially phenocopy the genetic defects.     

TNF-α Regulates the Effects of Irradiation in the Mouse Bone Marrow Microenvironment

Background

Secondary bone marrow (BM) myelodysplastic syndromes (MDS) are increasingly common, as a result of radio or chemotherapy administered to a majority of cancer patients. Patients with secondary MDS have increased BM cell apoptosis, which results in BM dysfunction (cytopenias), and an increased risk of developing fatal acute leukemias. In the present study we asked whether TNF-α, known to regulate cell apoptosis, could modulate the onset of secondary MDS.

Principal Findings

We show that TNF-α is induced by irradiation and regulates BM cells apoptosis in vitro and in vivo. In contrast to irradiated wild type (WT) mice, TNF-α deficient (TNF-α KO) mice or WT mice treated with a TNF-α-neutralizing antibody were partially protected from the apoptotic effects of irradiation. Next we established a 3-cycle irradiation protocol, in which mice were sub-lethally irradiated once monthly over a 3 month period. In this model, irradiated WT mice presented loss of microsatellite markers on BM cells, low white blood cell (WBC) counts, reduced megakaryocyte (MK) and platelet levels (thrombocytopenia) and macrocytic anemia, phenoypes that suggest the irradiation protocol resulted in BM dysfunction with clinical features of MDS. In contrast, TNF-α KO mice were protected from the irradiation effects: BM cell apoptosis following irradiation was significantly reduced, concomitant with sustained BM MK numbers and absence of other cytopenias. Moreover, irradiated WT mice with long term (≥5 months) BM dysfunction had increased BM angiogenesis, MMPs and VEGF and NFkB p65, suggestive of disease progression.

Conclusion

Taken together, our data shows that TNF-α induction following irradiation modulates BM cell apoptosis and is a crucial event in BM dysfunction, secondary MDS onset and progression.     

Acute exercise boosts cell proliferation and the heat shock response in lymphocytes: correlation with cytokine production and extracellular-to-intracellular HSP70 ratio

Exercise stimulates immune responses, but the appropriate “doses” for such achievements are unsettled. Conversely, in metabolic tissues, exercise improves the heat shock (HS) response, a universal cytoprotective response to proteostasis challenges that are centred on the expression of the 70-kDa family of intracellular heat shock proteins (iHSP70), which are anti-inflammatory. Concurrently, exercise triggers the export of HSP70 towards the extracellular milieu (eHSP70), where they work as pro-inflammatory cytokines. As the HS response is severely compromised in chronic degenerative diseases of inflammatory nature, we wondered whether acute exercise bouts of different intensities could alter the HS response of lymphocytes from secondary lymphoid organs and whether this would be related to immunoinflammatory responses. Adult male Wistar rats swam for 20 min at low, moderate, high or strenuous intensities as per an overload in tail base. Controls remained at rest under the same conditions. Afterwards, mesenteric lymph node lymphocytes were assessed for the potency of the HS response (42 °C for 2 h), NF-κB binding activity, mitogen-stimulated proliferation and cytokine production. Exercise stimulated cell proliferation in an “inverted-U” fashion peaking at moderate load, which was paralleled by suppression of NF-κB activation and nuclear location, and followed by enhanced HS response in relation to non-exercised animals. Comparative levels of eHSP70 to iHSP70 (H-index) matched IL-2/IL-10 ratios. We conclude that exercise, in a workload-dependent way, stimulates immunoinflammatory performance of lymphocytes of tissues far from the circulation and this is associated with H-index of stress response, which is useful to assess training status and immunosurveillance balance.     

Fine- and local- scale genetic structure of <Emphasis Type="Italic">Dysoxylum malabaricum,</Emphasis> a late-successional canopy tree species in disturbed forest patches in the Western Ghats,India

Dysoxylum malabaricum (white cedar) is an economically important tree species, endemic to the Western Ghats, India, which is the world’s most densely populated biodiversity hotspot. In this study, we used variation at ten nuclear simple sequence repeat loci to investigate genetic diversity and fine scale spatial genetic structure (FSGS) in seedlings and adults of D. malabaricum from four forest patches in the northern part of the Western Ghats. When genetic variation was compared between seedlings and adults across locations, significant differences were detected in allelic richness, observed heterozygosity, fixation index (F _IS), and relatedness (P < 0.05). Reduced genetic diversity and increased relatedness at the seedling stage might be due to fragmentation and disturbance. There was no FSGS at the adult stage and FSGS was limited to shorter distance classes at the seedling stage. However, there was clear spatial genetic structure at the landscape level (<50 km), regardless of age class, due to limited gene flow between forest patches. A comparison of the distributions of size classes in the four locations with published data from a more southern area, showed that large trees (diameter at breast height, DBH, >130 cm) are present in the southern sacred forests but not in the northern forest reserves. This pattern is likely due to stronger harvesting pressure in the north compared to the south, because in the north there are no cultural taboos regulating the extraction of natural resources. The implications for forest conservation in this biodiversity hotspot are discussed.     

The Role of Early Fungal Colonizers in Leaf‐Litter Decomposition in Portuguese Streams Impacted by Agricultural Runoff

We conducted a transplant experiment between two streams in NW Portugal impacted by agricultural runoff, mainly differing in phosphate concentration, to determine whether fungi on decomposing leaves would adapt to the new environment or would be replaced by fungi of the recipient stream. The most nutrient enriched stream had lower fungal diversity but faster leaf decomposition. Leaf transplantation did not alter fungal activity or species dominance. Multidimensional scaling ordination of fungal communities, from DNA fingerprint or conidial production, revealed that transplanted communities resembled more those of the original stream than the recipient stream. Results suggest that early fungal colonizers will determine the development and activity of fungal communities on decomposing leaves in streams impacted by agricultural practices. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

PKA compartmentalization links cAMP signaling and autophagy

Autophagy is a highly regulated degradative process crucial for maintaining cell homeostasis. This important catabolic mechanism can be nonspecific, but usually occurs with fine spatial selectivity (compartmentalization), engaging only specific subcellular sites. While the molecular machines driving autophagy are well understood, the involvement of localized signaling events in this process is not well defined. Among the pathways that regulate autophagy, the cyclic AMP (cAMP)/protein kinase A (PKA) cascade can be compartmentalized in distinct functional units called microdomains. However, while it is well established that, depending on the cell type, cAMP can inhibit or promote autophagy, the role of cAMP/PKA microdomains has not been tested. Here we show not only that the effects on autophagy of the same cAMP elevation differ in different cell types, but that they depend on a highly complex sub-compartmentalization of the signaling cascade. We show in addition that, in HT-29 cells, in which autophagy is modulated by cAMP rising treatments, PKA activity is strictly regulated in space and time by phosphatases, which largely prevent the phosphorylation of soluble substrates, while membrane-bound targets are less sensitive to the action of these enzymes. Interestingly, we also found that the subcellular distribution of PKA type-II regulatory PKA subunits hinders the effect of PKA on autophagy, while displacement of type-I regulatory PKA subunits has no effect. Our data demonstrate that local PKA activity can occur independently of local cAMP concentrations and provide strong evidence for a link between localized PKA signaling events and autophagy.Subject terms: Kinases, Autophagy