Journals
2009 EN
Pascale Seddas · Cecilia M. Arias · Christine Arnould
+7 more
To gain further insight into the role of the plant genome in arbuscular mycorrhiza (AM) establishment, we investigated whether symbiosis-related plant genes affect fungal gene expression in germinating spores and at the appressoria stage of root interactions. Glomus intraradices genes were identified in expressed sequence tag libraries of mycorrhizal Medicago truncatula roots by in silico expression analyses. Transcripts of a subset of genes, with predicted functions in transcription, protein synthesis, primary or secondary metabolism, or of unknown function, were monitored in spores and germinating spores and during interactions with roots of wild-type or mycorrhiza-defective (Myc-) mutants of M. truncatula. Not all the fungal genes were active in quiescent spores but all were expressed when G. intraradices spores germinated in wild-type M. truncatula root exudates or when appressoria or arbuscules were formed in association with wild-type M. truncatula roots. Most of the fungal genes were upregulated or induced at the stage of appressorium development. Inactivation of the M. truncatula genes DMI1, DMI2/MtSYM2, or DMI3/MtSYM13 was associated with altered fungal gene expression (nonactivation or inhibition), modified appressorium structure, and plant cell wall responses, providing first evidence that cell processes modified by symbiosis-related plant genes impact on root interactions by directly modulating AM fungal activity.
American Phytopathological Society
Journals
2009 EN
Sylvain La Camera · Claudine Balagué · Cornelia Göbel
+5 more
We previously reported that patatin-like protein 2 (PLP2), a pathogen-induced patatin-like lipid acyl hydrolase, promotes cell death and negatively affects Arabidopsis resistance to the fungus Botrytis cinerea and to the bacteria Pseudomonas syringae. We show here that, on the contrary, PLP2 contributes to resistance to Cucumber mosaic virus, an obligate parasite inducing the hypersensitive response. These contrasted impacts on different pathosystems were also reflected by differential effects on defense gene induction. To examine a possible link between PLP2 lipolytic activity and oxylipin metabolism, gene expression profiling was performed and identified B. cinerea among these pathogens as the strongest inducer of most oxylipin biosynthetic genes. Quantitative oxylipin profiling in wild-type and PLP2-modified, Botrytis-challenged plants established the massive accumulation of oxidized fatty acid derivatives in infected leaves. Several compounds previously described as modulating plant tissue damage and issued from the α-dioxygenase pathway were found to accumulate in a PLP2-dependent manner. Finally, the contribution of PLP2 to genetically controlled cell death was evaluated using PLP2-silenced or -overexpressing plants crossed with the lesion mimic mutant vascular-associated death 1 (vad1). Phenotypic analysis of double-mutant progeny showed that PLP2 expression strongly promotes necrotic symptoms in vad1 leaves. Collectively, our data indicate that PLP2 is an integral component of the plant cell death execution machinery, possibly providing fatty acid precursors for the biosynthesis of specific oxylipins and differentially affecting resistance to pathogens with distinct lifestyles.
American Phytopathological Society
Journals
2009 EN
Penelope A.R. Hawken · Trina Jorre de St Jorre · Jennifer Rodger
+3 more
In many species, the reproductive centers of the brain are profoundly affected by sociosexual stimuli. This is particularly evident in female ungulates such as sheep, in which exposure to males switches them from reproductively quiescent to fertile. In two experiments with female sheep, we tested whether the brain centers that control gonadotropin-releasing hormone (GnRH) neuronal activity respond differentially to "novel" vs. familiar males and whether the neuroendocrine response is associated with increased cell proliferation in the hippocampus, a site associated with memory formation. In experiment 1, groups of 10 female sheep that had previously been habituated to males for 3 mo were re-exposed to familiar males or were exposed to novel males. Only the novel males increased luteinizing hormone (LH) pulse frequency, indicating stimulation of GnRH neuronal activity. In experiment 2, groups of six female sheep were injected with bromodeoxyuridine (BrdU) and then maintained in isolation from males or exposed to novel males. Two days later, the hippocampus and hypothalamus were removed and processed for fluorescence immunohistochemistry. Again, exposure to males increased LH pulse frequency. Most important, male exposure also doubled the number of BrdU-positive cells in the dentate gyrus of the hippocampus. No BrdU-positive cells were detected in the hypothalamus. We conclude that the stimulus from novel males switches on the reproductive centers of the brain of female sheep and rapidly doubles the rate of cell proliferation in the hippocampus. The rapidity of this response contrasts with rodents, in which several days of exposure to male pheromones seem necessary for an effect on neurogenesis.
Journals
2009 EN
Lee Winston Y · Weber Dominique A · Laur Oskar
+1 more
Interaction of SIRPα with its ligand, CD47 regulates leukocyte functions including transmigration, phagocytosis, oxidative burst, and cytokine secretion. However, the molecular details of how this transmembrane glycoprotein interacts with CD47 on the cell surface are incomplete. Here we report that SIRPα forms noncovalently linked cis‐ homodimers and present evidence to suggest that homodimerization facilitates binding to CD47. Treatment of SIRPα expressing cells with a membrane impermeable crosslinker allowed detection of SIRPα dimers and oligomers on western blots. Biochemical analyses of soluble, recombinant extracellular regions of SIRPα, including domain‐truncation mutants, revealed that each of the three extracellular immunoglobulin loops of SIRPα formed dimers in solution. Co‐immunoprecipitation experiments using cells transfected with different affinity‐tagged SIRPα molecules revealed that SIRPα may form cis dimers. Notably, in cells treated with tunicamycin, SIRPα dimerization but not CD47 binding was inhibited, suggesting that a SIRPα dimer may be bivalent and capable of binding CD47 with increased avidity. Lastly, in adherent neutrophils stimulated with chemoattractant, dimerization of SIRPα was enhanced. Collectively, these data suggest that cis dimerization of SIRPα plays an important role in regulating CD47 binding and function in leukocytes.
Federation of American Societies for Experimental Biology
Journals
2009 EN
Bragulla Hermann H. · Homberger Dominique G.
Earlier authors (e.g., Boas, 1931) have tried to homologize the skin modifications in mammalian cornified digital end organs (claws, hooves, and nails), but have considered only major parts, such as the wall and sole. A recent analysis of the cat claw (Homberger et al., in press) and horse hoof (Bragulla & Homberger, 2007) revealed that the subcutis, dermis, and epidermis are regionally modified to meet functional demands. The structure of digital end organs is, hence, more complex than anticipated. For example, the subcutis can form a hydrostatic cushion or ossify to strengthen the attachment of the dermis to the bony distal phalanx. The dermis forms a papillary body, which increases the dermo‐epidermal interface, enhances the supply of the avascular epidermis via diffusion, and strengthens the dermo‐epidermal bond. In addition, the papillae, ridges, or lamellae of the dermal papillary body often influence the architecture of the modified epidermis that forms the cornified digital end organs. Furthermore, several hard‐ and soft‐cornified segments can be identified and distinguished within the cornified digital end organs. The local and integrated mechanical properties of the digital end organs result, therefore, from different combinations of comparable building blocks of modified skin. Hence, the traditional homologizations between claws, hooves, and nails need to be re‐evaluated at a more detailed level.
Federation of American Societies for Experimental Biology
Journals
2009 EN
Donato Dominique M. · Ryzhova Larisa · Meenderink Leslie M.
+2 more
P130Cas is a major Src substrate in cell‐ECM adhesions implicated in cell motility. The objective of this study was to measure the dynamics of p130Cas localization to adhesions, while also assessing domain requirements for this targeting. Variants of YFP‐tagged p130Cas were stably expressed in p130Cas‐null fibroblasts. In addition to full‐length p130Cas, deletion mutants lacking either the SH3 domain or the highly conserved "Cas‐family C‐terminal homology" (CCH) domain were analyzed. Total internal reflection fluorescence microscopy of live cells revealed that p130Cas localizes to adhesions with a kinetic profile similar to paxillin ‐ a standard marker for monitoring adhesion dynamics. Variants lacking either the SH3 or CCH domain localized poorly to adhesions, while deletion of both domains completely abrogated the localization. The CCH domain was also found to be sufficient for adhesion targeting. Deletion of the CCH domain was further linked to defects in p130Cas tyrosine phosphorylation and wound healing cell migration. Thus both the SH3 and CCH domains play important roles in p130Cas adhesion site targeting and this localization is critical for p130Cas signaling leading to cell motility.
Federation of American Societies for Experimental Biology
Journals
2009 EN
Ousley Dominique D. · Deering Scott J. · Spitsbergen John M.
Glial cell line‐derived neurotrophic factor (GDNF) has been shown to play a role in motor neuron survival and plasticity. As GDNF plays a role in neural development we were interested in determining where and when GDNF was expressed in skeletal muscle during the final stages of neuromuscular junction development. We hypothesized that GDNF protein would be expressed at high levels at the end‐plate in 3 week old rats and that levels of expression would decline with increased age (5 and 8 weeks). Soleus , extensor digitorum longus and extensor hallucis longus muscles were removed and GDNF protein localization was examined using immunocytochemical methods. Muscles taken from 3 week old rats contained end‐plate regions at various stages of development, while muscles taken from older rats contained no immature end‐plates. The results showed little or no staining for GDNF protein at end‐plate regions in muscles from 3 week old rats. Muscles taken from older rats showed clear staining for GDNF protein at the end‐plate region. The results suggest that GDNF is not expressed at higher levels at developing end‐plates. These observations may suggest that GDNF plays a role at the neuromuscular junction beyond initial development, possibly being necessary for maintenance and growth of junctions as muscles continue to enlarge with animal growth. Supported by NIH Grant 1R15AG022908‐01A2, MSU‐KCMS, and Western Michigan University.
Federation of American Societies for Experimental Biology
Journals
2009 EN
Lauf Peter K. · Adragorma C. · Goossens Dominique
+1 more
The Rh blood group is a macromolecular complex of protein subunits (D and CE), RhAG, a NH 3 /CO 2 gas transporter (PNAS 101:17222,2004; FASEB J 22;1,2008), and accessory proteins (ICAM‐4/LW, CD47 and GPB). In the mild hemolytic human Rh null disorder, the Rh complex is absent/severely deficient. Altered K fluxes have been found in Rh null RBC (Blood 48: 457, 1976). A double blind study on male (m) or female (f) wild type (WT), and Rh −/− and Rhag −/− KO mice (Transf Clin Biol 13:164,2006) studied: Total, ouabain (O)‐sensitive Na/K pump, O‐insensitive+bumetanide (B)‐sensitive Na‐K‐2Cl cotransport (NKCC), and O‐ and B‐insensitive KCC ± the thiol alkylant N‐ethylmaleimide (NEM), staurosporine (STP, a kinase inhibitor) and Mg removal by A23187. Results 1) Total, Na/K pump, NKCC and basal KCC influx were not different between groups. 2) NEM activated KCC by >100 % (p<0.001) in both m/f Rh −/− and RhAg −/− , compared to WT. KCC activation by both STP, and Mg depletion (except for m Rh −/− ), was similar in m Rh −/− , m/f Rhag −/− RBCs and WT. Conclusion Thiol modification reveals a Rh‐complex role in KCC regulation, i.e. via protein phosphorylation or protein/protein interactions. However, unlike in human Rh null RBCs, Na/K pump and passive K‐"leak" fluxes were normal, an apparent difference in the Rh complex‐K transport association between mice and man. Support in part by a WSU School of Medicine Grant.
Federation of American Societies for Experimental Biology
Journals
2009 EN
Duquesnes Nicolas · Li Lin · Morel Eric
+6 more
Protein kinase C (PKC) activation is classically considered as independent of the β‐adrenergic pathway. However, the cAMP‐ activated exchange factor Epac was recently shown to activate phospholipase C. βA stimulation is thus likely to stimulate PKC. We evaluated in cardiomyocytes whether βA stimulation could activate PKCε. Rat neonatal cardiomyocytes were subjected to isoproterenol stimulation (ISO). Inositol trisphosphate production was increased by 34% by 1µM ISO (p<0.05) and PKCε was translocated to particulate fractions (PF, western blot) in the perinuclear area (confocal microscopy) in a PKA‐independent manner since it was not inhibited by an infection with an adenovirus encoding a PKA inhibitor. Instead, PKCε activation was Epac dependent since 8‐CPT, an Epac activator, induced the same PKCε translocation as ISO and siRNAs of Epac completetly inhibited PKCε activation. The same translocation of PKCε in PF induced by βA stimulation was found in adult isolated rat hearts perfused by ISO with a sarcolemmmal membrane localization. This was associated with a phosphorylation of connexin‐43 on ser368 that was blocked by the PKC inhibitor BIM. In conclusion, these data demonstrate a new interconnection between β‐adrenergic and PKC pathways via Epac in cardiac cells with a potential role in cell‐to‐cell communications.
Federation of American Societies for Experimental Biology
Journals
2009 EN
Osborn Michelle L. · Homberger Dominique G.
In humans, the clavicle and scapula are suspended from the occiput and the uniquely large mastoid process by the upper trapezius and sternocleidomastoid (SCM) muscles, thereby creating the cranio‐cervico‐omo‐clavicular complex (CCOCC). While it is known that the humerus is more robust and the clavicle shorter on the dominant side of individuals, other skeletal characters of the CCOCC have not been analyzed in this respect. When weights are carried or lifted, the contracting upper trapezius muscle offsets the added weight on the arms and stabilizes the shoulder by using the clavicle as a lever arm. The SCM muscle counteracts the action of the upper trapezius on the skull, preventing the skull from being retroflexed. To test these premises using a natural experiment, we analyzed the skeletal asymmetry of 54 right‐handed individuals. 71% had a wider right mastoid process. 90% of a subsample of 9 individuals had a higher right superior nuchal line and a wider right mastoid process, thereby providing larger attachment sites for the larger trapezius and SCM muscles of right‐handed individuals. These results suggest that the upper trapezius and SCM muscles act as synergists to stabilize the head and shoulders when loads are carried. Our inference is supported by the innervation and developmental pattern of the trapezius and SCM muscles.
Federation of American Societies for Experimental Biology