SRFR1 Negatively Regulates Plant NB-LRR Resistance Protein Accumulation to Prevent Autoimmunity

Plant defense responses need to be tightly regulated to prevent auto-immunity, which is detrimental to growth and development. To identify negative regulators of Resistance (R) protein-mediated resistance, we screened for mutants with constitutive defense responses in the npr1-1 background. Map-based cloning revealed that one of the mutant genes encodes a conserved TPR domain-containing protein previously known as SRFR1 (SUPPRESSOR OF rps4-RLD). The constitutive defense responses in the srfr1 mutants in Col-0 background are suppressed by mutations in SNC1, which encodes a TIR-NB-LRR (Toll Interleukin1 Receptor-Nucleotide Binding-Leu-Rich Repeat) R protein. Yeast two-hybrid screens identified SGT1a and SGT1b as interacting proteins of SRFR1. The interactions between SGT1 and SRFR1 were further confirmed by co-immunoprecipitation analysis. In srfr1 mutants, levels of multiple NB-LRR R proteins including SNC1, RPS2 and RPS4 are increased. Increased accumulation of SNC1 is also observed in the sgt1b mutant. Our data suggest that SRFR1 functions together with SGT1 to negatively regulate R protein accumulation, which is required for preventing auto-activation of plant immunity.     

Verification and tissue-specific expression of nifU-like gene from the amphioxus Branchiostoma belcheri

A nifU-like gene exhibiting similarity to nifU of nitrogen fixation gene cluster was identified for the first time from the gut cDNA library of amphioxus Branchiostoma belcheri. Both RT-PCR and Northern blotting as well as in situ hybridization histochemistry verified that the cDNA represents an amphioxus nifU-like gene rather than a microbial contaminant. The nifU-like gene encodes a protein of 164 amino acid residues including a highly conserved U-type motif (C-X26-C-X43-C), and shares 66-86% identity to NifU-like proteins from a variety of species including vertebrates, invertebrates and microbes. It is expressed in a tissue-specific manner in the digestive system including epipharyngeal groove, endostyle, hepatic caecum and hind-gut and in the gill, ovary and testis. Taken together, it is highly likely that NifU-like protein plays some tissue-dependent and critical role in amphioxus.     

Analysis of DNA sequences of six chloroplast and nuclear genes suggests incongruence, introgression, and incomplete lineage sorting in the evolution of Lespedeza (Fabaceae)

The genus Lespedeza (Fabaceae) consists of 40 species disjunctively distributed in East Asia and eastern North America. Phylogenetic relationships of all Lespedeza species and closely related genera were reconstructed using maximum parsimony, maximum likelihood, and Bayesian analyses of sequence data from five chloroplast (rpl16, rpl32-trnL, rps16-trnQ, trnL-F, and trnK/matK) and one nuclear (ITS) DNA regions. All analyses yielded consistent relationships among major lineages. Our results suggested that Campylotropis, Kummerowia, and Lespedeza are monophyletic, respectively. Lespedeza is resolved as sister to Kummerowia and these two together are further sister to Campylotropis. Neither of the two subgenera, subgen. Lespedeza and subgen. Macrolespedeza, in Lespedeza based on morphological characters, is recovered as monophyletic. Within Lespedeza, the North American clade is retrieved as sister to the Asian clade. The nuclear and chloroplast markers showed incongruent phylogenetic signals at shallow-level phylogeny, which may point to either introgression or incomplete lineage sorting in Lespedeza. The divergence times within Lespedeza and among related genera were estimated using Bayesian approach with BEAST. It is assumed that following the divergence between Kummerowia and Lespedeza in Asia in the late Miocene, the ancestor of Lespedeza diverged into the North American and the Asian lineages. The North American ancestor quickly migrated to North America through the Bering land bridge in the late Miocene. The North American and Asian lineages started to diversify almost simultaneously in the late Miocene but resulted in biased numbers of species in two continents.     

A time–frequency approach to estimate critical time intervals in postural control

The critical time interval (CTI) is a parameter that has been used to distinguish open-loop from closed-loop control during upright stance. The aim of this study was to develop a new method to determine CTIs. The new approach, termed the intermittent critical time interval (ICTI) method, was motivated from evidence that upright standing is an intermittent rather than an asymptotic stability control process. For this ICTI method, center-of-pressure time series are first transformed to the time–frequency domain with a wavelet method. Subsequently, the CTI is assumed equal to the time span between two local maxima in the time–frequency domain within a distinct frequency band (i.e., 0.5–1.1 Hz). This new method may help facilitate better estimates of the transition time interval between open and closed-loop control during upright stance and can also be applied in future work such as in simulating postural control. In addition, this method can be used in future work to assess temporal changes in CTIs.     

A Dexamethasone Prodrug Reduces the Renal Macrophage Response and Provides Enhanced Resolution of Established Murine Lupus Nephritis

We evaluated the ability of a macromolecular prodrug of dexamethasone (P-Dex) to treat lupus nephritis in (NZB × NZW)F1 mice. We also explored the mechanism underlying the anti-inflammatory effects of this prodrug. P-Dex eliminated albuminuria in most (NZB × NZW)F1 mice. Furthermore, P-Dex reduced the incidence of severe nephritis and extended lifespan in these mice. P-Dex treatment also prevented the development of lupus-associated hypertension and vasculitis. Although P-Dex did not reduce serum levels of anti-dsDNA antibodies or glomerular immune complexes, P-Dex reduced macrophage recruitment to the kidney and attenuated tubulointerstitial injury. In contrast to what was observed with free dexamethasone, P-Dex did not induce any deterioration of bone quality. However, P-Dex did lead to reduced peripheral white blood cell counts and adrenal gland atrophy. These results suggest that P-Dex is more effective and less toxic than free dexamethasone for the treatment of lupus nephritis in (NZB × NZW)F1 mice. Furthermore, the data suggest that P-Dex may treat nephritis by attenuating the renal inflammatory response to immune complexes, leading to decreased immune cell infiltration and diminished renal inflammation and injury.     

A predominantly nuclear protein affecting cytoplasmic localization of beta-actin mRNA in fibroblasts and neurons.

The localization of beta-actin mRNA to the leading lamellae of chicken fibroblasts and neurite growth cones of developing neurons requires a 54-nt localization signal (the zipcode) within the 3' untranslated region. In this study we have identified and isolated five proteins binding to the zipcode. One of these we previously identified as zipcode binding protein (ZBP)1, a 4-KH domain protein. A second is now investigated in detail: a 92-kD protein, ZBP2, that is especially abundant in extracts from embryonic brain. We show that ZBP2 is a homologue of the human hnRNP protein, KSRP, that appears to mediate pre-mRNA splicing. However, ZBP2 has a 47-amino acid (aa) sequence not present in KSRP. Various portions of ZBP2 fused to GFP indicate that the protein most likely shuttles between the nucleus and the cytoplasm, and that the 47-aa insert promotes the nuclear localization. Expression of a truncated ZBP2 inhibits the localization of beta-actin mRNA in both fibroblast and neurons. These data suggest that ZBP2, although predominantly a nuclear protein, has a role in the cytoplasmic localization of beta-actin mRNA.     

Aldosterone induction of hepatic stellate cell contraction through activation of RhoA/ROCK-2 signaling pathway

The RhoA/ROCK-2 signaling pathway is necessary for activated hepatic stellate cell (HSC) contraction. HSC contraction plays an important role in the pathogenesis of cirrhosis and portal hypertension. This study investigated whether aldosterone contributes to HSC contraction by activation of the RhoA/ROCK-2 signaling pathway. Primary HSCs were isolated from Sprague-Dawley rats via in situ pronase/collagenase perfusion. We found that aldosterone enhanced the contraction of a collagen lattice seeded with HSCs. This induced contraction was suppressed by the mineralcorticoid receptor (MR) inhibitor spironolactone, the ROCK-2 inhibitor Y27632, and the angiotensin II type 1 receptor (AT(1)R) inhibitor irbesartan. Moreover, actin fiber staining showed that aldosterone significantly increased actin fiber formation in HSCs. Pre-incubating with spironolactone, Y27632, or irbesartan inhibited the aldosterone-induced actin fiber reorganization. Molecularly, the effect of aldosterone on activation of HSC contraction was mediated by phosphorylated myosin light chain (P-MLC) through the RhoA/ROCK-2 signaling pathway. All these inhibitors had the ability to block aldosterone-induced protein expressions in the RhoA/ROCK-2/P-MLC cascade in HSCs. Taken together, our current study suggests that aldosterone induces contraction of activated HSCs through the activation of the RhoA/ROCK-2 signaling pathway. This finding may provide a potential therapeutic target for control of cirrhosis and portal hypertension.     

Determinants of the interaction of the spinal muscular atrophy disease protein SMN with the dimethylarginine-modified box H/ACA small nucleolar ribonucleoprotein GAR1

Deletion or mutation of the SMN1 (survival of motor neurons) gene causes the common, fatal neuromuscular disease spinal muscular atrophy. The SMN protein is important in small nuclear ribonucleoprotein (snRNP) assembly and interacts with snRNP proteins via arginine/glycine-rich domains. Recently, SMN was also found to interact with core protein components of the two major families of small nucleolar RNPs, fibrillarin and GAR1, suggesting that SMN may also function in the assembly of small nucleolar RNPs. Here we present results that indicate that the interaction of SMN with GAR1 is mediated by the Tudor domain of SMN. Single point mutations within the Tudor domain, including a spinal muscular atrophy patient mutation, impair the interaction of SMN with GAR1. Furthermore, we find that either of the two arginine/glycine-rich domains of GAR1 can provide for interaction with SMN, but removal of both results in loss of the interaction. Finally, we have found that unlike the interaction of SMN with the Sm snRNP proteins, interaction with GAR1 and fibrillarin is not enhanced by arginine dimethylation. Our results argue against post-translational arginine dimethylation as a general requirement for SMN recognition of proteins bearing arginine/glycine-rich domains.