Divergent RNA binding specificity of yeast Puf2p

PUF proteins bind mRNAs and regulate their translation, stability, and localization. Each PUF protein binds a selective group of mRNAs, enabling their coordinate control. We focus here on the specificity of Puf2p and Puf1p of Saccharomyces cerevisiae, which copurify with overlapping groups of mRNAs. We applied an RNA-adapted version of the DRIM algorithm to identify putative binding sequences for both proteins. We first identified a novel motif in the 3' UTRs of mRNAs previously shown to associate with Puf2p. This motif consisted of two UAAU tetranucleotides separated by a 3-nt linker sequence, which we refer to as the dual UAAU motif. The dual UAAU motif was necessary for binding to Puf2p, as judged by gel shift, yeast three-hybrid, and coimmunoprecipitation from yeast lysates. The UAAU tetranucleotides are required for optimal binding, while the identity and length of the linker sequences are less critical. Puf1p also binds the dual UAAU sequence, consistent with the prior observation that it associates with similar populations of mRNAs. In contrast, three other canonical yeast PUF proteins fail to bind the Puf2p recognition site. The dual UAAU motif is distinct from previously known PUF protein binding sites, which invariably possess a UGU trinucleotide. This study expands the repertoire of cis elements bound by PUF proteins and suggests new modes by which PUF proteins recognize their mRNA targets.     

The Arabidopsis Rho of Plants GTPase AtROP6 Functions in Developmental and Pathogen Response Pathways

How plants coordinate developmental processes and environmental stress responses is a pressing question. Here, we show that Arabidopsis (Arabidopsis thaliana) Rho of Plants6 (AtROP6) integrates developmental and pathogen response signaling. AtROP6 expression is induced by auxin and detected in the root meristem, lateral root initials, and leaf hydathodes. Plants expressing a dominant negative AtROP6 (rop6^DN) under the regulation of its endogenous promoter are small and have multiple inflorescence stems, twisted leaves, deformed leaf epidermis pavement cells, and differentially organized cytoskeleton. Microarray analyses of rop6^DN plants revealed that major changes in gene expression are associated with constitutive salicylic acid (SA)-mediated defense responses. In agreement, their free and total SA levels resembled those of wild-type plants inoculated with a virulent powdery mildew pathogen. The constitutive SA-associated response in rop6^DN was suppressed in mutant backgrounds defective in SA signaling (nonexpresser of PR genes1 [npr1]) or biosynthesis (salicylic acid induction deficient2 [sid2]). However, the rop6^DN npr1 and rop6^DN sid2 double mutants retained the aberrant developmental phenotypes, indicating that the constitutive SA response can be uncoupled from ROP function(s) in development. rop6^DN plants exhibited enhanced preinvasive defense responses to a host-adapted virulent powdery mildew fungus but were impaired in preinvasive defenses upon inoculation with a nonadapted powdery mildew. The host-adapted powdery mildew had a reduced reproductive fitness on rop6^DN plants, which was retained in mutant backgrounds defective in SA biosynthesis or signaling. Our findings indicate that both the morphological aberrations and altered sensitivity to powdery mildews of rop6^DN plants result from perturbations that are independent from the SA-associated response. These perturbations uncouple SA-dependent defense signaling from disease resistance execution.Rho of Plants (ROPs), also known as RACs (for clarity, the ROP nomenclature will be used throughout this article), comprise a plant-specific group of Rho family small G proteins. Like other members of the Ras superfamily of small G proteins, ROPs function as molecular switches, existing in a GTP-bound “on” state and a GDP-bound “off” state. In the GTP-bound state, ROPs interact with specific effectors that transduce downstream signaling or function as scaffolds for interaction with additional effector molecules (Berken and Wittinghofer, 2008). Conserved point mutations in the G1 (P loop) Gly-15 or the G3 (switch II) Gln-64, which abolish GTP hydrolysis, or the G1 Thr-20 or G4 Asp-121 that compromise GDP/GTP exchange, can form either constitutively active or dominant negative mutants, respectively (Feig, 1999; Berken et al., 2005; Berken and Wittinghofer, 2008; Sorek et al., 2010). Primarily based on studies with neomorphic mutants, ROPs have been implicated in the regulation of cytoskeleton organization and dynamics, vesicle trafficking, auxin transport and response, abscisic acid (ABA) response, and response to pathogens (Nibau et al., 2006; Yalovsky et al., 2008; Yang, 2008; Lorek et al., 2010; Wu et al., 2011; and refs. therein).In Arabidopsis (Arabidopsis thaliana), there are 11 ROP proteins (Winge et al., 1997). Assigning specific functions to individual members of this family is difficult, however, because ROPs are functionally redundant. A ROP10 loss-of-function mutant was reported to be ABA hypersensitive (Zheng et al., 2002), displaying enhanced expression of tens of genes in response to ABA treatments (Xin et al., 2005). However, in the absence of exogenous ABA, gene expression in the rop10 mutant was similar to that in wild-type plants (Xin et al., 2005). Loss of leaf epidermis pavement cell polarity was reported for rop4 rop2-RNAi (for RNA interference) double mutant plants (Fu et al., 2005). Mild changes in pavement and hypocotyl cell structure and microtubule (MT) organization were reported for a rop6 loss-of-function mutant (Fu et al., 2009).The involvement of ROPs in auxin-regulated development has been addressed in several studies (Wu et al., 2011). Ectopic expression of a dominant negative ROP2 (rop2^DN) mutant under regulation of the 35S promoter resulted in a loss of apical dominance and a reduction in the number of lateral roots. In contrast, ectopic expression of constitutively active ROP2 (rop2^CA) caused an increase in the number of lateral roots and an enhanced decrease in primary root length in response to auxin. Consistent with these findings, the expression of a constitutively active NtRAC1 in tobacco (Nicotiana tabacum) protoplasts induced the expression of auxin-regulated genes in the absence of auxin and promoted the formation of protein nuclear bodies containing components of the proteasome and COP9 signalosome (Tao et al., 2002, 2005; Wu et al., 2011). The ROP effector ICR1 (for interactor of constitutively active ROP1) regulates polarized secretion and is required for polar auxin transport (Lavy et al., 2007; Bloch et al., 2008; Hazak et al., 2010; Hazak and Yalovsky, 2010). In the root, local auxin gradients induce the accumulation of ROPs in trichoblasts at the site of future root hair formation (Fischer et al., 2006). Recently, it was shown that interdigitation of leaf epidermis pavement cells depends on Auxin-Binding Protein1 (ABP1)-mediated ROP activation (Xu et al., 2010). Taken together, these data indicate that ROPs are involved in both mediating the auxin response and facilitating directional auxin transport. It is still unclear, however, which ROPs function in these processes.ROP function was linked to plant defense responses in several studies. In rice (Oryza sativa), OsRAC1 is a positive regulator of the hypersensitive response, possibly through interactions with the NADPH oxidase RbohB, Required for Mla12 Resistance, and Heat Shock Protein90 (Ono et al., 2001; Thao et al., 2007; Wong et al., 2007). Interestingly, other members of the rice ROP family, namely RAC4 and RAC5, are negative regulators of resistance to the rice blast pathogen Magnaporthe grisea (Chen et al., 2010). Similar to rice, when expressed in tobacco, dominant negative OsRAC1 suppressed the hypersensitive response (Moeder et al., 2005). In barley (Hordeum vulgare), several constitutively active ROP/RAC mutants and a MT-associated ROPGAP1 loss-of-function mutant enhanced susceptibility to the powdery mildew Blumeria graminis f. sp. hordei (Bgh). The activated ROP-enhanced susceptibility to Bgh was attributed to disorganization of the actin cytoskeleton and was shown to depend on Mildew Resistance Locus O (MLO; Schultheiss et al., 2002, 2003; Opalski et al., 2005; Hoefle et al., 2011). In barley, three ROP proteins, HvRACB, HvRAC1, and HvRAC3, were linked to both development and pathogen response (Schultheiss et al., 2005; Pathuri et al., 2008; Hoefle et al., 2011).We have analyzed the function of the Arabidopsis AtROP6 (ROP6) by characterizing its expression pattern and its regulation by auxin and the phenotype of plants that express rop6^DN under the regulation of its endogenous promoter. The utilization of the dominant negative mutant overcame functional redundancy, while expression under the regulation of the endogenous promoter enabled the analysis of ROP6 function in a developmental context. Phenotypic and gene expression analyses indicate that ROP6 functions in developmental, salicylic acid (SA)-dependent, and SA-independent defense response pathways.     

Investigating Thought Disorder in Schizophrenia: Evidence for Pathological Activation

Background

Previous research has yielded evidence for enhanced semantic priming in formal thought-disordered schizophrenia patients, a result that fits well with the hypothesis of disinhibited processes of spreading activation in this population.

Objective

The current study examined whether hyper priming among schizophrenia patients is an outcome of further spreading of activation of a node or a result of farther activation of nodes in the semantic network. We also try to shed light on the fate of this activation.

Methods

The present study tested this hypothesis by using semantic and identical priming in two different experiments. SOA (stimulus onset asynchrony) was manipulated (240 ms vs. 740 ms) within block. It is assumed that among healthy individuals, performance relies on a balance between activation and inhibition processes, contrary to in schizophrenic individuals. In order to examine this hypothesis, we compared formal thought-disordered schizophrenia patients, non thought-disordered schizophrenia patients, and healthy controls.

Results

For thought-disordered schizophrenia patients, we found a large positive semantic effect and identical priming effect (129 ms and 154 ms, respectively) only with short SOA. SOA and type of priming did not modulate priming effects in the control groups.

Conclusions

This result supports the claim that there is a lack of inhibitory processes among thought-disordered patients. Hyper priming in the thought-disorder group may be an outcome of hyper activation followed by rapid decay below baseline threshold.     

Selective loss of glycogen synthase kinase-3α in birds reveals distinct roles for GSK-3 isozymes in tau phosphorylation

Mammalian glycogen synthase kinase-3 (GSK-3), a critical regulator in neuronal signaling, cognition, and behavior, exists as two isozymes GSK-3α and GSK-3β. Their distinct biological functions remains largely unknown. Here, we examined the evolutionary significance of each of these isozymes. Surprisingly, we found that unlike other vertebrates that harbor both GSK-3 genes, the GSK-3α gene is missing in birds. GSK-3-mediated tau phosphorylation was significantly lower in adult bird brains than in mouse brains, a phenomenon that was reproduced in GSK-3α knockout mouse brains. Tau phosphorylation was detected in brains from bird embryos suggesting that GSK-3 isozymes play distinct roles in tau phosphorylation during development. Birds are natural GSK-3α knockout organisms and may serve as a novel model to study the distinct functions of GSK-3 isozymes.     

Distinct differentiation potential of blood monocyte subsets in the lung

Peripheral blood monocytes are a population of circulating mononuclear phagocytes that harbor potential to differentiate into macrophages and dendritic cells. As in humans, monocytes in the mouse comprise two phenotypically distinct subsets that are Gr1(high)CX(3)CR1(int) and Gr1(low)CX(3)CR1(high), respectively. The question remains whether these populations contribute differentially to the generation of peripheral mononuclear phagocytes. In this study, we track the fate of adoptively transferred, fractionated monocyte subsets in the lung of recipient mice. We show that under inflammatory and noninflammatory conditions, both monocyte subsets give rise to pulmonary dendritic cells. In contrast, under the conditions studied, only Gr1(low)CX(3)CR1(high) monocytes, but not Gr1(high)CX(3)CR1(int) cells, had the potential to differentiate into lung macrophages. However, Gr1(high)CX(3)CR1(int) monocytes could acquire this potential upon conversion into Gr1(low)CX(3)CR1(high) cells. Our results therefore indicate an intrinsic dichotomy in the differentiation potential of the two main blood monocyte subsets.     

Lung macrophages serve as obligatory intermediate between blood monocytes and alveolar macrophages

Alveolar macrophages are a unique type of mononuclear phagocytes that populate the external surface of the lung cavity. Early studies have suggested that alveolar macrophages originate from tissue-resident, local precursors, whereas others reported their derivation from blood-borne cells. However, the role of circulating monocytes as precursors of alveolar macrophages was never directly tested. In this study, we show through the combined use of conditional cell ablation and adoptive cell transfer that alveolar macrophages originate in vivo from blood monocytes. Interestingly, this process requires an obligate intermediate stage, the differentiation of blood monocytes into parenchymal lung macrophages, which subsequently migrate into the alveolar space. We also provide direct evidence for the ability of both lung and alveolar macrophages to proliferate.     

Mathematical model of excitation-contraction in a uterine smooth muscle cell

Uterine contractility is generated by contractions of myometrial smooth muscle cells (SMCs) that compose most of the myometrial layer of the uterine wall. Calcium ion (Ca²⁺) entry into the cell can be initiated by depolarization of the cell membrane. The increase in the free Ca²⁺ concentration within the cell initiates a chain of reactions, which lead to formation of cross bridges between actin and myosin filaments, and thereby the cell contracts. During contraction the SMC shortens while it exerts forces on neighboring cells. A mathematical model of myometrial SMC contraction has been developed to study this process of excitation and contraction. The model can be used to describe the intracellular Ca²⁺ concentration and stress produced by the cell in response to depolarization of the cell membrane. The model accounts for the operation of three Ca²⁺ control mechanisms: voltage-operated Ca²⁺ channels, Ca²⁺ pumps, and Na⁺/Ca²⁺ exchangers. The processes of myosin light chain (MLC) phosphorylation and stress production are accounted for using the cross-bridge model of Hai and Murphy (Am J Physiol Cell Physiol 254: C99–C106, 1988) and are coupled to the Ca²⁺ concentration through the rate constant of myosin phosphorylation. Measurements of Ca²⁺, MLC phosphorylation, and force in contracting cells were used to set the model parameters and test its ability to predict the cell response to stimulation. The model has been used to reproduce results of voltage-clamp experiments performed in myometrial cells of pregnant rats as well as the results of simultaneous measurements of MLC phosphorylation and force production in human nonpregnant myometrial cells. cellular calcium control mechanisms; myometrial contractions; myosin light chain phosphorylation     

25 hydroxy-vitamin D(3)-1alpha hydroxylase expression and activity in cultured human osteoblasts and their modulation by parathyroid hormone, estrogenic compounds and dihydrotestosterone

Human osteoblasts (hOB) produce and respond to 1,25(OH)(2)D(3) (1,25D), suggesting an autocrine/paracrine system. We therefore examined hormonal modulation of the expression and activity of 25 hydroxy-vitamin D(3)-1alpha hydroxylase (1-Ohase) in hOB. Cells from pre- and post-menopausal women or men, were treated with estrogenic compounds and 1-OHase expression and activity were measured. 1-OHase mRNA expression was highest in pre-menopausal women hOB and was increased by all hormones tested. In post-menopausal hOB all hormones except biochainin A (BA) and genistein (G) increased 1-OHase mRNA expressions to less extent. In male-derived hOB only dihydrotestosterone (DHT) and carboxy BA (cBA) increased 1-OHase mRNA expression. 1,25D production from 25(OH)D(3) had a K(m) of approximately 769-400 ng/ml (1.92-1.07 microM) and V(max) of 31.3-17.4 ng/ml (0.078-0.044 microM/60 min/5 x 10(6)cells) respectively, and was increased by all hormones except raloxifene (Ral) with higher stimulation in pre- than in post-menopausal cells. Only BA was almost five times more potent in pre- rather than post-menopausal hOBs. In male hOB only DHT and cBA increased 1,25D production whereas estradiol-17beta (E(2)) had no effect and BA decreased it. These results provide evidence for the expression of 1-OHase mRNA and production of 1,25D in hOBs, which are age and sex dependent and are hormonally modulated. The role of this local autocrine/paracrine 1,25D system in bone physiology deserves further investigation.