Complete Atrial-Specific Knockout of Sodium-Calcium Exchange Eliminates Sinoatrial Node Pacemaker Activity

The origin of sinoatrial node (SAN) pacemaker activity in the heart is controversial. The leading candidates are diastolic depolarization by “funny” current (I_f) through HCN4 channels (the “Membrane Clock“ hypothesis), depolarization by cardiac Na-Ca exchange (NCX1) in response to intracellular Ca cycling (the "Calcium Clock" hypothesis), and a combination of the two (“Coupled Clock”). To address this controversy, we used Cre/loxP technology to generate atrial-specific NCX1 KO mice. NCX1 protein was undetectable in KO atrial tissue, including the SAN. Surface ECG and intracardiac electrograms showed no atrial depolarization and a slow junctional escape rhythm in KO that responded appropriately to β-adrenergic and muscarinic stimulation. Although KO atria were quiescent they could be stimulated by external pacing suggesting that electrical coupling between cells remained intact. Despite normal electrophysiological properties of I_f in isolated patch clamped KO SAN cells, pacemaker activity was absent. Recurring Ca sparks were present in all KO SAN cells, suggesting that Ca cycling persists but is uncoupled from the sarcolemma. We conclude that NCX1 is required for normal pacemaker activity in murine SAN.     

Effects of a murine germ cell-specific knockout of Connexin 43 on Connexin expression in testis and fertility

Connexin 43 (Cx 43)—expressed by germ cells (GC), Sertoli cells (SC) and Leydig cells—is one of at least eleven Cx in the murine testis. A general knockout (KO) of Cx 43 in mice results in perinatal death and a SC-specific KO of Cx 43 (SCCx43KO) causes infertility of male mice by preventing the initiation of spermatogenesis. To further elucidate the role of Cx 43 in the testis, a new mouse model with a GC-specific KO of Cx 43 (GCCx43KO) was created by using the Cre/loxP recombination system. A transgenic mouse line expressing the Cre gene under the tissue non-specific alkaline phosphatase promoter and a transgenic floxed Cx 43-LacZ mouse line were mated. The resulting F1-generation was backcrossed with homozygous Cx 43 floxed mice, and offspring was genotyped. Immunohistochemical analysis of testes of different aged homozygous mice revealed normal spermatogenesis and reduced Cx 43 immunoreactions. RT-qPCR and Western blots showed a downregulation of Cx 43 mRNA and protein, and a nearly unchanged mRNA expression of Cx 26, Cx 33 and Cx 45 in pubertal and adult KO mice. Western blots revealed considerable immunoreactive bands for Cx 26 and Cx 45. Male and female homozygous GCCx43KO mice were viable and fertile. Our data suggest, in contrast to inter SC and inter SC–GC cross talk in SCCx43KO mice which depends selectively on Cx 43 expression, that Cx 43 in GC seems not to be essential in GC–SC communication, when other Cx persist to be expressed.     

No adaptation to altitude in the invasive plant Erigeron annuus in the Swiss Alps

The altitudinal distribution of plants is restricted by various environmental factors, with climatic conditions being one of the primary constraints. Here, we investigate what limits the altitudinal range of the introduced species Erigeron annuus in the Swiss Alps. We planted offspring of E. annuus plants originating from different altitudes into two common gardens, one located at an altitude representing the main area of distribution (400 m) and the other close to the current altitudinal limit of E. annuus in Switzerland (1000 m). In both common gardens all established plants survived and grew vigorously during the growing season. However, there was high winter mortality of seedlings at 1000 m. Furthermore, plant phenology was delayed and reproductive output was reduced at 1000 m, although the seeds produced were larger. The general lack of adaptation to altitude and only moderate levels of plasticity suggest that there is little potential for E. annuus to persist beyond its current altitudinal limit in the Swiss Alps. However, climate warming might promote the upward range expansion of E. annuus by reducing winter mortality and by increasing the chance of producing seeds within the growing season.     

CIN85 regulates dopamine receptor endocytosis and governs behaviour in mice

Despite extensive investigations of Cbl‐interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85^Δex2) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85^Δex2 animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post‐synaptic compartment of striatal neurons in which it co‐clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85^Δex2 mice.     

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.     

Altered dose response to beta-agonists in SERCA1a-expressing hearts ex vivo and in vivo

In this study we evaluated the contractile characteristics of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)1a-expressing hearts ex vivo and in vivo and in particular their response to beta-adrenergic stimulation. Analysis of isolated, work-performing hearts revealed that transgenic (TG) hearts develop much higher maximal rates of contraction and relaxation than wild-type (WT) hearts. Addition of isoproterenol only moderately increased the maximal rate of relaxation (+20%) but did not increase contractility or decrease relaxation time in TG hearts. Perfusion with varied buffer Ca(2+) concentrations indicated an altered dose response to Ca(2+). In vivo TG hearts displayed fairly higher maximal rates of contraction (+ 25%) but unchanged relaxation parameters and a blunted but significant response to dobutamine. Our study also shows that the phospholamban (PLB) level was decreased (-40%) and its phosphorylation status modified in TG hearts. This study clearly demonstrates that increases in SERCA protein level alter the beta-adrenergic response and affect the phosphorylation of PLB. Interestingly, the overall cardiac function in the live animal is only slightly enhanced, suggesting that (neuro)hormonal regulations may play an important role in controlling in vivo heart function.     

Ten-year survival analysis for renal carcinoma patients treated with an autologous tumour lysate vaccine in an adjuvant setting

About 30% of renal cell carcinomas (RCC) will develop recurrence after surgery. Despite evidence for a significantly improved survival by autologous tumour cell vaccination therapy, the procedure has not become standard. Between August 1993 and December 1996, 1,267 RCC patients undergoing radical nephrectomy in 84 German hospitals were subsequently treated by autologous tumour cell vaccination therapy. The study group comprised 692 patients with complete follow-up (stages pT2-3, pNx-2, M0 based on the TNM classification, 4th edition). Subsequent propensity-score matching according to 7 defined criteria with 861 control patients undergoing nephrectomy alone without adjuvant treatment at the Carl-Thiem-Hospital Cottbus, resulted in 495 matched pairs. Overall and stage-specific survival rates were analysed after a median follow-up of 131 months. The 5- and 10-year overall survival (OS) rates were 80.6 and 68.9% in the vaccine group and 79.2 and 62.1% in the control group (p = 0.066). Patients with pT3 stage RCC revealed 5- and 10-year OS rates of 71.3 and 53.6% in the study group and 65.4 and 36.2% in the control group (p = 0.022). In multivariable analysis, patients in the vaccine group showed a significantly improved survival both in the whole study group (HR = 1.28, p = 0.030) and in the subgroup presenting with pT3 stage tumours (HR = 1.67, p = 0.011). Adjuvant treatment with autologous vaccination therapy resulted in a significantly improved overall survival in pT3 stage RCC patients, suggesting benefit especially in this subgroup. However, controlled clinical trials integrating the recent TNM classification and further risk constellations are required to define additional patient groups that may derive benefit from this treatment.     

Structural Determination of Functional Units of the Nucleotide Binding Domain (NBD94) of the Reticulocyte Binding Protein Py235 of Plasmodium yoelii

Background

Invasion of the red blood cells (RBC) by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH) plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94) of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.

Methodology/Principal Findings

In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94_444–547, NBD94_566–663 and NBD94_674–793, respectively. Using fluorescence correlation spectroscopy NBD94_444–547 has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94_444–547 in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 Å in length. The high quality of the constructs, forming the hinge-region, NBD94_566–663 and NBD94_674–793 enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94_566–663 consists of two helices with 97.8 Å and 48.6 Å in length, linked by a loop. By comparison, the low resolution structure of NBD94_674–793 in solution represents a chair–like shape with three architectural segments.

Conclusions

These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the potential use of this region as a novel drug target.     

A Matter of Contrast: Yellow Flower Colour Constrains Style Length in Crocus species

Most flowers display distinct colour patterns comprising two different areas. The peripheral large-area component of floral colour patterns attracts flower visitors from some distance and the central small-area component guides flower visitors towards landing sites. Whereas the peripheral colour is largely variable among species, the central colour, produced mostly by anthers and pollen or pollen mimicking floral guides, is predominantly yellow and UV-absorbing. This holds also for yellow flowers that regularly display a UV bull’s eye pattern. Here we show that yellow-flowering Crocus species are a noticeable exception, since yellow-flowering Crocus species–being entirely UV-absorbing–exhibit low colour contrast between yellow reproductive organs and yellow tepals. The elongated yellow or orange-yellow style of Crocus flowers is a stamen-mimicking structure promoting cross-pollination by facilitating flower visitors’ contact with the apical stigma before the flower visitors are touching the anthers. Since Crocus species possess either yellow, violet or white tepals, the colour contrast between the stamen-mimicking style and the tepals varies among species. In this study comprising 106 Crocus species, it was tested whether the style length of Crocus flowers is dependent on the corolla colour. The results show that members of the genus Crocus with yellow tepals have evolved independently up to twelve times in the genus Crocus and that yellow-flowering Crocus species possess shorter styles as compared to violet- and white-flowering ones. The manipulation of flower visitors by anther-mimicking elongated styles in Crocus flowers is discussed.     

IP3 receptor-dependent Ca2+ release modulates excitation-contraction coupling in rabbit ventricular myocytes

Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-dependent Ca(2+) signaling exerts positive inotropic, but also arrhythmogenic, effects on excitation-contraction coupling (ECC) in the atrial myocardium. The role of IP(3)R-dependent sarcoplasmic reticulum (SR) Ca(2+) release in ECC in the ventricular myocardium remains controversial. Here we investigated the role of this signaling pathway during ECC in isolated rabbit ventricular myocytes. Immunoblotting of proteins from ventricular myocytes showed expression of both type 2 and type 3 IP(3)R at levels approximately 3.5-fold less than in atrial myocytes. In permeabilized myocytes, direct application of IP(3) (10 microM) produced a transient 21% increase in the frequency of Ca(2+) sparks (P < 0.05). This increase was accompanied by a 13% decrease in spark amplitude (P < 0.05) and a 7% decrease in SR Ca(2+) load (P < 0.05) and was inhibited by IP(3)R antagonists 2-aminoethoxydiphenylborate (2-APB; 20 microM) and heparin (0.5 mg/ml). In intact myocytes endothelin-1 (100 nM) was used to stimulate IP(3) production and caused a 38% (P < 0.05) increase in the amplitude of action potential-induced (0.5 Hz, field stimulation) Ca(2+) transients. This effect was abolished by the IP(3)R antagonist 2-APB (2 microM) or by using adenoviral expression of an IP(3) affinity trap that buffers cellular IP(3). Together, these data suggest that in rabbit ventricular myocytes IP(3)R-dependent Ca(2+) release has positive inotropic effects on ECC by facilitating Ca(2+) release through ryanodine receptor clusters.