Fourth Loop Research Articles

A Power-Efficient Multiband Planar USB Dongle Antenna for Wireless Sensor Networks.

Wireless Sensor Networks (WSNs) had been applied in Internet of Things (IoT) and in Industry 4.0. Since a WSN system contains multiple wireless sensor nodes, it is necessary to develop a low-power and multiband wireless communication system that satisfies the specifications of the Federal Communications Commission (FCC) and the Certification European (CE). In a WSN system, many devices are of very small size and can be slipped into a Universal Serial Bus (USB), which is capable of connecting to wireless systems and networks, as well as transferring data. These devices are widely known as USB dongles. This paper develops a planar USB dongle antenna for three frequency bands, namely 2.30–2.69 GHz, 3.40–3.70 GHz, and 5.15–5.85 GHz. This study proposes a novel antenna design that uses four loops to develop the multiband USB dongle. The first and second loops construct the low and intermediate frequency ranges. The third loop resonates the high frequency property, while the fourth loop is used to enhance the bandwidth. The performance and power consumption of the proposed multiband planar USB dongle antenna were significantly improved compared to existing multiband designs.

PorA-Loop4 derived peptides of Neisseria meningitidis cause a G1 cell cycle arrest through the Akt signalling pathway in human brain microvascular endothelial cells

Neisseria meningitidis (meningococcus) is a major meningitis-causing bacteria and is known for its ability to breach blood-brain barrier (BBB). Meningococcus binds to Laminin receptor (LAMR) on the surface of endothelium, which is part of the BBB. The meningococcal surface proteins PorA and PilQ were previously identified as bacterial ligands responsible for binding and, subsequently, the LAMR-binding moiety of PorA was localised to its fourth extracellular loop (PorA-Loop4). Using a circularised peptide corresponding to PorA-Loop 4 from N. meningitidis MC58, the PorA-LAMR interaction induced specific cellular responses in human brain microvascular endothelial cells (HBMECs) including G1 cell cycle arrest. Flow cytometric analysis indicated that the treatment of HBMECs with PorA-Loop4 for 24 h caused a significant reduction of cells (20 %) at S-phase and a corresponding increase (23 %) in G1 population. Immunoblotting and quantitative real time PCR (qRT-PCR) analysis suggested that a blockade in Akt signalling (key proteins including Akt, GSK-3β, CyclinD1, and CDK4) contributes to the G1 arrest. Immunoblotting showed that the expression of phosphorylated GSK-3β and CDK4 were significantly increased in treated HBMECs. In contrast, the expression of phosphorylated Akt and Cyclin D1 were decreased following treatment. Transcriptome analysis using qRT-PCR confirmed that treatment of HBMECs with PorA-Loop4 peptide for 2, 4, 8, or 24 h increased gene expression of CDK4, and decreased expression of Cyclin D1. Immunofluorescent imaging of Akt, GSK-3β, CyclinD1, and CDK4 in Loop4-treated HBMECs are consistent with qRT PCR and immunoblot results. The data confirm that PorA-Loop4 induce G1 arrest through the Akt signalling pathway via Akt/GSK-3β/CyclinD1/CDK4.

CB1 cannabinoid receptor-phosphorylated fourth intracellular loop structure-function relationships.

A peptide comprising the juxtamembrane C-terminal intracellular loop 4 (IL4) of the CB1 cannabinoid receptor possesses three Serine residues (Ser402, Ser411 and Ser415). Here we report the effect of Ser phosphorylation on the CB1 IL4 peptide conformation and cellular signaling functions using nuclear magnetic resonance spectroscopy, circular dichroism, G protein activation and cAMP production. Circular dichroism studies indicated that phosphorylation at various Ser residues induced helical structure in different environments. NMR data indicates that helical content varies in the order of IL4pSer411 > IL4pSer415 > IL4 > IL4pSer402. The efficacy of phosphorylated IL4 peptides in activating Go and Gi3 ([35S]GTPγS binding) and inhibiting cAMP accumulation in N18TG2 cells were correlated with helicity changes. Treatment of cells with bradykinin, which activates PKC, augmented CB1-mediated inhibition of cAMP accumulation, and this was reversed by a PKC inhibitor, suggesting that phosphorylation of serine might be a physiologically relevant modification in vivo. We conclude that phosphorylation-dependent alterations of helicity of CB1 IL4 peptides can increase efficacy of G protein signaling.

Confronting the fourth generation two-Higgs-doublet model with the phenomenology of heavy Higgs bosons

A sequential fourth generation is known to be excluded because the non-decoupling contribution to $\kappa_g$, the Higgs coupling modifier with a gluon pair, is unacceptably large. Recently a new way to save the model was suggested in the Type-II two Higgs doublet model: if the Yukawa couplings of down-type fermions have wrong-sign, the contributions from $t'$ and $b'$ to $\kappa_g$ are cancelled. We study the theoretical and experimental constraints on this model, focusing on the heavy Higgs bosons. Two constraining features are pointed out. First the exact wrong-sign limit does not allow the alignment, which makes the perturbative unitarity for the scalar-scalar scattering put the upper bounds on the heavy Higgs boson masses like $M_H, M_A \lesssim 920$ GeV and $M_{H^\pm} \lesssim 620$ GeV. Secondly, the Yukawa couplings of the fourth generation fermions to the heavy Higgs bosons are generically large as being proportional to the heavy fermion mass and, for the down-type fermions, to $\tan\beta$ as well. The gluon fusion productions of $H$ and $A$ through the fourth generation quark loops become significant. We found that the current LHC data on $pp \to Z Z$ for $H$ along with the theoretical and indirect constraints exclude the model at leading order.

Synthesis and In Vitro Neuroprotector Activity of Diastereoisomers of a Dimeric Dipeptide Mimetic of Nerve Growth Factor GK-2

Previously, a dimeric dipeptide mimetic bis-(N-monosuccinyl-L-glutamyl-L-lysine) hexamethylenediamide (GK-2) based on the β-turn of the fourth loop of nerve growth factor was prepared by us, activated TrkA, and exhibited neuroprotective activity in vitro (10–5 – 10–9 M) and in vivo (0.05 – 5 mg/kg i.p.). The present work reports the synthesis of two of its diastereomers, bis-(N-monosuccinyl-L-glutamyl-D-lysine) (GK-2LD) and bis-(N-monosuccinyl-D-glutamyl-L-lysine) hexamethylenediamides (GK-2DL). Studies of their neuroprotective activities using HT-22 neuronal culture under oxidative stress showed that switching L-lysine to D-lysine led to a significant activity decrease whereas switching L-glutamic acid to the D-stereoisomer caused it to disappear completely. Both dipeptide residues were concluded to be involved in interactions with the TrkA receptor.

Dipeptide Mimetic of the BDNF GSB-106 with Antidepressant-Like Activity Stimulates Synaptogenesis.

Dipeptide mimetic of the brain-derived neurotrophic factor bis(N-monosuccinyl-L-seryl-L-lysine) hexamethylenediamide (working name GSB-106), which reproduces the homodimeric structure of BDNF and the beta-turn of its fourth loop, activates TrkB, AKT, and ERK, exhibits neuroprotective and antidepressant activity, and is able to stimulate neurogenesis in the hippocamp of stressed mice. Using Western blot hybridization and synaptophysin (synaptogenesis marker), we showed the ability of chronically administered GSB-106 to stimulate synaptogenesis, increasing the synaptic density in the hippocamp by 50%. Under the same conditions, GSB-106 exhibited antidepressant activity (decreased (by 18%) immobility of animals in Porsolt test), which may be associated with the stimulation of neurogenesis and synaptogenesis in the hippocamp.

DEVELOPMENT OF THE TECHNIQUE FOR DETERMINATION OF NATURAL IMPURITIES IN THE SUBSTANCE OF A NEW CONNECTION OF GK-2 PEPTIDE STRUCTURE

The study is devoted to the development of related substances determination technique in the original dipeptide, which is fourth loop of nerve growth factor mimetic - GK-2, and was carried out using methods of thin-layer chromatography and high-performance liquid chromatography with spectrophotometric detection (HPLC and TLC). The technique for determining of the related impurities content includes two stages: analysis by thin-layer chromatography and analysis by high-performance liquid chromatography. The developed technique allows detecting impurities in the substance in the content of 0,1 % and more.

Phosphatidylinositol (4, 5)‐bisphosphate coordinates functional interactions in the dopamine transporter to promote amphetamine behaviors

The psychostimulant amphetamine (AMPH) mainly mediates its pharmacological and behavioral effects by increasing extracellular dopamine (DA) availability. DA homeostasis is maintained by the dopamine transporter (DAT), a presynaptic membrane protein that mediates the high‐affinity reuptake of released DA from the synaptic cleft. We have previously demonstrated that AMPH induces N‐term phosphorylation of the DAT, which leads to transport‐mediated efflux of DA. Furthermore, we have shown that phosphatidylinositol (4, 5)‐bisphosphate (PIP2) directly interacts with the DAT and facilitates AMPH‐induced DA efflux, but is not required for DA uptake. Specially, PIP2 binds DAT through electrostatic interactions with positively charged DAT N‐terminal residues. Disrupting the interactions between DAT and PIP2 or depleting PIP2 diminishes reverse transport (efflux) of DA. Previous studies on the human serotonin transporter show that non‐N‐terminal PIP2 binding sites (within the fourth intracellular loop) are essential for AMPH‐induced reverse transport of serotonin, but not substrate uptake. Together, these data led us to hypothesize that PIP2 coordinates functional interactions between the N‐terminus and the fourth intracellular loop to promote reverse transport of DA. Here, we demonstrate that depletion of PIP2 after DAT N‐terminus phosphorylation does not affect AMPH‐induced DA efflux, suggesting that N‐terminus phosphorylation is the limiting step of reverse transport of DA. Notably, we show that residue R443, which resides in the fourth intracellular loop, also modulates AMPH‐induced DA efflux. Thus, we focus on the role of the interaction between the DAT N‐terminus and R443 in mediating the reverse transport of DA. Furthermore, we translate our molecular discoveries in vivo by using a coordinated genetic and pharmacological approach to study AMPH‐induced behaviors in Drosophila melanogasterSupport or Funding Information1F31MH114316 (JIA), AHA Predoctoral Award (AS), DA035263(AG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Loops and Recursions in Cognitive Science: Cross-Roads between Methodology and Epistemology

This article addresses the need for cognitive science to loop back and examine its roots and presuppositions, pointing out the three recursive issues: 1.) The observer effect or how observing a phenomenon affects the phenomenon that is being observed, an issue that has been acknowledged by natural science, which cognitive science attempts to emulate, and empirical phenomenology, but not cognitive science itself; 2.) Human kinds or how our research affects us, the researchers, and society (people’s self-understandings), an issue which forms a loop with the observer effect – observation thus changing the observed, the observer, as well as itself, and 3.) The dangers of over-eager extrapolation or how complexity is lost during shifts in explanatory level, issues pertaining to using findings from studies of one explanatory level (e.g. experiments with rats) to inform a different explanatory level (issues within human society). Finally, the article presents a fourth recursive loop which presents a potential solution to the above: a self-correcting mechanism that allows science to recursively correct its mistakes and improve on its own work.

Structure of C-terminal Domain of Peptidyl-prolyl cis-trans Isomerase from Pseudomonas syringae pv. Tomato str. DC3000 at 1.6Å Resolution.

Peptidyl-prolyl cis-trans isomerase (PPIase) accelerates the intrinsically slow conversion between cis- and trans- configurations of proline, thus affecting backbone conformation and altering the direction of peptide chains. PPIase from Pseudomonas syringae pv. Tomato (PSPTO) DC3000 (PSPTO-PPIase) is considered to belong to the FKBP subfamily of PPIase. To solve the high resolution structure of the PSPTO-PPIase, and to explore its potential function in plants pathogen PSPTO DC3000. The PSPTO-PPIase was expressed in E.coli and purified through ion exchange and size exclusion chromatography. While only the C-terminal domain of PSPTO-PPIase was successfully crystalized, and its structure was solved to 1.6 Å resolution by molecular replacement method. Structural comparison showed that PSPTO-PPIase adopts a similar overall fold with microphage infectivity potentiators (MIPs), which also belong to the FKBP subfamily of PPIase. In addition, the BIAcore result confirmed that PSPTO-PPIase can bind an immunosuppressive drug FK506 as some other FKBP subfamily members do. Our results suggested that PSPTO-PPIase may function in a similar manner to virulent factor MIPs during pathogenesis. And the immunosuppressive drugs FK506 and rapamycin binding to PSPTO-PPIase potentially interferes and inhibits the plant pathogen PSPTO DC3000. In addition, the amino acids with short side chains in the fourth loop (L4) of PSPTO-PPIase may account for its variable roles in the respective pathogen.

A single amino acid in MRGPRX2 necessary for binding and activation by pruritogens

A single amino acid in MRGPRX2 necessary for binding and activation by pruritogens

Design, synthesis, and neuroprotective effects of a dimeric dipeptide mimetic of the third loop of the nerve growth factor

Previously, we prepared dimeric dipeptide mimetics of the first and the fourth loops of the nerve growth factor (NGF): hexamethylenediamides of bis(N-aminocaproyl-glycyl-L-lysine) (GK-6) and bis(N-monosuccinyl-L-glutamyl-L-lysine) (GK-2). Both mimetics activated TrkA-receptors, but induced different postreceptor signal pathways. GK-2 selectively activated PI3K/AKT, whereas GK-6 activated both PI3K/AKT and MAPK/ERK. Both mimetics exhibited a neuroprotective activity. In this study, we continued the investigation of a contribution of separate loop-like structures in the NGF functions and created and studied dimeric dipeptide mimetics based on a beta-turn of the NGF third loop: hexamethylenediamides of bis(N-gamma-hydroxybutyryl-L-lysyl-L-histidine) (GTS-115) and bis(N-acetyl-L-lysyl-L-histidine) (GTS-113). GTS-115 was shown to exhibit the neuroprotective activity in the concentration range from 10–5 to 10–7 М towards the HT-22 cell culture under the conditions of oxidative stress. The acetyl-containing GTS-113 mimetic proved to be inactive. GTS-115 (1 mg/kg/day intraperitoneally, for 7 days, the administration was started 4 h after the operation) exhibited the neuroprotective properties and decreased the infarction volume by 25% on the model of a stroke that was induced by a transient occlusion of the medial cerebral artery of rats. The action mechanism of GTS-115 was studied by Western-blot analysis and this mimetic in a concentration of 10–6 М was shown to activate the TrkA-receptor and both MAPK/ERK and PI3K/AKT basic postreceptor signal pathways. The inhibitory analysis revealed different contributions of these pathways into the GTS-115 neuroprotective effect. The LY294002 selective inhibitor of PI3K completely blocked the neuroprotective effect of GTS-115 in vitro, whereas the PD98059 specific inhibitor of MEK1 and MEK2 decreased this effect only by 10–15%. GTS-115 peptide stimulated a differentiation of the PC12 cells and caused a hyperalgesia in rats. These facts were in a good agreement with the literature data on the participation of the MAP-kinase pathway in these effects. Thus, the third NGF loop and the neighboring first NGF loop activated the postreceptor pathways in a similar way and exhibited the similar activities.

Formation of a DNA Mini-Dumbbell with a Quasi-Type II Loop.

Quasi-loops, wherein the backbone has a discontinuous site in the loop, have been found to provide structural flexibility in the formation of DNA three-way junctions. Recently, a highly compact mini-dumbbell (MDB) structure composed of two adjacent CCTG or TTTA type II loops has been reported. Yet, it remains elusive if the presence of a quasi-loop will also facilitate the formation of an MDB. In this study, the possibility of whether an MDB can be formed containing a quasi-type II loop has been investigated. We first demonstrate that two adjacent CTTG type II loops can also form an MDB, which is thermodynamically more stable than those formed by CCTG and TTTA loops. Then, we systematically introduce quasi-type II loops with their discontinuous sites at different backbone positions in the CTTG MDB. Our results show that an MDB can be formed with a quasi-type II loop, in which there is a discontinuous site between the third thymine and the fourth guanine loop residues. The possible inclusion of a quasi-loop in MDBs expands the sequence criteria for the formation of MDBs by natural DNA sequences.

Improved fluorescence assays to measure the defects associated with F508del-CFTR allow identification of new active compounds.

Cystic fibrosis (CF) is a debilitating disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for a Cl-/HCO3 - channel. F508del, the most common CF-associated mutation, causes both gating and biogenesis defects in the CFTR protein. This paper describes the optimization of two fluorescence assays, capable of measuring CFTR function and cellular localization, and their use in a pilot drug screen. HEK293 cells expressing YFP-F508del-CFTR, in which halide sensitive YFP is tagged to the N-terminal of CFTR, were used to screen a small library of compounds based on the VX-770 scaffold. Cells expressing F508del-CFTR-pHTomato, in which a pH sensor is tagged to the fourth extracellular loop of CFTR, were used to measure CFTR plasma membrane exposure following chronic treatment with the novel potentiators. Active compounds with efficacy ~50% of VX-770, micromolar potency, and structurally distinct from VX-770 were identified in the screen. The F508del-CFTR-pHTomato assay suggests that the hit compound MS131A, unlike VX-770, does not decrease membrane exposure of F508del-CFTR. Most known potentiators have a negative influence on F508del-CFTR biogenesis/stability, which means membrane exposure needs to be monitored early during the development of drugs targeting CFTR. The combined use of the two fluorescence assays described here provides a useful tool for the identification of improved potentiators and correctors. The assays could also prove useful for basic scientific investigations on F508del-CFTR, and other CF-causing mutations.

Evaluation of the Effect of Thermal Stratification on the Residual Life of the Surge Line of the Pressure Compensator in the No. 5 Unit of the Novovoronezh NPP

This article is concerned with investigations of temperature stratifi cation of the fi rst-loop coolant in the surge line of the pressure compensator in the No. 5 unit of the Novovoronezh NPP. The actual stress-strain state and accumulated structural damage in the surge line in different operating regimes was analyzed by means of multiparametric computational and experimental monitoring. The experimental data demonstrate thermal stratifi cation, giving rise to a temperature differential over the cross section ranging from 30°C under normal operating conditions to 150°C, as a result of disruption of the normal operating conditions, and high thermal stresses. The analysis shows that the cyclic strength of the weld connection of the surge line can be exhausted by the end of the 60-yr life of the No. 5 unit. It is concluded that the actual residual stresses must be measured and calculations of the cyclic strength and the residual life of the surge line must be performed. It is suggested that a technology be developed to strengthen the weld-joint metal in order to reduce the formation of operational surface defects. Since 1980, tens of operating defects and even through cracks formed in pipelines because of stratifi cation were discovered in NPP with PWR and VVER [1, 2]. Defects in the form of shallow surface cracks in transitional surfacing were found in the piping of the pressure compensation system of the No. 5 unit of the Novovoronezh NPP during operational monitoring of the weld connections. According to the recommendations of the International Insurance Inspectorate the infl uence of thermal stratifi cation on the damage rate for the surge line of the pressure compensator should be determined. The function of the pressure compensation system is to create and maintain the coolant pressure in the fi rst loop in order to prevent the coolant from boiling in stationary regimes and to limit the deviation in transitional and emergency regimes. The pressure compensator communicates with the coolant in the hot string of the fourth loop of the main circulation pipeline (MSP) via the 426 × 40 mm connecting surge line (Fig. 1). While the reactor is operating, the surge line is heated owing to the closed circulation of the coolant. When the average temperature of the fi rst-loop coolant changes in transitional regimes associated with the disruption of equipment operation and accompanying a change in the load, part of the coolant fl ows from the pressurizer into the loop or from the loop into the pressurizer along the surge line. In addition, a limitation on the deviation of the pressure from the nominal value is attained by compression or expansion of the steam blanket in the pressurizer. Thus, the integrity of the surge line is directly related with the reliability of fi rst-loop operation. In the course of operation the following loading factors act on the pipeline: mass of the structure, internal pressure, temperature change, and temperature stratifi cation in transient regimes. Thermal stratifi cation is associated with the separation of the coolant fl ow over the section of the pipeline into a cold layer at the bottom and hot layer at the top. The temperature gradients in the coolant result in beyond design-basis stresses in

Hepatitis B virus nuclear export elements: RNA stem-loop α and β, key parts of the HBV post-transcriptional regulatory element

ABSTRACTMany viruses contain RNA elements that modulate splicing and/or promote nuclear export of their RNAs. The RNAs of the major human pathogen, hepatitis B virus (HBV) contain a large (~600 bases) composite cis-acting 'post-transcriptional regulatory element' (PRE). This element promotes expression from these naturally intronless transcripts. Indeed, the related woodchuck hepadnavirus PRE (WPRE) is used to enhance expression in gene therapy and other expression vectors. These PRE are likely to act through a combination of mechanisms, including promotion of RNA nuclear export. Functional components of both the HBV PRE and WPRE are 2 conserved RNA cis-acting stem-loop (SL) structures, SLα and SLβ. They are within the coding regions of polymerase (P) gene, and both P and X genes, respectively. Based on previous studies using mutagenesis and/or nuclear magnetic resonance (NMR), here we propose 2 covariance models for SLα and SLβ. The model for the 30-nucleotide SLα contains a G-bulge and a CNGG(U) apical loop of which the first and the fourth loop residues form a CG pair and the fifth loop residue is bulged out, as observed in the NMR structure. The model for the 23-nucleotide SLβ contains a 7-base-pair stem and a 9-nucleotide loop. Comparison of the models with other RNA structural elements, as well as similarity searches of human transcriptome and viral genomes demonstrate that SLα and SLβ are specific to HBV transcripts. However, they are well conserved among the hepadnaviruses of non-human primates, the woodchuck and ground squirrel.

Synthesis of GK-2, a Dimeric Dipeptide Nerve Growth Factor Mimetic and Potential Neuroprotective Agent

A series of dipeptide mimetics of nerve growth factor were synthesized, one of which, bis-(monosuccinyl-Lglutamyl-L-lysine) hexamethylenediamide (GK-2), constructed on the basis of the β-turn of its fourth loop, was selected for development as a potential neuroprotective drug. This study describes the first phase in the creation of a technology for producing GK-2 – selection of the optimum synthesis scheme. Two nine-stage synthesis schemes were assessed, consisting of lengthening the peptide chain from the N or C terminal. Condensation in both cases was performed using N-hydroxysuccinimide esters. Both schemes yielded product with identical optical and diastereomeric purity, not requiring complex purification and involving similar time input, though the scheme growing the peptide chain from the C terminal was 1.2 times more economical in terms of reagent costs per kg of product and was selected as the main method for developing the laboratory methodology.

Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-β assembly

Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid β-protein (Aβ) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aβ oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific Na(+)/K(+)-ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aβ-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn(879) and Trp(880) is essential for ASPD-NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 interaction.

Morphological and molecular investigations of the holocephalan elephant fish nephron: the existence of a countercurrent-like configuration and two separate diluting segments in the distal tubule.

In marine cartilaginous fish, reabsorption of filtered urea by the kidney is essential for retaining a large amount of urea in their body. However, the mechanism for urea reabsorption is poorly understood due to the complexity of the kidney. To address this problem, we focused on elephant fish (Callorhinchus milii) for which a genome database is available, and conducted molecular mapping of membrane transporters along the different segments of the nephron. Basically, the nephron architecture of elephant fish was similar to that described for elasmobranch nephrons, but some unique features were observed. The late distal tubule (LDT), which corresponded to the fourth loop of the nephron, ran straight near the renal corpuscle, while it was convoluted around the tip of the loop. The ascending and descending limbs of the straight portion were closely apposed to each other and were arranged in a countercurrent fashion. The convoluted portion of LDT was tightly packed and enveloped by the larger convolution of the second loop that originated from the same renal corpuscle. In situ hybridization analysis demonstrated that co-localization of Na(+),K(+),2Cl(-) cotransporter 2 and Na(+)/K(+)-ATPase α1 subunit was observed in the early distal tubule and the posterior part of LDT, indicating the existence of two separate diluting segments. The diluting segments most likely facilitate NaCl absorption and thereby water reabsorption to elevate urea concentration in the filtrate, and subsequently contribute to efficient urea reabsorption in the final segment of the nephron, the collecting tubule, where urea transporter-1 was intensely localized.

Highly homologous proteins exert opposite biological activities by using different interaction interfaces.

We present a possible molecular basis for the opposite activity of two homologues proteins that bind similar ligands and show that this is achieved by fine-tuning of the interaction interface. The highly homologous ASPP proteins have opposite roles in regulating apoptosis: ASPP2 induces apoptosis while iASPP inhibits it. The ASPP proteins are regulated by an autoinhibitory interaction between their Ank-SH3 and Pro domains. We performed a detailed biophysical and molecular study of the Pro – Ank-SH3 interaction in iASPP and compared it to the interaction in ASPP2. We found that iASPP Pro is disordered and that the interaction sites are entirely different: iASPP Ank-SH3 binds iASPP Pro via its fourth Ank repeat and RT loop while ASPP2 Ank-SH3 binds ASPP2 Pro via its first Ank repeat and the n-src loop. It is possible that by using different moieties in the same interface, the proteins can have distinct and specific interactions resulting in differential regulation and ultimately different biological activities.