Central Loop Research Articles

PIN phosphorylation is sufficient to mediate PIN polarity and direct auxin transport.

The plant hormone auxin plays a crucial role in regulating plant development and plant architecture. The directional auxin distribution within tissues depends on PIN transporters that are polarly localized on the plasma membrane. The PIN polarity and the resulting auxin flow directionality are mediated by the antagonistic actions of PINOID kinase and protein phosphatase 2A. However, the contribution of the PIN phosphorylation to the polar PIN sorting is still unclear. Here, we identified an evolutionarily conserved phosphorylation site within the central hydrophilic loop of PIN proteins that is important for the apical and basal polar PIN localizations. Inactivation of the phosphorylation site in PIN1(Ala) resulted in a predominantly basal targeting and increased the auxin flow to the root tip. In contrast, the outcome of the phosphomimic PIN1(Asp) manipulation was a constitutive, PINOID-independent apical targeting of PIN1 and an increased auxin flow in the opposite direction. Furthermore, the PIN1(Asp) functionally replaced PIN2 in its endogenous expression domain, revealing that the phosphorylation-dependent polarity regulation contributes to functional diversification within the PIN family. Our data suggest that PINOID-independent PIN phosphorylation at one single site is adequate to change the PIN polarity and, consequently, to redirect auxin fluxes between cells and provide the conceptual possibility and means to manipulate auxin-dependent plant development and architecture.

Structural insights into the dual nucleotide exchange and GDI displacement activity of SidM/DrrA

GDP-bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub-cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP-to-GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA-1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure-based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein.

An Intersubunit Signaling Network Coordinates ATP Hydrolysis by m-AAA Proteases

Ring-shaped AAA+ ATPases control a variety of cellular processes by substrate unfolding and remodeling of macromolecular structures. However, how ATP hydrolysis within AAA+ rings is regulated and coupled to mechanical work is poorly understood. Here we demonstrate coordinated ATP hydrolysis within m-AAA protease ring complexes, conserved AAA+ machines in the inner membrane of mitochondria. ATP binding to one AAA subunit inhibits ATP hydrolysis by the neighboring subunit, leading to coordinated rather than stochastic ATP hydrolysis within the AAA ring. Unbiased genetic screens define an intersubunit signaling pathway involving conserved AAA motifs and reveal an intimate coupling of ATPase activities to central AAA pore loops. Coordinated ATP hydrolysis between adjacent subunits is required for membrane dislocation of substrates, but not for substrate processing. These findings provide insight into how AAA+ proteins convert energy derived from ATP hydrolysis into mechanical work.

TEM Response of a Large Loop Source over a Homogeneous Earth Model: A Generalized Expression for Arbitrary Source-Receiver Offsets

In the present research, an attempt is made to derive the generalized expressions for the transient electromagnetic response of a large loop source over the surface of a homogeneous earth model for arbitrary receiver positions inside or outside the source loop. Expressions are derived for the impulse as well as step excitations of the source loop. As a cross check for validity of expressions, the step response expression is obtained from the impulse response expression and vice versa. Computations are performed for the TEM response over a homogeneous earth model for source-receiver offset (r = 0) pertinent to the central loop configuration and the results are compared with the published results for the central loop TEM responses. The results are in well coincidence with each other and thus provide the check for the authenticity of the expressions. To exemplify the nature of TEM response at various source receiver offsets, results are presented for the TEM response at source receiver offsets r = 0, r = a/2, r = a and r = 2a, 6a, 12a pertaining to the central loop, in-loop, on-loop and offset loop configurations, respectively. The results depict their characteristic variations. At receiver positions inside the loop source, both the impulse as well as step responses are of same sign, whereas at the receiver positions outside the loop source, both the curves exhibit a change of sign that shifts towards the later times with increase in the offset distances. The change of sign in impulse response occurs at a relatively later time than that in the step response. This is the initial presentation of TEM response expressions for the large loop source over a homogeneous earth model for arbitrary receiver position inside and/ or outside the loop source except for the case of receiver at the center of the loop and at the coincident loop point. This research would be of immense use in the development and use of the large loop TEM method in its various configurations and thus would enhance the applicability and cost effectiveness of the large loop source TEM method.

Stability of intramolecular quadruplexes: sequence effects in the central loop.

Hundreds of thousands of putative quadruplex sequences have been found in the human genome. It is important to understand the rules that govern the stability of these intramolecular structures. In this report, we analysed sequence effects in a 3-base-long central loop, keeping the rest of the quadruplex unchanged. A first series of 36 different sequences were compared; they correspond to the general formula GGGTTTGGGHNHGGGTTTGGG. One clear rule emerged from the comparison of all sequence motifs: the presence of an adenine at the first position of the loop was significantly detrimental to stability. In contrast, adenines have no detrimental effect when present at the second or third position of the loop. Cytosines may either have a stabilizing or destabilizing effect depending on their position. In general, the correlation between the Tm or ΔG° in sodium and potassium was weak. To determine if these sequence effects could be generalized to different quadruplexes, specific loops were tested in different sequence contexts. Analysis of 26 extra sequences confirmed the general destabilizing effect of adenine as the first base of the loop(s). Finally, analysis of some of the sequences by microcalorimetry (DSC) confirmed the differences found between the sequence motifs.

Flux noise in ion-implanted nanoSQUIDs

Focused ion-beam (FIB) technology has been used to fabricate miniature Nb DC SQUIDs(superconducting quantum interference devices) which incorporate resistively shuntedmicrobridge junctions and a central loop with a hole diameter ranging from 1058 to 50 nm.The smallest device, with a 50 nm hole diameter, has a white flux noise level of2.6 µΦ0 Hz−1/2 at104 Hz. The scaling of the flux noise properties and focusing effect of the SQUID with the holesize were examined. The observed low frequency flux noises of different devices werecompared with the contribution due to the spin fluctuation of defects introduced duringFIB processing and of thermally activated flux hopping in the SQUID washer.

The Probing Depth of Transient Electromagnetic Field Method

AbstractTaking central loop configuration excited by a step pulse on the ground as an example, we study the probing depth of electromagnetic sounding with analytical method, finite difference time domain and timefrequency methods. The aim is to provide a basis for field work design in order to reach the expected target. Analytical calculation proves that the transient electromagnetic field propagates in the earth at a limited speed, and numerical simulation exhibits reflection of the field under the quasi‐static condition. The results show that the probing depth in transient electromagnetic exploration is mainly determined by measuring time because the time‐domain electromagnetic field follows the law of causality. The initial propagation velocity of the transient electromagnetic field is not related to earth resistivity. Due to earth dispersion the front edge of the impulse becomes smoothed gradually and every frequency component has its velocity related with resistivity, and longer measuring time will be required for the same depth in lower resistivity stratum. The maximum probing depth is the distance over which an electromagnetic wave goes to and back from some depth in the earth in a given time. The minimum depth is limited by apparatus's performance. However, the anomalous bodies embedded in the upper subsurface may be detected at a late time. The relation between time and frequency can be obtained from the time‐frequency density spectrum of transient field signals.

Clock genes regulate circadian rhythmical change of sglt1 gene expression according to feeding schedule through binding of BMAL1 on the promoter region

Sodium‐D‐glucose cotransporter SGLT1 plays an important role in the absorption of glucose in the small intestine. To examine whether the circadian rhythmical expression of the intestinal sglt1 gene is regulated by clock genes, we determined the levels of mRNA and BMAL1 binding on the promoter regions of the sglt1 gene by real‐time RT‐PCR and chromatin immunoprecipitation assay, respectively, in the jejunum of mice receiving free access to diet and those with restricted feeding from 9:00 to 17:00 under light (7:00‐19:00)‐dark (19:00‐7:00) cycle. Jejunal expression of sglt1 gene gradually increased from 7:00, and reached a maximal level at 19:00 in mice with free access to diet. The peak was shifted to 7:00 in mice with restricted feeding schedule. The gene expression of bmal1, a transcription factor driving the central feedback loop of clock genes, displayed a maximal expression at the period when sglt1 gene expression started to increase. The gene expression of per1‐3, the clock genes coding negative regulators of BMAL1, was highest at or soon after the time of maximal expression of sglt1 gene in both groups. The binding of BMAL1 to the promoters of sglt1 and per2 genes was associated with their expressional changes. The results suggest that the circadian expressional change of sglt1 gene in the jejunum of mice which is cued by the feeding schedule is directly regulated by a feedback loop of clock genes.

Folding of single-stranded DNA quadruplexes containing an autonomously stable mini-hairpin loop

The single-stranded DNA quadruplex motif TG(3)-L(1)-G(3)-L(2)-G(3)-L(3)-G(3)T (where L(1), L(2) and L(3) are the three loop sequences) was used as a template for probing the effects of the loop sequences on stability and folding topology. An autonomously stable mini-hairpin sequence (ACGTAGT) was inserted into the central loop (L(2)) of different sequences with intrinsic propensities to form either parallel or anti-parallel structures. Single nucleotides (T) at positions L(1) and L(3) strongly favour the formation of a parallel structure with the L(2) hairpin insert affecting stability in the same way as a T(7) loop. However, in the context of an anti-parallel quadruplex with T(3) loops in positions L(1) and L(3), the mini-hairpin in the central loop forms a stable structure which enhances the T(m) of the quadruplex by approximately 10 degrees C when compared with the T(7) insert. The CD and UV melting data show that base pairing interactions within the ACGTAGT hairpin loop sequence, when accommodated as a diagonal loop in an anti-parallel structure, can enhance stability and lead to novel quadruplex structures, adding complexity to the folding landscape and expanding the potential repertoire of sequences that are able to regulate gene expression in vivo.

Structural and Functional Requirements for Activity of the Tim9–Tim10 Complex in Mitochondrial Protein Import

The Tim9-Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space. How the complex interacts with precursors is not clear, although it has been proposed that Tim10 acts in substrate recognition, whereas Tim9 acts in complex stabilization. In this study, we report the structure of the yeast Tim9-Tim10 hexameric assembly determined to 2.5 A and have performed mutational analysis in yeast to evaluate the specific roles of Tim9 and Tim10. Like the human counterparts, each Tim9 and Tim10 subunit contains a central loop flanked by disulfide bonds that separate two extended N- and C-terminal tentacle-like helices. Buried salt-bridges between highly conserved lysine and glutamate residues connect alternating subunits. Mutation of these residues destabilizes the complex, causes defective import of precursor substrates, and results in yeast growth defects. Truncation analysis revealed that in the absence of the N-terminal region of Tim9, the hexameric complex is no longer able to efficiently trap incoming substrates even though contacts with Tim10 are still made. We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space.

Y-junction of superconducting Josephson chains

We show that, for pertinent values of the fabrication and control parameters, an attractive finite coupling fixed point emerges in the phase diagram of a Y-junction of superconducting Josephson chains. The new fixed point arises only when the dimensionless flux f piercing the central loop of the network equals π and, thus, does not break time-reversal invariance; for f ≠ π , only the strongly coupled fixed point survives as a stable attractive fixed point. Phase slips (instantons) have a crucial role in establishing this transition: we show indeed that, at f = π , a new set of instantons—the W-instantons—comes into play to destabilize the strongly coupled fixed point. Finally, we provide a detailed account of the Josephson current–phase relationship along the arms of the network, near each one of the allowed fixed points. Our results evidence remarkable similarities between the phase diagram accessible to a Y-junction of superconducting Josephson chains and the one found in the analysis of quantum Brownian motion on frustrated planar lattices.

Phosphorylation of the Yeast Nitrate Transporter Ynt1 Is Essential for Delivery to the Plasma Membrane during Nitrogen Limitation

Ynt1 is the sole high affinity nitrate transporter of the yeast Hansenula polymorpha. It is highly regulated by the nitrogen source, by being down-regulated in response to glutamine by repression of the YNT1 gene and Ynt1 ubiquitinylation, endocytosis, and vacuolar degradation. On the contrary, we show that nitrogen limitation stabilizes Ynt1 levels at the plasma membrane, requiring phosphorylation of the transporter. We determined that Ser-246 in the central intracellular loop plays a key role in the phosphorylation of Ynt1 and that the nitrogen permease reactivator 1 kinase (Npr1) is necessary for Ynt1 phosphorylation. Abolition of phosphorylation led Ynt1 to the vacuole by a pep12-dependent end4-independent pathway, which is also dependent on ubiquitinylation, whereas Ynt1 protein lacking ubiquitinylation sites does not follow this pathway. We found that, under nitrogen limitation, Ynt1 phosphorylation is essential for rapid induction of nitrate assimilation genes. Our results suggest that, under nitrogen limitation, phosphorylation prevents Ynt1 delivery from the secretion route to the vacuole, which, aided by reduced ubiquitinylation, accumulates Ynt1 at the plasma membrane. This mechanism could be part of the response that allows nitrate-assimilatory organisms to cope with nitrogen depletion.

Overview of human tremor physiology.

The physiology differs in the many forms of human tremor. Tremors may derive from mechanical oscillations, mechanical reflex oscillations, normal central oscillators, and pathologic central oscillators. Methods of studying tremor include accelerometry and electromyography (EMG). An excellent method consists of accelerometry and EMG combined with spectral analysis and weighting of the body part, which allows separation of tremors coming from mechanical reflex and central oscillators. Physiologic tremor is a mechanical tremor with a possible contribution of the normal 8-12 Hz central oscillator; exaggerated physiologic tremor is a mechanical reflex tremor. Essential tremor (ET) comes from a central oscillator that can be easily influenced with sensory input. The classic rest tremor of Parkinson's disease (PD) comes from a central oscillator that seems less easily influenced with sensory input but can be affected by transcranial magnetic stimulation. Other tremors with central oscillators are palatal tremor and orthostatic tremor. Other tremors whose physiology involves central loops includes cerebellar tremor and cortical tremor. Neuropathic tremors may be a result of delays in peripheral loops, but central oscillators play a role in some.

Differential Effect of Morphine and Morphine-6-glucuronide on the Control of Breathing in the Anesthetized Cat

Morphine's metabolite, morphine-6-glucuronide (M6G), activates the mu-opioid receptor. Previous data suggest that M6G activates a unique M6G receptor that is selectively antagonized by 3-methoxynaltrexome (3mNTX). The authors compared the effects of M6G and morphine on breathing in the anesthetized cat and assessed whether 3mNTX reversal was selective for M6G. Step changes in end-tidal carbon dioxide concentration were applied in cats anesthetized with alpha-chloralose-urethane. In study 1, the effect of the 0.15 mg/kg morphine followed by 0.2 mg/kg 3mNTX and next 0.8 mg/kg M6G was assessed in six cats. In study 2, the effect of 0.8 mg/kg M6G followed by 0.2 mg/kg 3mNTX and 0.15 mg/kg morphine was tested in another six cats. The ventilatory carbon dioxide responses were analyzed with a two-compartment model of the ventilatory controller, which consists of a fast peripheral and a slow central component. Both opioids shifted the ventilatory carbon dioxide responses to higher end-tidal carbon dioxide levels. Morphine had a preferential depressant effect within the central chemoreflex loop. In contrast, M6G had a preferential depressant effect within the peripheral chemoreflex loop. Irrespective of the opioid, 3mNTX caused full reversal of and prevented respiratory depression. In anesthetized cats, the mu-opioids morphine and M6G induce respiratory depression at different sites within the ventilatory control system. Because 3mNTX caused full reversal of the respiratory depressant effects of both opioids, it is unlikely that a 3mNTX-sensitive unique M6G receptor is the cause of the differential respiratory behavior of morphine and M6G.

Different Interactions between MT7 Toxin and the Human Muscarinic M1 Receptor in Its Free and N-Methylscopolamine-Occupied States

Muscarinic MT7 toxin is a highly selective and potent antagonist of the M(1) subtype of muscarinic receptor and acts by binding to an allosteric site. To identify the molecular determinants by which MT7 toxin interacts with this receptor in its free and NMS-occupied states, the effect on toxin potency of alanine substitution was evaluated in equilibrium and kinetic binding experiments as well as in functional assays. The determination of the crystallographic structure of an MT7-derivative (MT7-diiodoTyr51) allowed the selection of candidate residues that are accessible and present on both faces of the three toxin loops. The equilibrium binding data are consistent with negative cooperativity between N-methylscopolamine (NMS) and wild-type or modified MT7 and highlight the critical role of the tip of the central loop of the toxin (Arg34, Met35 Tyr36) in its interaction with the unoccupied receptor. Examination of the potency of wild-type and modified toxins to allosterically decrease the dissociation rate of [(3)H]NMS allowed the identification of the MT7 residues involved in its interaction with the NMS-occupied receptor. In contrast to the results with the unoccupied receptor, the most important residue for this interaction was Tyr36 in loop II, assisted by Trp10 in loop I and Arg52 in loop III. The critical role of the tips of the MT7 loops was also confirmed in functional experiments. The high specificity of the MT7-M(1) receptor interaction exploits several MT7-specific residues and reveals a different mode of interaction of the toxin with the free and NMS-occupied states of the receptor.

CDI Versus inversão: aplicação a dados reais

The aim of this paper is to carry out a comparison between two types of 2D electromagnetic modeled sections normally used on AEM data quantitative interpretation especially on those obtained from ATDEM measurements (ATDEM). One is obtained from fast modeling of the secondary magnetic flux decay and is the most common quantitative interpretation tool. The other is of more restrict usage and use 1D inversion techniques. In the first case, the technique transforms the ATDEM responses on a conductivity depth image (CDI) and a glimpse of the subsurface conductivity distribution can be obtained with a fast processing approach. In the second case, the use of a 1D inversion, based on the NLSTCI computational algorithm (short for nonlinear least-squares inversion of transient soundings for a central induction loop) allows to get a similar result but now relying on a wave equation development. It is a mathematically and computationally complex, and more time consuming technique. The two approaches were applied to airborne GEOTEMTM system real data, collected on a survey at San Pedro Valley (Southeast Arizona) in 1997.The available data from this region were made available by the USGS and were used in this quantitative analysis. As such, 2D sections were obtained, and from these, maps were generated. The results from 2D sections - conductivity spatial distribution obtained from CDIs and 1D inversions - were volumetrically interpolated (voxels) allowing a 3D representation of the conductivity spatial distribution for the surveyed area.

Methionine oxidation impairs reverse cholesterol transport by apolipoprotein A-I

HDL protects against vascular disease by accepting free cholesterol from macrophage foam cells in the artery wall. This pathway is critically dependent on lecithin:cholesterol acyltransferase (LCAT), which rapidly converts cholesterol to cholesteryl ester. The physiological activator of LCAT is apolipoprotein A-I (apoA-I), the major HDL protein. However, cholesterol removal is compromised if apoA-I is exposed to reactive intermediates. In humans with established cardiovascular disease, myeloperoxidase (MPO) oxidizes HDL, and oxidation by MPO impairs apoA-I's ability to activate LCAT in vitro. Because a single methionine residue in apoA-I, Met-148, resides near the center of the protein's LCAT activation domain, we determined whether its oxidation by MPO could account for the loss of LCAT activity. Mass spectrometric analysis demonstrated that oxidation of Met-148 to methionine sulfoxide associated quantitatively with loss of LCAT activity in both discoidal HDL and HDL(3), the enzyme's physiological substrates. Reversing oxidation with methionine sulfoxide reductase restored HDL's ability to activate LCAT. Discoidal HDL prepared with apoA-I containing a Met-148-->Leu mutation was significantly resistant to inactivation by MPO. Based on structural data in the literature, we propose that oxidation of Met-148 disrupts apoA-I's central loop, which overlaps the LCAT activation domain. These observations implicate oxidation of a single Met in apoA-I in impaired LCAT activation, a critical early step in reverse cholesterol transport.

Flexibility and charge asymmetry in the activation loop of Src tyrosine kinases

Regulated activity of Src kinases is critical for cell growth. Src kinases can be activated by trans-phosphorylation of a tyrosine located in the central activation loop of the catalytic domain. However, because the required exposure of this tyrosine is not observed in the down-regulated X-ray structures of Src kinases, transient partial opening of the activation loop appears to be necessary for such processes. Umbrella sampling molecular dynamics simulations are used to characterize the free energy landscape of opening of the hydrophilic part of the activation loop in the Src kinase Hck. The loop prefers a partially open conformation where Tyr416 has increased accessibility, but remains partly shielded. An asymmetric distribution of the charged residues in the sequence near Tyr416, which contributes to shielding, is found to be conserved in Src family members. A conformational equilibrium involving exchange of electrostatic interactions between the conserved residues Glu310 and Arg385 or Arg409 affects activation loop opening. A mechanism for access of unphosphorylated Tyr416 into an external catalytic site is suggested based on these observations.

Opalin, a Transmembrane Sialylglycoprotein Located in the Central Nervous System Myelin Paranodal Loop Membrane

In contrast to compact myelin, the series of paranodal loops located in the outermost lateral region of myelin is non-compact; the intracellular space is filled by a continuous channel of cytoplasm, the extracellular surfaces between neighboring loops keep a definite distance, but the loop membranes have junctional specializations. Although the proteins that form compact myelin have been well studied, the protein components of paranodal loop membranes are not fully understood. This report describes the biochemical characterization and expression of Opalin as a novel membrane protein in paranodal loops. Mouse Opalin is composed of a short N-terminal extracellular domain (amino acid residues 1-30), a transmembrane domain (residues 31-53), and a long C-terminal intracellular domain (residues 54-143). Opalin is enriched in myelin of the central nervous system, but not that of the peripheral nervous system of mice. Enzymatic deglycosylation showed that myelin Opalin contained N- and O-glycans, and that the O-glycans, at least, had negatively charged sialic acids. We identified two N-glycan sites at Asn-6 and Asn-12 and an O-glycan site at Thr-14 in the extracellular domain. Site-directed mutations at the glycan sites impaired the cell surface localization of Opalin. In addition to the somata and processes of oligodendrocytes, Opalin immunoreactivity was observed in myelinated axons in a spiral fashion, and was concentrated in the paranodal loop region. Immunogold electron microscopy demonstrated that Opalin was localized at particular sites in the paranodal loop membrane. These results suggest a role for highly sialylglycosylated Opalin in an intermembranous function of the myelin paranodal loops in the central nervous system.

Calculation of conductivity and depth correction factors for the S-layer differential transform

The VTEM system developed and operated by Geotech Limited and Geotech Airborne Limited is a central loop configuration system lending itself to many traditional ground interpretation strategies. One of these is the S-layer (thin, conductive layer) differential transform that is used to generate resistivity-depth sections. An empirical study indicated that delineating conductors in a conductive half space necessitates the implementation of a scale factor in order to obtain the correct depths and conductivity values when applying the S-layer differential transform.Based on an empirical approach, there was found to be an infinite number of depth correction factors that will still yield acceptable conductivity values, and the need arose to explain the origin of this discrepancy and to find the correct depth correction factor. A correction strategy was followed, based on scaling results to yield exact conductivities when applied to half-space models. Assuming that the equivalent filament for the S-layer behaviour, as with the equivalent filament for the half-space behaviour, does not coincide with the electric field maxima in the subsurface led to a plausible depth correction factor which was validated on various synthetic models.